key: cord- -gktnz authors: abreu, maria t; peyrin-biroulet, laurent title: providing guidance during a global viral pandemic for the care of patients with inflammatory bowel disease date: - - journal: j crohns colitis doi: . /ecco-jcc/jjaa sha: doc_id: cord_uid: gktnz nan the covid- pandemic has taken both physicians and patients by surprise. never in the history of man have we witnessed such a rapid spread of a virus, which highlights the vulnerability and interconnectivity of the world. the international organization for the study of inflammatory bowel disease [ioibd] is a society composed of ibd specialists from around the world. the members are gastroenterologists, both adult and paediatric, colorectal surgeons, and gastrointestinal pathologists. members are chosen on the strength of their contributions to the field of inflammatory bowel disease and because of their regional reputation as the leaders in ibd of their region. we have partnered closely with the european crohn's and colitis organisation because of our shared desire to provide the best guidance to patients with inflammatory bowel disease at a global level, as well as to the physicians that are caring for these patients. we have tried to remain cognisant of the fact that the majority of ibd patients are being cared for by general gastroenterologists who are all doing their best during this epidemic to keep their patients, staff, and themselves safe from infection. early on when this epidemic began, we chose to put our minds together and use what information we had at the time to develop guidance for patients. the process is called a rand panel wherein the members of ioibd, as well as other very knowledgeable practitioners of ibd, voted on a series of statements largely having to do with the risk of infection with the sars-cov- virus and the development of covid- in patients who have ibd. in particular, patients who are taking immunomodulators and biologic drugs are understandably concerned about the risk of contracting sars-cov- and developing more severe covid- . our first article in this online issue of jcc, dedicated to covid- and ibd, shares the information from that rand panel and goes further to describe how we resume medications, especially once patients who do have covid- or have presumed covid- have cleared the infection. it goes without saying that this is a moving target and, as we become more sophisticated in our ability to test accurately for covid- and/or to test accurately for resolution of the infection with antibody testing, we will again see changes in how we manage these ibd patients. we urge you to continue to check the ioibd or the ecco websites for the most up-to-date information, and we hope to keep updating the guidance in all of the various dimensions of ibd care. ecco has also developed guidance that is practical and described the do's and don't's of ibd care during this pandemic. like the ioibd guidance, it is an attempt to combine the available data with high-level ibd clinical experience. in many countries around the world, but particularly in northern italy and in new york where the pandemic has hit the hardest, there were many patients who required hospitalisation and who had ibd. patients with ibd may be hospitalised either because of a flare of their disease or because they have contracted covid- . one of the articles in this issue deals with guidance having to do with the hospitalised patients with ibd and how best to manage them. this article also has important information having to do with surgery for ibd and the timing of surgery for ibd. it provides a very nice structure for thinking about how these patients must be managed and what to be on the lookout for, including testing for covid, since between % and % of patients with covid- present with diarrhoea which may be confused with a flare of their ibd. for all intents and purposes, in many hospitals covid testing is occurring when the patient is in the emergency room. pregnant patients with ibd represent a special population because of the risks posed by active ibd on the developing fetus and the concerns surrounding medications' effects on the developing infant. in the era of covid- , pregnant women with ibd are particularly stressed about the potential for infection. dr de lima-karagiannis and colleagues from ecco have developed a very thorough document that describes what is known about pregnancy outcomes in relationship to covid- and the few cases of pregnant ibd patients with covid- . in their thoughtful guidance document, they present data that shows pregnancy outcomes are worse in patients that develop covid- , with higher rates of prematurity and spontaneous abortions. they acknowledge that there is likely to be reporting bias. in high-risk areas, a high rate of covid- positivity has been seen in pregnant patients who present to the hospital-thus making the case for universal testing in all pregnant women at the time of hospitalisation, although most will be asymptomatic from a covid- perspective. the article also provides useful information with respect to medications in the face of pregnancy with or with covid- infection. one of the sea changes that has occurred in the way we care for patients is the dialing on of telemedicine as a way to see our patients with ibd. this has been a giant step forward because for many of our patients with ibd, especially those who are on maintenance therapies and doing well, these televisits are a very compelling way to stay on top of what is happening with the patients. there are obviously nuances with respect to how different countries deal with this, especially as it relates to insurance and payers. we have done worldwide surveys of gastroenterologists with respect to how much telemedicine they were doing before and then after this pandemic, and the results are striking. we will never go back to the way things were and we will have telemedicine to stay. as time goes on, we will find new and improved ways to make this a very effective and efficient way to follow our patients. very early on, most of our patients wanted to know if it was safe to continue on their medications and was it safe to come into infusion centres. obviously, many of our patients with ibd are receiving intravenous medications including infliximab, vedolizumab, and the first infusion of ustekinumab. in many places, patients are also receiving these infusions at home. by the time you see this article, most home infusion centres and certainly all hospital-based infusion centres have developed protocols to keep patients and staff safe from getting covid- using the proper personal protective equipment [ppe] . we have an article devoted to infusion centre guidance and, again, this will continue to evolve as it becomes easier to test patients and see if they are positive. one can imagine if we had a very rapid accurate test, we could be performing this on patients who have not yet converted just before they are to receive an infusion. at the time we write this, this is not the case. as gastroenterologists, endoscopy is a very important part of what we do. in particular in patients with ibd, it has been the mainstay of documenting mucosal healing, not to mention looking for dysplasia in patients with chronic colitis. endoscopy runs the risk of aerosolising covid- in patients who are positive. although that risk is probably higher when doing upper endoscopies because of the airway, covid- has been found in stool and is known to be able to infect the lining epithelial cells. we have recently published a study looking at the expression of the two receptors that are required for viral entry and found that, in ibd patients and in healthy people, these receptors called ace and tmprss are both expressed in the gut. the ileum has the highest expression of ace and the colon has the highest expression of the tmprss . we did not see an increase in expression in ibd patients. if anything, inflammation lowers the expression of ace . we saw a slight protective effect or lower levels in patients who were on infliximab. although these studies were all done from tissue collected previously and not from patients infected with covid- , they provide reassurance that patients with ibd are not at a greater risk of either shedding it from their stool or of contracting it through a faecal-oral route. our colleagues provide guidance as to how best to organise an endoscopy unit and who to prioritise within the ibd space for doing procedures. for those of us who perform clinical trials in ibd, most of the pharmaceutical companies transiently put on hold enrolling new patients into the study and are dealing with what to do with patients who are already in the study, especially those that require in-person visits or colonoscopies, stool collection, or blood collection as part of the study. we provide a general overview of how the food and drug administration and the european medicines agency have dealt with this crisis and how best to continue clinical trials and to be ready, when things get better, to start up again with clinical trials. and finally, this pandemic in many ways has given us an opportunity to think and analyse what we have been doing. it has energised all of us to think about how this can be a platform for investigation. the manuscripts provide many important, unanswered questions that as a community of physicians caring for ibd patients we should work together to address. we hope that you will find the information in this issue helpful. please stay tuned and continue to visit our websites at [ioibd.org] or [ecco-ibd.eu] for the latest information on all these topics. we have also provided on the websites downloadable content, in particular infusion centre, endoscopy, and telemedicine guidelines that can be printed and posted in your centres. management of patients with crohn's disease and ulcerative colitis during the covid- pandemic: results of an international meeting gastrointestinal and hepatic manifestations of novel coronavirus disease in a large cohort of infected patients from new york: clinical implications sars-cov- productively infects human gut enterocytes expression of sars-cov- entry molecules ace and tmprss in the gut of patients with ibd this paper was published as part of a supplement financially supported by ecco and ioibd. mta has consulted for janssen, prometheus bioscience, takeda, focus medical communications, pfizer, boehringer ingelheim pharmaceuticals, gilead, imedex, cornerstone health, inc, landos biophama, ucb biopharma srl, eli lilly, and cosmo biopharma. mta has received grant support from prometheus bioscience, takeda, and pfizer. l.p.b. has received personal fees from abbvie, janssen, genentech, ferring, tillots, pharmacosmos, celltrion, takeda, boerhinger ingelheim, pfizer, index pharmaceuticals, sandoz, celgene, biogen, samsung bioepis, alma, sterna, nestle, enterome, allergan, msd, roche, arena, gilead, hikma, amgen, bms, vifor, norgine; mylan, lilly, fresenius kabi, oppilan pharma, sublimity therapeutics, applied molecular transport, ose immunotherapeutics, enthera and theravance; grants from abbvie, msd and takeda. key: cord- -qsfykh k authors: nedelsky, natalia b.; kuballa, petric; castoreno, adam b.; xavier, ramnik j. title: inflammatory bowel disease at the intersection of autophagy and immunity: insights from human genetics date: - - journal: molecular genetics of inflammatory bowel disease doi: . / - - - - _ sha: doc_id: cord_uid: qsfykh k studies using human genetics have identified more than loci that affect the risk of developing inflammatory bowel disease (ibd), including crohn’s disease (cd) and ulcerative colitis (uc). several of these genes have been found to play key roles in the process of autophagy, a lysosome-based degradation pathway. although historically considered to be a relatively nonselective process of degradation of cytosolic contents, autophagy has recently been revealed to have several selective and immune-specific functions that are relevant to the maintenance of intestinal homeostasis, including xenophagy, mitophagy, antigen presentation, secretion, and inflammasome regulation. in this chapter, we review the evidence that links autophagy-related genes, their immune-specific functions, and possible mechanisms of ibd pathogenesis. we summarize the basic molecular events underlying general and selective autophagy and present evidence suggesting possible pathogenic mechanisms revealed by studies of ibd-associated risk alleles of atg l and irgm. finally, we review chemical biology-based experimental approaches for identifying autophagy regulatory pathways that may have implications for the development of therapeutics. genome-wide association studies (gwas) of ibd have been a major success story for genome-level studies of complex human disease. one of the particularly exciting results from these studies was the identifi cation of autophagy as an unanticipated player in determining risk of ibd. this pathway, long considered to be a relatively nonselective process of bulk cytoplasmic degradation by the lysosome, is thought to have evolutionary roots as a mechanism to maintain metabolic homeostasis in response to decreased nutrient levels [ ] . however, studies over the last years have expanded the functions of autophagy to include roles in organelle clearance, antigen presentation, cell death, development, and the degradation of intracellular pathogens, among others. the role of autophagy in immune-related processes [ - ] was particularly unexpected and has served as a guide in many studies seeking to connect human genetics to mechanisms of ibd pathogenesis [ - ] . these genetic studies have identifi ed multiple genes within the autophagy network as playing a role in cd, including atg l , irgm , nod , and lrrk [ - ] . in the case of uc, the autophagy-related genes dap and smurf have also been identifi ed as determinants of risk [ , ] . further studies subsequently identifi ed polymorphisms in ulk , atg a , and gabarapl as harboring significant associations with cd [ , ] . although autophagy has been implicated in the disease process in a range of other disorders, including neurodegenerative disease [ , ] , atherosclerosis, and cancer, to date no compelling genetic association has been found between autophagy and many immune-related diseases, including multiple sclerosis, celiac disease, rheumatoid arthritis, and psoriasis. however, targeting sequencing studies have also begun to identify coding mutations in essential autophagy genes as playing roles in disease, raising the possibility that more diseaseassociated autophagy-related polymorphisms have yet to be uncovered [ , ] . in this chapter, we review studies that have employed human genetics to understand the link between autophagy and ibd. we begin by examining the idea of the intestinal mucosa as a physiological system wherein homeostasis is particularly vulnerable to disruptions in autophagy. we then present a basic overview of the molecular events involved in autophagy, followed by discussions of how autophagy may function as a selective process with critical roles in ibd-related immunity. these roles include ( ) degradation of intracellular pathogens (xenophagy), ( ) mitophagy, ( ) antigen presentation, ( ) secretion, and ( ) infl ammasome regulation. in the cases of xenophagy, mitophagy, and antigen presentation, we present detailed evidence of how autophagy functions in these pathways. next, we review results from studies of two particularly well-examined cd risk single-nucleotide polymorphisms (snps), rs (atg l , coding variant t a) and rs (irgm). finally, we discuss approaches from the fi eld of chemical biology that may be used to identify autophagy regulatory pathways with possible implications for therapeutics development. as mentioned above, autophagy has been implicated in several immune-related processes that infl uence ibd pathogenesis, including xenophagy, mitophagy, antigen presentation, secretion and vesicular traffi cking, and cytokine-based regulation of infl ammasome activity. one major point of intersection between these processes is their critical importance in the intestinal mucosa. this complex environment must maintain a delicate balance of immune responses, which are continuously stimulated by metabolic stresses and antigens from food. microbes also play a major role in this milieu, which carries the highest bacterial load in the body. this tissue, therefore, may be especially susceptible to disruption in autophagy, with consequent alteration of homeostatic processes. for example, mice with conditional knockout of atg in intestinal epithelial cells show increased severity of colitis when exposed to the infectious pathogen citrobacter rodentium , while mice with defi ciency in the core autophagy gene atg l in hematopoietic cells show increased susceptibility to chemically induced models of colitis [ , ] . studies in human genetics have provided evidence that microbial stimulation may exist as a node of functional interaction between cd susceptibility genes, including atg l and nod . for example, expression of the atg l cd-associated risk allele (atg l t a) results in impairment of autophagy and antigen presentation, both of which are enhanced by muramyl dipeptide (mdp), the bacterial ligand for the receptor nod [ , ] . although no evidence has been found for an epistatic interaction between these two genes, this type of functional interaction suggests that autophagy may operate as a key point of intersection among multiple susceptibility genes. the process of autophagy begins with an appearance of a fl at membrane sheet termed the isolation membrane (also known as the phagophore assembly site in yeast), the origin of which remains unknown. the membrane elongates and expands to encompass its cargo, eventually fusing to form a double-membrane structure known as an autophagosome. the outer autophagosomal membrane then fuses with the lysosomal membrane, forming a degradative autolysosome. it should be noted that the source of the autophagosomal membrane is still a highly debated topic and the existence of multiple sources cannot be excluded. in the case of the er, a novel Ω (omega)-shaped vesicle called the omegasome has been identifi ed, which resides on the er and might precede the appearance of the isolation membrane [ ] . many of the molecular events underlying the assembly and elongation of autophagy membranes were initially identifi ed by genetic screens in saccharomyces cerevisiae [ , ] . these screens have identifi ed approximately core autophagy proteins, about half of which have clear homologues in mammals. for the purposes of this chapter, we divide these genes into fi ve functional categories: ( ) the ulk complex (atg complex in yeast); ( ) atg ; ( ) vps complexes; and ( , ) two ubiquitin-like conjugation systems anchored by atg and atg , respectively. the ulk complex (composed of atg , fip , and either ulk or ulk ) plays a central role in starvation-induced autophagy and is thought to be involved in the initiation of the autophagic cascade. the complex is recruited to sites of autophagosome formation, where it likely acts to recruit and disassemble other atg protein complexes [ - ] . atg , a transmembrane protein, shuttles between autophagosomes, the trans-golgi network, and late endosomes in a cycling process that may involve the ulk complex. atg is of special interest to studies of xenophagy, since a recent report suggested that atg is essential for the formation of the isolation membrane in anti-salmonella autophagy in mouse embryonic fi broblasts (mefs) [ ] . in mammals, the vps complex is likely required for the recruitment of several autophagy proteins to sites of autophagosome formation. this complex plays a critical role in supplying phosphatidylinositol -phosphate (pi p) to the growing autophagosome, where it acts as an essential driving force in autophagosome assembly [ - ] . furthermore, depending on the identity of its protein components, some alternative versions of the vps complex may serve regulatory roles in autophagosome maturation and autophagosome-lysosome fusion [ - , - ] . two ubiquitin-like conjugation systems play important roles in autophagosome formation and maturation. the atg -based conjugation system is initiated by the e -like atg , which activates and transfers atg to the e -like atg . atg is then subsequently conjugated to atg [ , ] . atg and atg then bind with atg l , forming a complex that is essential for autophagosome formation [ ] . in addition to a key role in autophagosome formation, the second ubiquitin-like conjugation system may play additional roles in cargo recognition [ ] . in this system, atg is activated by the e -like atg , is transferred to the e -like atg , and is conjugated to phosphatidylethanolamine through the e -like action of the atg -atg complex. the conjugation of phosphatidylethanolamine to atg allows subsequent incorporation of atg into the inner and outer membranes of autophagosomes [ - ] . mammals appear to have two families of atg homologues, known as lc s and gabaraps. while all atg homologues can be incorporated into the autophagosomal membrane, the incorporation of lc s and gabaraps might occur at different stages, with lc s acting at the stage of biogenesis and gabaraps playing a more important role in autophagosome maturation [ ] . given that atg remains associated with autophagosomes even after their fusion with lysosomes, tracking atg and its homologues allows for localization of autophagosomes. furthermore, since the inner membranes of autophagosomes are degraded upon formation of autolysosomes, tracking atg enables the measurement of autophagic fl ux as refl ected by atg degradation [ , ] . several layers of regulation exist in the initiation and progression through the steps of autophagic degradation. one important regulatory infl uence is the cytoskeleton. autophagosome traffi cking involves movement along microtubules, which facilitates fusion with lysosomes [ , ] . changes in lysosome positioning regulate mtorc signaling, since peripheral localization of lysosomes and mtorc increases mtorc activity, while perinuclear lysosomal clustering decreases activity [ ] . heat shock protein beta- (hspb ), a protein that interacts with and mediates reorganization of the actin cytoskeleton, is also linked to mitophagy [ ] . disruption of hspb signaling can lead to accumulation of dysfunctional mitochondria, mitochondrial fragmentation, and effects in mitochondrial respiration in mefs [ ] . transcription also serves as a regulatory infl uence in the autophagic process. autophagosome and lysosome biogenesis appear to be transcriptionally coregulated by the transcription factor eb (tfeb) [ ] . overexpression of tfeb induces autophagy, while a block of tfeb expression by rnai decreases autophagy. interestingly, tfeb action is implicated in basal as well as starvation-induced autophagy, and therefore tfeb might represent a transcriptional master regulator of the autophagy pathway. furthermore, translocation of tfeb from the cytosol to the nucleus is strongly enhanced upon removal of nutrients, suggesting that tfeb function is sensitive to nutrients and growth factors. under fed conditions, phosphorylation on serine of tfeb can be mediated by the extracellular signal-regulated kinase (erk ) and might be suffi cient to retain tfeb in the cytosol. a comprehensive analysis of genes with known functions in the autophagy pathway sheds some light on transcriptional regulation of autophagy in the context of starvation and tfeb function. strikingly, tfeb-dependent transcription patterns generally correlate with starvation effects, further emphasizing the role of tfeb as an autophagy regulator that can directly or indirectly sense nutritional status [ ] . however, the transcription of the majority of genes is not signifi cantly affected by starvation or tfeb expression levels [ ] , suggesting that transcription factors other than tfeb control the expression of most autophagy and autophagy-related genes, possibly in a nutrient-insensitive fashion. autophagy has now been well described as playing a key role in the degradation of intracellular pathogens, including bacteria, in a specialized process known as xenophagy. however, the environment of the intestinal mucosa provides special challenges to this function. as this tissue is exposed to both commensal and pathogenic bacteria, the interactions between these fl ora and host immune responses must be tightly controlled. the molecular events surrounding the process of xenophagy follow the same basic pattern as those involved in autophagic degradation of other cellular contents, such as organelles or aggregated protein deposits. however, the mechanisms underlying the selection of cargo for degradation by xenophagy remain largely unknown. components of the innate immune response can interact with autophagy proteins to target bacteria for autophagic degradation. mechanisms involved in xenophagy of other species of bacteria may share some components with anti-salmonella xenophagy, but to date, detailed molecular mechanisms have been best explored for salmonella . upon infection of epithelial cells, salmonella enterica serotype typhimurium ( s . typhimurium) resides in salmonella -containing vacuoles (scvs). the environment of the scv promotes bacterial survival and replication. the scv protective niche is established by effectors of two type iii secretion systems (t sss) encoded by salmonella pathogenicity islands and (sp- and sp- ). the secretion and activity of t ss components is highly regulated both temporally and spatially. for example, ph sensing regulates the transition from translocon protein secretion and pore formation to effector secretion [ ] . once secreted into the host cell, the activity of effectors can be modulated. for example, the t ss sp- effectors sopa, sope, sptp, and sopb are all ubiquitinated shortly after infection by the host cell machinery [ ] . the phosphoinositide phosphatase activity of sopb regulates several processes that promote internalization and survival of s . typhimurium, including cytoskeletal rearrangements that promote invasion, scv biogenesis and maturation, and prosurvival akt activation [ ] . the ability of sopb to regulate these functions is dependent upon its ubiquitination status [ , ] . a sopb mutant that cannot be ubiquitinated has a number of defects, including a failure to relocalize from the plasma membrane to scvs, disrupted recruitment of rab to scvs, and perturbed s . typhimurium replication [ ] . thus, ubiquitination-dependent relocalization of sopb to scvs promotes the establishment of scvs as a niche environment for replication. this is due in part to the ability of sopb to reduce the net negative charge of pi( , )p and phosphatidylserine lipids on scv membranes, thereby disrupting maturation and fusion with lysosomes [ ] . although most s . typhimurium resides in scvs, a small but signifi cant fraction is released into the cytosol from damaged scvs. this cytosolic s . typhimurium rapidly becomes ubiquitinated. ubiquitinated bacteria may be recognized by selective cargo receptors, including p and nbr , which act as bridging factors that bind to both ubiquitin and the autophagosome. p and nbr share a similar protein structure and domain composition and may show some level of redundancy, particularly in degradation of protein aggregates (aggrephagy) and mitochondria (mitophagy). however, while p is important for the autophagic clearance of salmonella , nbr is not [ ] . therefore, the mechanism of xenophagy, which relies on ubiquitination events on or around intracellular bacteria, might differ from other ubiquitin-dependent selective autophagy events. interestingly, nuclear dot protein (ndp ), in addition to p , has been implicated in the recognition of ubiquitin-coated invasive bacteria, but not in other forms of selective autophagy [ ] . ndp contains a putative lir motif at its n terminus, can directly bind ubiquitin through a c-terminal ubiquitin-binding zinc fi nger, and can form homo-oligomers via its coiled-coil domain [ - ] . p and ndp appear to act cooperatively in the recognition and killing of s . typhimurium, listeria monocytogenes , and shigella fl exneri [ , ] . however, p and ndp are recruited to distinct microdomains of invading salmonella , suggesting that ubiquitin coats on salmonella might be heterogeneous [ ] . interestingly, in macrophages but not in epithelial cells, proteasomes localize to ubiquitin-coated salmonella with potential effects on effi cient rupture of damaged scvs and major histocompatibility complex (mhc) class i antigen presentation [ , ] . taken together, ubiquitin-dependent autophagic targeting of intracellular pathogens and by-products has implications for pathogen growth and the regulation of infl ammatory responses. however, in most cases, the e ubiquitin ligases, the nature of ubiquitin modifi cations, and the substrates involved in ubiquitin-dependent xenophagy are not known and may be pathogen-as well as cell type-specifi c. one important exception is lrsam , an lrr-and ring-domain protein that was recently identifi ed as an e ubiquitin ligase crucial for ubiquitin-dependent autophagy of s . typhimurium [ ] . interestingly, lrsam was required for ubiquitination of intracellular bacteria but dispensable for ubiquitination of aggregated proteins, confi rming that lrsam serves as a selective and novel antibacterial sensor that mediates target selection for the xenophagy pathway. to date, however, lrsam remains the only known ubiquitin ligase associated with antibacterial autophagy, and a detailed biochemical characterization of the aforementioned factors will yield critical insights into host-pathogen interactions and may boost the development of novel pharmaceuticals aimed to combat infectious diseases. as mentioned above, intestinal tissues are in constant interaction with both commensal and pathogenic bacteria. to prevent inappropriate responses to commensal bacteria, the cells of the intestinal mucosa must be educated to tolerate these microbes and their products [ ] . breakdown of this tolerance, with consequent inappropriate infl ammation, has been proposed to play a major role in the pathogenesis of ibd. this model is supported by the fi nding that the ibd-associated genes atg l , irgm , and ulk all play roles in the process of xenophagy. atg l , irgm, and ulk each function in the degradation of s . typhimurium, while atg l and irgm also play a role in antibacterial autophagy of a cd-relevant strain of escherichia coli (adherent-invasive e. coli , or aiec) [ , - ] . ulk may also function in the degradation of l. monocytogenes . furthermore, the function of lrrk in reactive oxygen species (ros)-driven bacterial killing, as well as its role in autophagy, raises the possibility that this protein may also be involved in xenophagy of gut bacteria [ - ] . defects in these gene products might lead to impaired bacterial handling, promoting an environment of chronic infl ammation and inappropriate host responses to commensal bacteria. some intracellular pathogens have also been highlighted as potential contributors to risk of cd, including mycobacteria. genetic loci that include irgm , lrrk , and nod have been associated with increased susceptibility to leprosy and tuberculosis, suggesting an interesting point of interaction between autophagy genes, cd, and mycobacteria. these studies, combined with reports that autophagy may play a key role in control of mycobacterium tuberculosis , have led to the suggestion that control of mycobacteria might be altered in cd [ , ] . genetic studies appear to support this idea, especially in the context of granulomas, a histological hallmark of both cd and tuberculosis: polymorphisms in loci containing the autophagy genes atg a and gabarapl have been linked to granulomas in cd patients. to date, however, connections between cd and the mycobacterioses remain largely speculative, to be answered perhaps by future well-powered gwas for tuberculosis [ ] . the recent wealth of data from the human microbiome project has also highlighted how little we understand of the complex relationship between ibd and microbial stimulation. certainly the diversity and composition of the gut microbiota are major factors infl uencing gut homeostasis, and particular dietary nutrients and metabolites are likely to interact with host genetics to infl uence host-microbiome interactions. an imbalance in the composition of the gut microbiome, termed dysbiosis, has been associated with ibd, including shifts in relative abundances of bacterial taxa, decreases in the diversity of the bacterial community, and alterations in the functional composition of the microbiome. the autophagic processing of mitochondria (mitophagy) was directly implicated in the context of ibd by a recent study that identifi ed smurf as a susceptibility gene for uc [ ] . this gene is an ubiquitin ligase that was recently identifi ed, through an image-based genome-wide sirna screen, as an important mediator of selective viral autophagy and mitophagy [ ] . in contrast to other substrates for selective autophagy, for example, surplus peroxisomes or ribosomes, mitophagy is well studied and underlying biochemical mechanisms for this process are emerging. upon loss of mitochondrial membrane potential, pten-induced putative kinase (pink ) accumulates on the outer mitochondrial membrane [ , ] . this step is accompanied by stabilization of pink protein and may involve a membrane potential-dependent block of pink proteolysis by the mitochondrial inner membrane protease presenilin-associated rhomboidlike protein (parl). this protease is effective in healthy mitochondria but impaired in damaged mitochondria [ , ] . pink then recruits parkin to damaged mitochondria, which results in parkin -dependent ubiquitination of some mitochondrial proteins such as voltage-dependent anion channel (vdac ) and mitofusins [ - ] . p accumulates around ubiquitin-decorated damaged mitochondria and pb -domain-mediated oligomerization of p results in mitochondrial clustering, eventually leading to autophagic degradation of mitochondria [ ] . it should be noted that the mitochondrial ubiquitination event required for mitophagy may involve multiple substrates and/or may be cell type-specifi c, as p recruitment to damaged mitochondria and mitophagy is not impaired in vdac -defi cient mefs [ ] . in addition, activating molecule in beclin -regulated autophagy (ambra ), an autophagy-regulating protein initially identifi ed in neuronal cells [ ] , is recruited to damaged mitochondria in a parkin -dependent fashion [ ] and knockdown of ambra expression results in impairment of mitophagy. interestingly, ambra recruitment most likely involves direct binding to parkin , but ambra is not a substrate for parkin -mediated ubiquitination [ ] . thus, parkin -mediated mitophagy relies at least in part on effects other than ubiquitination. as mentioned above, the best evidence linking mitophagy to ibd comes from the identifi cation of smurf as a gene associated with susceptibility to uc. smurf is a hect domain-containing e ubiquitin ligase that interacts with the selective autophagy factor p [ ] . smurf −/− mefs show defects in the degradation of sindbis and herpes simplex virus as well as impaired clearance of damaged mitochondria. supporting this observation, smurf -defi cient mice display an accumulation of damaged mitochondria in the heart, brain, and liver. interestingly, smurf −/− mefs are competent for starvation-induced bulk autophagy, indicating that smurf is a specifi c mediator of selective autophagy. however, a mechanistic link between smurf polymorphisms and ibd pathogenesis remains to be examined. evidence for a link between ibd, antigen presentation, and autophagy originates from studies of cd-associated risk variants in nod and atg l . although a role for autophagy in antigen presentation has been well described in basic studies, how this function may be altered in the context of ibd remains relatively unknown. mhc molecules on the cell surface present peptide antigens to t cells. mhc class i molecules interact with the t cell receptor (tcr) of cd + cytotoxic t cells, while mhc class ii molecules interact with the tcr of cd + helper t cells. mhc class i antigens are derived from proteasomal degradation of cytosolic proteins. in contrast, mhc class ii antigens are delivered to the lysosome before transfer to the mhc class ii loading compartment and transport to the cell surface. delivery of these antigens to the lysosome can occur either via endocytosis of protein antigens from the extracellular space or by autophagy of cytoplasmic material [ - ] . autophagy also plays an important role in the presentation of pathogen antigens by dendritic cells (dcs). mice lacking atg specifi cally in dcs show impaired cd + t cell priming following infection with herpes simplex virus, an impairment that is due to a decreased ability of atg -defi cient dcs to process and present antigens for presentation on mhc class ii molecules [ ] . furthermore, these cells show delayed fusion of phagosomes to lysosomes. autophagic degradation may also combine with vesicular traffi cking to facilitate presentation of citrullinated self-peptides by dcs to cd + t cells. this fi nding is particularly relevant to ibd, since immune responses against citrullinated self-proteins are associated with autoimmunity [ , ] . interestingly, dcs from patients with cd-associated risk variants of atg l or nod show defects in autophagy induction, bacterial traffi cking, and mhc class ii antigen presentation to cd + t cells [ ] . furthermore, autophagy was recently reported to destabilize the immune synapse between dcs and t cells; in this report, decreased expression of atg l or irgm resulted in hyperstable interactions between dcs and t cells as well as increased activation of t cells, suggesting a mechanism by which adaptive immunity might be increased in patients with cd who carry atg l risk alleles [ ] . the suggestion that the secretory pathway might be modulated by autophagy in the context of cd arose from the gwas-based identifi cation of atg l as a common cd-associated risk gene. patients carrying the cd-associated variant of this gene (atg l t a), as well as atg l hypomorphic (atg l hm ) mice, were found to show abnormalities in specialized epithelial cells called paneth cells. these cells play a central role in innate immunity and are an important source of antimicrobial peptides in the small intestine, serving to prevent microbial invasion and control the composition of the gut microfl ora [ ] . atg l t a patients and atg l hm mice exhibit decreased numbers of granules and diffuse staining for lysozyme in their paneth cells, raising the possibility that autophagy plays a key role in secretion in these cells [ , ] . supporting this hypothesis, mice lacking atg or atg in intestinal epithelial cells also show paneth cell defects [ ] . interestingly, the paneth cell phenotype in atg l hm mice can be infl uenced by exposure to a pathogen, since the cellular defects can be eliminated by maintaining mice in a virus-free environment [ ] . paneth cell defects have also been observed in mice lacking other cd-associated genes, including nod and xbp [ , ] . more recently, a role was reported for paneth cells in sensing nutrient availability, demonstrating that caloric restriction reduces paneth cell-specifi c signaling by mtorc , a regulator of autophagy [ ] . taken together, these reports suggest that paneth cell homeostasis and function are intimately linked to autophagy. however, whether autophagy directly regulates vesicular traffi cking and secretion remains unknown, since the molecular mechanisms by which atg l might regulate these cellular functions are poorly understood. infl ammasomes are molecular scaffolds that activate caspase and maturation of the proinfl ammatory cytokines interleukin (il)- β and il- . several types of infl ammasomes have been described, which are activated by a number of endogenous and exogenous signals. recent studies have shown that autophagy defects can lead to infl ammasome hyperactivation. for example, stimulation of atg l −/− fetal macrophages with lipopolysaccharide (lps) induces elevated levels of activate caspase and enhanced secretion of il- β and il- [ ] . mice with specifi c deletion of atg l in hematopoietic cells also show higher serum levels of il- β and il- in response to dextran sulfate sodium (dss), accompanied by increased infl ammation and mortality. the underlying mechanism linking autophagy to regulation of the infl ammasome remains unknown. one possibility is that defects specifi cally in mitophagy result in increased levels of ros, which are known to cause hyperactivation of infl ammatory activity [ - ] . supporting this idea, atg l −/− macrophages treated with a ros scavenger did not show the increased il- β secretion described above [ ] . samples from patients have also shown atg l coding variant-dependent alterations in il- β secretion. in these studies, stimulation of peripheral blood mononuclear cells with mdp, but not lps, resulted in a relative increase of il- β secretion from atg l t a-expressing cells. although no difference was found in levels of activated caspase , protein levels of pro-il- β and mrna levels of il- β were increased in the context of the atg l risk allele [ ] . the autophagy adapter p may also provide a connection between il- β signaling and atg l [ ] . in a recent study, we showed that levels of p are normally regulated by atg l -based activation of the ubiquitin ligase cullin- , which promotes proteasomal degradation of p . loss of atg l , however, can result in decreased levels of cullin- and decreased degradation of p . as p can act as a scaffold in the il- β signaling pathway, elevated levels of p may result in amplifi ed il- β signaling [ ] . one of the best-studied cd-associated risk alleles is the t a coding polymorphism in atg l . atg l −/− mice are not viable [ ] , but an alternative hypomorphic model (atg l hm ) has yielded important clues to how the t a coding polymorphism may result in altered autophagy and increased infl ammation [ , ] . the site of the t a polymorphism is near a wd-repeat domain, which is present in mammalian atg l but absent in yeast atg . this domain is not required for autophagy, consistent with the fi nding that the t a polymorphism does not affect classical autophagy [ , ] . as a core autophagy protein, atg l has been shown to play multiple roles in ibd-relevant processes, including xenophagy, antigen presentation, il- β production, and secretion (see fig. . ) . interestingly, the cd-associated risk allele another role for autophagy in secretion is suggested by observations in macrophages, in which secretion of active il- β (as well as active il- , not shown) upon lps stimulation is regulated by autophagy at two levels. in this pathway, loss of atg l expression is associated with increased cytokine secretion. ( b ) autophagy can also act as a sensor for bacterial products. in dendritic cells, activation of nod by mdp induces autophagy that leads to mhc class ii antigen presentation. this pathway appears to be altered in the context of cd-associated snps in atg l and nod . ( c ) autophagy plays a role in targeting bacteria and bacterial products for degradation/killing. in one pathway, observed in primary blood mononuclear cells, activation of nod by mdp induces autophagy that leads to production of pro-il- β and secretion of active il- β. the cd-associated variant atg l t a is associated with increased amounts of active il- β upon mdp stimulation. autophagy can also directly degrade bacteria. in epithelial cells, bacteria become ubiquitinated and targeted for autophagic degradation in a process that requires the cd-associated genes atg l and irgm appears to have different effects on these functions depending on the cell type examined [ , ] . it is perhaps not surprising, then, that t a has also been associated with defects in antigen presentation increases in il- β production and abnormalities in paneth cell secretion in patient samples [ , , ] . in addition, antibacterial autophagy of salmonella is affected by the presence of the atg l cd risk allele [ ] , and patients with the t a allele show increased susceptibility to infection by helicobacter pylori [ ] . these fi ndings illustrate that disease genes are likely to have specifi c functions in specifi c cell types, and researchers must consider the cell type-and stimulation-specifi c contexts used to examine disease-associated phenotypes. irgm (immunity-related gtpase family m) is a human gene recently identifi ed as playing an important role in antibacterial and antiviral autophagy. to date, xenophagy of pathogens including s . typhimurium, m . tuberculosis , and cd-associated aiec has been reported to be dependent on irgm [ , , , ] . a synonymous snp within the human irgm locus (rs ) is associated with cd, and a kb deletion polymorphism within the ′ untranslated region of irgm is in perfect linkage disequilibrium with this snp [ , ] . however, both rs and the deletion polymorphism have also been reported to be in perfect linkage disequilibrium with a synonymous exonic snp (rs ). this snp is associated with decreased binding of the microrna mir- to the ′ utr of irgm , an event that downregulates expression of irgm [ ] . this fi nding is consistent with the observation that expression of mir- is increased in epithelial cells of the infl amed ileum and colon of cd patients compared with controls [ ] . this result may be relevant to autophagic targeting of pathogenic bacteria, since knockdown of irgm or overexpression of mir- affects the autophagic targeting of aeic. furthermore, an additional irgm polymorphism (− tt) is associated with an increase of irgm expression and protection against m . tuberculosis [ ] . despite the approaches described in the studies above, in which individual autophagy genes are examined in relative isolation, it is important to note that a complex network of autophagy-related proteins exists in the cell and that cellular functions captured within this network likely extend beyond the strict bounds of lysosomal degradation. as mentioned above, autophagy is implicated in many cellular pathways, including adaptations to changes in environmental factors such as nutrient availability. many autophagy and autophagy-related proteins have been characterized, and distinct autophagic complexes with functions in autophagosome assembly and maturation have been identifi ed and analyzed in great detail. the current picture suggests a stepwise process, but the precise regulation of autophagy in changing environmental settings-including fl uctuations in growth factor supply, cytokine signaling, and the presence of pathogens-likely involves complex cross talk between pathway components. behrends and colleagues recently used an approach which might be described as "pathway proteomics" to investigate the network organization of the human autophagy system [ ] . in doing so, they provided a basis for understanding the autophagy network on a protein level. in brief, interaction partners for epitope-tagged stably expressed proteins, representing core autophagy as well as autophagy-related processes, were identifi ed using mass spectrometry following immunoprecipitation of the respective tagged protein. a subset of proteins interacting with primary baits were used as baits in secondary screens, leading to the identifi cation of high-confi dence candidate interactions between a total of baits and an additional proteins. a high coverage of known interactions, a high level of validation (reciprocal immunoprecipitations and in vitro assays), and validation of autophagy functions of network components via rnai allow for the designation of these results as a true autophagy protein interaction network. interestingly, different autophagy subnetworks such as the ulk complex, vps complex, atg conjugation system, and pi p signaling members were revealed to have an underappreciated high level of interconnectivity. novel interaction partners for the mammalian atg homologues were also identifi ed. these interactors may be shared among all atg homologues, specifi c for lc or gabarap subfamily members or specifi c for individual atg homologues. however, differential cellular abundance of distinct atg homologues could in part account for different effi ciencies with respect to the co-immunoprecipitation of atg interaction partners [ ] . nevertheless, differences in lc -interacting region (lir) motif composition among different atg homologues can affect binding to p , nbr [ ] , and possibly control differential requirements of lc and gabarap subfamily members in early or late phases of autophagosome biogenesis [ ] . in addition, it will be interesting to see if some of the novel atg interactors might function as receptors for certain types of selective autophagy. good candidates might be recovered by a comparison of atg interactors identifi ed by these proteomics studies and a list of proteins predicted to combine lir and ubiquitin-binding motifs (kay hofmann, unpublished data in [ ] ). interestingly, pharmacological induction of autophagy led to alterations of protein-protein interactions within some, but not all, subnetworks [ ] . it will be interesting to see if such clusters might be targets of micrornas, which generally modify the translation of multiple genes at the same time. in addition, it is possible that different subnetworks are altered by different stimuli, possibly in a cell typespecifi c fashion, which could ultimately help in the design of disease-specifi c autophagy-enhancing or autophagy-inhibiting drugs. as highlighted throughout this chapter, many unanswered questions remain regarding the mechanistic basis of multiple autophagy functions. we believe that the fi eld of chemical biology is poised to help answer these questions, providing the tools to dissect the regulatory nodes that exist at the intersections of autophagy and immunity. furthermore, studies in this fi eld may help to identify druggable targets within the autophagy pathway and its regulatory infl uences. small molecules have been proven to be useful tools in autophagy research, demonstrating success by increasing our knowledge of autophagy regulation while simultaneously identifying potential therapeutic compounds. small-molecule screens are particularly important in identifying probes for dissecting complex biological processes such as autophagy, as they allow the dissection of multiple steps in a pathway, provide temporal control over target function, and are often reversible. these screens are also particularly powerful in enabling researchers to identify molecules that parallel gene activity. furthermore, by pursuing the mechanism of action (moa) of active compounds, novel regulatory pathways can be identifi ed. target identifi cation and moa determination is a rate-limiting step to smallmolecule discovery in phenotype-based screens and relies on the integration of multiple complementary approaches. one such approach is candidate based, which can be used to determine whether novel small molecules target known moas. several unbiased approaches to determine small-molecule moas can also be used, including ( ) combining quantitative proteomics (silac) with affi nity enrichment to identify proteins that interact with the small molecule and yield the observed phenotype, ( ) compound profi ling and connectivity analysis using gene expression signatures, and ( ) genetic complementation of small-molecule effects by rnai or overexpression screening to identify genes that function in the same pathway as the small molecule [ , ] . several forward chemical screens have been used to identify compounds with relevance to autophagy. these cell-based phenotypic screens employed libraries of fda-approved drugs and known bioactive compounds to identify chemical modulators of autophagy [ - ] . various readouts were used to measure autophagic activity, including gfp-lc puncta formation, the clearance of mutant huntingtin and a t α (alpha)-synuclein aggregates, and degradation of luciferase-fused lc . autophagy-related small molecules discovered in these screens can be broadly classifi ed into two groups: ( ) mtor-dependent molecules, which consist of compounds that induce autophagy, and ( ) mtor-independent molecules, which can be either inducers or suppressors of autophagy. the fi rst group includes inhibitors of class i pi k/akt/mtor signaling, such as allosteric and atp-competitive mtor kinase, akt, pi k, and dual mtor/pi k inhibitors. the second (mtorindependent) group includes inhibitors of camp-epac-plc-ε (epsilon)-ip and ca + -calpain-gs-α (alpha) signaling [ ] . results from such chemical screens have been carried forward into cell and animal models of autophagy-related diseases. for example, (smer) compounds were originally identifi ed in a screen for enhancers of rapamycin-induced growth defects in s. cerevisiae [ ] . smers , , or were found to be active in mammalian cell models of huntington's and parkinson's disease, promoting autophagic clearance of mutant huntingtin and a t α (alpha)-synuclein aggregates [ ] . in vivo, smers protect against neurodegeneration in a drosophila model of huntington's disease [ ] . like the smers, the pan-nitric oxide synthase (nos) inhibitor nω (omega)nitro-l -arginine methyl ester hydrochloride (l-name) can block neurodegeneration in the same drosophila huntington's disease model and, similar to rapamycin, can clear mutant huntingtin aggregates in a zebrafi sh huntington's disease model, suggesting that nos inhibitors have therapeutic potential [ ] . l-name triggers functional autophagy in a variety of cell types as measured by lc processing, the accumulation of rfp-lc puncta, and the atg -dependent clearance of mutant huntingtin aggregates [ ] . while nos inhibitors such as l-name decrease no, complementary experiments to increase no levels using no donors and no synthase overexpression revealed an inhibitory role for no in autophagy [ ] . the moa of no was found to involve two different mechanisms of inhibiting autophagy. first, s-nitrosylation of jnk impairs phosphorylation of bcl , leading to increased bcl -belin interaction. second, s-nitrosylation of ikkβ promotes the tsc -dependent activation of mtorc [ ] . the role of hmgb in regulating mitophagy following rotenone-induced disruption of oxidative phosphorylation is a further example that highlights the utility of small-molecule-gene interactions in identifying key autophagy pathways [ ] . mitochondrial stress mediated by rotenone, a small molecule that inhibits complex i in the electron transport chain, led to mitophagy that was facilitated by hmgb and its transcriptional target hspb . hmgb and hspb were found to control mitochondrial homeostasis, as knockdown of either hmgb or hspb resulted in perturbed morphology, glycolysis, atp production, and mitochondrial fragmentation [ , ] . furthermore, rotenone-induced mitophagy was disrupted by cytochalasin d, suggesting a role for the actin cytoskeleton in hmgb -hspb -mediated mitophagy [ ] . to enhance the therapeutic utility of small molecules, the relationships between compound structure and biological activity (structure-activity relationships or sars) can be studied to determine which chemical groups present in the compound are important for activity and how modifi cations of the structure can improve selectivity and potency. in one example, chen et al. synthesized a novel set of diphenylbutylpiperidines that demonstrated tenfold improved potency over lead compounds fl uspirilene and penfl uridol as measured by lc -gfp puncta [ ] . in addition to autophagy activators, autophagy inhibitors can also be useful as probes in determining whether a given state is dependent on classical autophagy or whether a compound truly induces autophagic fl ux. pi k inhibitors -ma and wortmannin and the vacuolar h + atpase inhibitor bafi lomycin a are typically used for these purposes. autophagy inhibitors can also have therapeutic potential. chou and colleagues identifi ed dbeq, a reversible atp-competitive inhibitor of the p atpase [ ] . inhibition of p following dbeq treatments triggers diverse phenotypes, including a blockage of autophagosome maturation, activation of caspase and , and inhibition of cancer cell proliferation, suggesting a potential therapeutic utility for p inhibitors in cancer [ ] . unlike the pi k class i inhibitors discussed above, the discovery of potent and selective inhibitors of class iii vps has proven diffi cult. class iii vps contains a smaller, more rigid atp binding pocket compared to class i p γ, possibly adding to the diffi culty in fi nding inhibitors [ ] . -ma, a widely used inhibitor of vps , is typically used at millimolar concentrations, thus likely triggering many off-target effects that complicate results. to gain insight into developing vps selective inhibitors, the structures of vps in complexes with -ma, pik- , pik- , and pi were solved [ ] . by employing structure-based design, miller and colleagues were able to synthesize pt , an analog of pik- , with tenfold more selectivity for vps than for class i p γ [ ] . improved vps inhibitors will be useful to probe the role of vps in autophagy with more selectivity and without confounding effects of class i p γ inhibition. the success of small-molecule screens in identifying and dissecting autophagy regulatory pathways is notable for its repercussions for therapeutics. the identifi cation of new druggable targets may lead, after extensive optimization, to potential therapeutics in human diseases with autophagy-dependent and/or autophagymodulatory features. experimental results such as those obtained using smers and nos inhibitors suggest that small molecules may also be employed to target antibacterial autophagy at multiple stages. recent research has provided signifi cant insight into autophagic functions in immunity, including xenophagy, mitophagy, antigen presentation, vesicular traffi cking and secretion, and cytokine activity. however, substantial questions remain regarding the mechanistic basis of such functions as well as how individual disease risk alleles modulate these functions. in particular, studies of the microbiome may be critically important in understanding the relationship between xenophagy, immunity, and ibd. we anticipate that microbiome-wide studies (mwas), in combination with detailed insights into the function of autophagy in health and disease, will help inform the development of novel biological and chemical entities for the treatment of patients suffering from these infl ammatory disorders. the role of atg proteins in autophagosome formation autophagy genes function sequentially to promote apoptotic cell corpse degradation in the engulfi ng cell autophagy genedependent clearance of apoptotic cells during embryonic development role of autophagy in immunity and autoimmunity, with a special focus on systemic lupus erythematosus autophagy in immunity and infl ammation autophagy and the immune system autophagy in immunity and cell-autonomous defense against intracellular microbes genome-wide meta-analysis increases to the number of confi rmed crohn's disease susceptibility loci genome-wide association defi nes more than distinct susceptibility loci for crohn's disease genomewide association study identifi es new susceptibility loci for crohn disease and implicates autophagy in disease pathogenesis a genome-wide association scan of nonsynonymous snps identifi es a susceptibility variant for crohn disease in atg l metaanalysis identifi es additional ulcerative colitis risk loci, increasing the number of confi rmed associations to hostmicrobe interactions have shaped the genetic architecture of infl ammatory bowel disease genetic variation in the autophagy gene ulk and risk of crohn's disease genetic variants in autophagy-related genes and granuloma formation in a cohort of surgically treated crohn's disease patients mitochondrial importance in alzheimer's, huntington's and parkinson's diseases mechanisms of mitophagy de novo mutations in the autophagy gene wdr cause static encephalopathy of childhood with neurodegeneration in adulthood recessive mutations in epg cause vici syndrome, a multisystem disorder with defective autophagy autophagy in the intestinal epithelium regulates citrobacter rodentium infection loss of the autophagy protein atg l enhances endotoxin-induced il- beta production nod and nod direct autophagy by recruiting atg l to the plasma membrane at the site of bacterial entry nod stimulation induces autophagy in dendritic cells infl uencing bacterial handling and antigen presentation the origin of the autophagosomal membrane eaten alive: a history of macroautophagy isolation and characterization of autophagy-defective mutants of saccharomyces cerevisiae ulk-atg -fip complexes mediate mtor signaling to the autophagy machinery tor directly controls the atg kinase complex to regulate autophagy the atg kinase complex is involved in the regulation of protein recruitment to initiate sequestering vesicle formation for nonspecifi c autophagy in saccharomyces cerevisiae kinase-inactivated ulk proteins inhibit autophagy via their conserved c-terminal domains using an atg -independent mechanism the lc recruitment mechanism is separate from atg l -dependent membrane formation in the autophagic response against salmonella beclin forms two distinct phosphatidylinositol -kinase complexes with mammalian atg and uvrag two beclin -binding proteins, atg l and rubicon, reciprocally regulate autophagy at different stages distinct regulation of autophagic activity by atg l and rubicon associated with beclin -phosphatidylinositol- -kinase complex identifi cation of barkor as a mammalian autophagy-specifi c factor for beclin and class iii phosphatidylinositol -kinase autophagy requires endoplasmic reticulum targeting of the pi -kinase complex via atg l bif- interacts with beclin through uvrag and regulates autophagy and tumorigenesis autophagic and tumour suppressor activity of a novel beclin -binding protein uvrag beclin -binding uvrag targets the class c vps complex to coordinate autophagosome maturation and endocytic traffi cking a protein conjugation system essential for autophagy a new protein conjugation system in human. the counterpart of the yeast apg p conjugation system essential for autophagy differential involvement of atg l in crohn disease and canonical autophagy: analysis of the organization of the atg l complex in fi broblasts atg -family interacting motif crucial for selective autophagy lc , a mammalian homologue of yeast apg p, is localized in autophagosome membranes after processing lc , gabarap and gate localize to autophagosomal membrane depending on form-ii formation the structure of atg b-lc complex reveals the mechanism of lc processing and delipidation during autophagy lc and gate- / gabarap subfamilies are both essential yet act differently in autophagosome biogenesis dynein-dependent movement of autophagosomes mediates effi cient encounters with lysosomes lysosomal positioning coordinates cellular nutrient responses modulation of actin microfi lament dynamics and fl uid phase pinocytosis by phosphorylation of heat shock protein highmobility group box is essential for mitochondrial quality control tfeb links autophagy to lysosomal biogenesis ph sensing by intracellular salmonella induces effector translocation diversifi cation of a salmonella virulence protein function by ubiquitin-dependent differential localization the phosphoinositide phosphatase sopb manipulates membrane surface charge and traffi cking of the salmonella-containing vacuole the adaptor protein p /sqstm targets invading bacteria to the autophagy pathway the tbk adaptor and autophagy receptor ndp restricts the proliferation of ubiquitin-coated bacteria molecular characterization of ndp , a novel protein of the nuclear domain , which is redistributed upon virus infection and interferon treatment cellular localization, expression, and structure of the nuclear dot protein the ubiquitin-binding adaptor proteins p / sqstm and ndp are recruited independently to bacteria-associated microdomains to target salmonella to the autophagy pathway ) p and ndp target intracytosolic shigella and listeria to different autophagy pathways ubiquitination of intracellular bacteria: a new bacteria-sensing system? autophagy controls salmonella infection in response to damage to the salmonella-containing vacuole the lrr and ring domain protein lrsam is an e ligase crucial for ubiquitin-dependent autophagy of intracellular salmonella typhimurium the role of the intestinal context in the generation of tolerance and infl ammation crohn's diseaseassociated adherent-invasive e. coli are selectively favoured by impaired autophagy to replicate intracellularly impaired autophagy of an intracellular pathogen induced by a crohn's disease associated atg l variant deletion polymorphism upstream of irgm associated with altered irgm expression and crohn's disease a synonymous variant in irgm alters a binding site for mir- and causes deregulation of irgm-dependent xenophagy in crohn's disease loss of leucine-rich repeat kinase causes age-dependent bi-phasic alterations of the autophagy pathway role of autophagy in g s-lrrk -associated neurite shortening in differentiated sh-sy y cells lrrk is involved in the ifn-gamma response and host response to pathogens genome-wide analysis of the host intracellular network that regulates survival of mycobacterium tuberculosis autophagy gene variant irgm - t contributes to protection from tuberculosis caused by mycobacterium tuberculosis but not by m. africanum strains crohn's disease and the mycobacterioses: a quarter century later. causation or simple association? image-based genomewide sirna screen identifi es selective autophagy factors stabilized by mitochondrial depolarization recruits parkin to damaged mitochondria and activates latent parkin for mitophagy /sqstm is required for parkin-induced mitochondrial clustering but not mitophagy; vdac is dispensable for both mitochondrial membrane potential regulates pink import and proteolytic destabilization by parl pink / parkin-mediated mitophagy is dependent on vdac and p /sqstm proteasome and p mediate mitophagy and degradation of mitofusins induced by parkin mitofusin and mitofusin are ubiquitinated in a pink /parkin-dependent manner upon induction of mitophagy drosophila parkin requires pink for mitochondrial translocation and ubiquitinates mitofusin ambra regulates autophagy and development of the nervous system parkin interacts with ambra to induce mitophagy autophagy promotes mhc class ii presentation of peptides from intracellular source proteins autophagic compartments gain access to the mhc class ii compartments in thymic epithelium antigen-loading compartments for major histocompatibility complex class ii molecules continuously receive input from autophagosomes in vivo requirement for atg in antigen presentation by dendritic cells autophagy in antigen-presenting cells results in presentation of citrullinated peptides to cd t cells endogenous mhc class ii processing of a viral nuclear antigen after autophagy autophagy attenuates the adaptive immune response by destabilizing the immunologic synapse paneth cells, antimicrobial peptides and maintenance of intestinal homeostasis virus-plussusceptibility gene interaction determines crohn's disease gene atg l phenotypes in intestine a key role for autophagy and the autophagy gene atg l in mouse and human intestinal paneth cells a common role for atg l , atg and atg in small intestinal paneth cells and crohn disease induction and rescue of nod -dependent th -driven granulomatous infl ammation of the ileum xbp links er stress to intestinal infl ammation and confers genetic risk for human infl ammatory bowel disease ) mtorc in the paneth cell niche couples intestinal stem-cell function to calorie intake a role for mitochondria in nlrp infl ammasome activation mitochondrial reactive oxygen species promote production of proinfl ammatory cytokines and are elevated in tnfr -associated periodic syndrome (traps) fatty acid-induced nlrp -asc infl ammasome activation interferes with insulin signaling crohn's disease-associated atg l polymorphism modulates pro-infl ammatory cytokine responses selectively upon activation of nod autophagy suppresses interleukin- beta (il- beta) signaling by activation of p degradation via lysosomal and proteasomal pathways vacuolating cytotoxin and variants in atg l that disrupt autophagy promote helicobacter pylori infection in humans human irgm regulates autophagy and cell-autonomous immunity functions through mitochondria sequence variants in the autophagy gene irgm and multiple other replicating loci contribute to crohn's disease susceptibility network organization of the human autophagy system characterization of the interaction of gabarapl- with the lir motif of nbr selective autophagy: ubiquitin-mediated recognition and beyond identifying the proteins to which small-molecule probes and drugs bind in cells the connectivity map: using gene-expression signatures to connect small molecules, genes, and disease small molecule regulators of autophagy identifi ed by an image-based high-throughput screen screen for chemical modulators of autophagy reveals novel therapeutic inhibitors of mtorc signaling small molecules enhance autophagy and reduce toxicity in huntington's disease models identifi cation of novel autophagy regulators by a luciferase-based assay for the kinetics of autophagic fl ux chemical modulators of autophagy as biological probes and potential therapeutics complex inhibitory effects of nitric oxide on autophagy metabolic regulation by hmgb -mediated autophagy and mitophagy synthesis and sar study of diphenylbutylpiperidines as cell autophagy inducers reversible inhibitor of p , dbeq, impairs both ubiquitin-dependent and autophagic protein clearance pathways shaping development of autophagy inhibitors with the structure of the lipid kinase vps acknowledgments this work was supported by funding from the crohn's and colitis foundation of america and nih grants dk , dk , and dk to r.j.x. key: cord- - u a jqz authors: grassia, roberto; soro, sara; conti, clara benedetta title: inflammatory bowel diseases and biological treatment in sars-cov- era. why not? date: - - journal: inflamm bowel dis doi: . /ibd/izaa sha: doc_id: cord_uid: u a jqz nan in december , a new β-coronavirus (sars-cov- ) and its related disease, covid- , spread first in china and then worldwide, becoming a pandemic. the patients affected by chronic diseases seem to be at a higher risk to develop severe pneumonia. in this setting, the initial indication from the inflammatory bowel disease (ibd) center in wuhan was to discontinue all biological and immunosuppressive treatments. there, among ibd patients, none developed covid- . subsequent guidelines suggested for patients with ibd to continue all treatments to avoid relapse. in an italian observational study, among ibd patients ( % adults), % under biological therapy, none was hospitalized for covid- . we confirm this observation. on april , , italy officially registered . sars-cov- infected people. particularly, our province of cremona reports the highest rate of infected people in the world: of inhabitants. however, the prevalence could be even higher, as these data are based on hospitalized and symptomatic patients only. our ibd cohort includes patients, ( . %) under biological treatment (median time of treatment, months), males ( . %), with crohn's disease (cd), and ulcerative colitis (rcu) (mean age . ; sd± . ). thirty patients are treated with antitumor necrosis factor alpha (anti-tnfα), with vedolizumab, and with ustekinumab. none of the patients under biological treatment reported covid- symptoms. some ibd constitutional and therapeutic aspects could support these data. first, sars-cov- binds to the cells through angiotensin-converting enzyme (ace ). two distinct formsfull-length and soluble ace -exist. the soluble form circulates in the blood, acting as a competitive interceptor of sars-cov- . interestingly, soluble ace is upregulated in the blood of ibd patients. again, the severity of covid- seems to be linked to the "cytokines storm" syndrome, with massive production of interleukin (il)- , il- , tnf, and interferon-γ. in this direction, tocilizumab, a il- receptor antagonist, has been included in the covid- therapy trials. the suppression of the inflammatory response in ibd by biological drugs could help against both the mucosal inflammation and the covid- pneumonia, sustained by the abnormal "cytokine storm." on the other hand, we anecdotally report only case of covid- in our ibd cohort in a patient under azathioprine therapy. further studies are needed to investigate if ibd patients under biological treatment could be protected against the covid- pneumonia, whereas those under immunosuppressive therapy (ie, thiopurines) could remain at higher risk of developing the disease due to the different mechanism of action. clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study protection of inflammatory bowel disease patients from the outbreak and rapid spread of covid- infection in wuhan aga clinical practice update on management of inflammatory bowel disease during the covid- pandemic: expert commentary uneventful course in ibd patients during sars-cov- outbreak in northern italy are patients with inflammatory bowel disease at increased risk for covid- infection? key: cord- -mjtxiu t authors: occhipinti, vincenzo; pastorelli, luca title: challenges in the care of ibd patients during the covid- pandemic: report from a “red zone” area in northern italy date: - - journal: inflamm bowel dis doi: . /ibd/izaa sha: doc_id: cord_uid: mjtxiu t nan in december , the first, troublesome news concerning a novel and highly infective viral pneumonia spreading from wuhan (china) came to our attention. it was difficult at that time to predict, let alone imagine, the magnitude of how severe acute respiratory syndrome coronavirus (sars-cov- ) would change the way we practice medicine (not to mention our lives) only months later. the first report on february of a severe case of coronavirus disease (covid- ) in a fit, young male from codogno, a town just south of milan, was rapidly followed by the identification of clusters of infected patients in the densely populated lombardia region (approximately million inhabitants with a density of . inhabitants/sq km) and in nearby regions within northern italy. our hospital, policlinico san donato research hospital, is a university hospital located in the southeastern part of the milan metropolitan area, where it represents one of the largest clinics in the territory. our daily activity as gastroenterologists was immediately affected. we promptly implemented a strategy of phone call screenings before any gastrointestinal (gi) consults or endoscopic procedures, questioning patients about their geographic provenance (prohibiting patients from coming from the so-called red zone, the area surrounding codogno), suspicious symptoms, and high-risk contacts. we had just started to contemplate how to safely continue our endoscopic activities in the absence of clear indications regarding the use of personal protective equipment (ppe) when within a few days the regional health system was overwhelmed by a massive number of covid- patients. lombardia rapidly became one of the most severely affected regions of the world, with , infected individuals, identified by performing , nasopharyngeal swabs, and , deaths by april . on march , the entire region was declared a red zone, and days later a complete national lockdown was declared. within the entire national territory, all nonurgent consults and procedures were cancelled and indefinitely postponed. with routine activities halted, all gastroenterologists from our gi unit except one have been reassigned to provide care to covid- patients. one after another, seven covid- units were created and staffed with doctors of various specialties to manage > dedicated hospital beds. a small internal medicine ward, dedicated to care for the few noninfected patients accessing the hospital for urgent medical conditions, was preserved and also used for gi emergencies. in weeks our hospital, like many others in northern italy, was almost completely converted into a covid- hospital. every possible effort was made to quickly increase the capacity of intensive care units (icus) to accommodate the alarming numbers of very sick covid- patients, including constructing new units in unused areas of the hospital or converting surgical rooms into icus. these drastic measures were implemented in a very short period of time, and although necessary to counteract the devastation brought about by the outbreak, they also posed tremendous challenges to the care of patients with gi conditions, including those with inflammatory bowel diseases (ibd). indeed, with severely reduced resources in regard to personnel, equipment, and space to manage patients, our ibd center had to face several critical issues. as a tertiary care center for ibd patients and one of the most prominent in the milan metropolitan area, our ibd center from the very beginning of the covid- pandemic has been hit with a daily onslaught of phone calls and e-mails from our patients with concerns as to how the situation may affect them. many were anxious about the necessity to postpone their already planned consults or endoscopies, and others were truly terrified of the idea of coming to the hospital for scheduled infusions or visits. most of the questions, however, were related to patients' risk of infection, especially while on immunosuppressive therapies. indeed, to date, available evidence-based information regarding the risk of covid- infection in ibd is scarce. anecdotally, ibd patients do not appear to be at higher risk of developing covid- compared with the general population. in fact, the chinese elite ibd union did not identify any sars-cov- infected patients in their patient registry, and to date only a few infected ibd patients have been reported in the literature. nonetheless, although these data are quite encouraging, solid epidemiologic evidence from areas with high ibd prevalence, such as in western countries, is lacking at the present time and it is unclear whether the observations in china can be extrapolated to the rest of the ibd patient population worldwide. as such, although it is reasonable to reassure patients, this does not mean that they can ignore general precautions. indeed, the single most important intervention we can engage in to protect our patients with ibd is to emphasize the importance of "primary prevention": that is, avoiding, as much as possible, the risk of getting infected. this is especially true in frail patients-specifically, elderly or malnourished patients, those with several comorbidities, and those with long-standing aggressive disease-who respond poorly to current treatments. these particular ibd patients, with the aforementioned conditions, are highly likely to be at significant increased risk of severe sars-cov- infections. the importance of hand-washing and other simple measures, such as avoiding touching the nose, ears, and eyes with dirty hands and covering the mouth and nose with a bent elbow when coughing or sneezing, is often underestimated. the possibility of orofecal transmission, suggested by the findings of viral rna in stool even after patients have tested negative by nasopharyngeal swab, has to be considered. routine use of ppe is still a matter of debate. although not currently recommended by the world health organization because of the lack of evidence, the use of masks, even in the absence of symptoms, appears reasonable to us. in any case, the use of ppe (eg, masks and gloves) has rapidly and widely become commonplace among the general population. from this perspective, our role is also to inform patients how to correctly use these ppe devices, because incorrect behavior may paradoxically increase the risk of infection: for example, by touching the outer side of the mask, reusing the same mask for several days, touching the face with gloves, and removing gloves improperly. in addition, the need to comply with social distancing recommendations, avoiding crowded places, and not leaving home unless necessary must be stressed as much as possible. it is important to explain that coming to hospitals for nonurgent procedures or consultations is an unnecessary risk that should be absolutely avoided. as a matter of fact, while reducing access to hospitals, we have had to rely as much as possible on telemedicine. likely similar to most centers in italy, our center does not use any specific system or dedicated equipment for this purpose. we have tried to do our best to provide adequate service for our ibd patients, despite the marked reduction of dedicated gi personnel, now largely reassigned to covid- units. we have managed to have at least one person (gi-or ibd-dedicated nurse) available / to answer e-mails and phone calls. this more "passive" approach may be adequate for patients with mild, stable disease. however, for patients on biologic therapies, we have implemented a mandatory phone call-in the day before any planned hospital visit to screen for possible covid- symptoms or contact with infected individuals and to reassure patients that all possible precautions are being taken by the ibd center to reduce the risk of infection. clinical conditions of patients on subcutaneous biologics can usually be checked by phone call; in addition, whenever possible, we use drug delivery services, established by the national health system, specifically to provide hospital-dispensed medications to patients during this pandemic. of course, in centers with greater numbers of available and dedicated personnel, an "active" approach, which means reaching out to every patient with ibd to provide instructions and reassurance, should be preferred. from this point of view, specialized personnel, such as ibd-dedicated nurses, are of paramount importance. despite the fact that data from the sars and middle east respiratory syndrome coronaviruses did not show a higher risk in immunosuppressed patients, the impact of immunosuppressive therapies on covid- is largely unknown, and no evidence-based recommendations can be provided at this time. with only a few cases of infected ibd patients reported, this situation is particularly true in our field. interestingly, however, immunosuppressive drugs such as hydroxychloroquine and tocilizumab have been proposed as therapies for covid- . thus, it is possible that targeting the immune system with specific compounds will reduce the immune-related lung damage characteristic of sars-cov- infection and dampen the severity of the disease. on the other hand, immunosuppressive drugs are usually associated with more severe viral infections because they may increase viral load. undeniably, whereas nonurgent consults and procedures in ibd patients with mild disease can safely be postponed, those on biologics are at risk of disease relapse if therapies are suddenly stopped. accordingly, the international organization for the study of inflammatory bowel disease recommends not stopping biologic therapies, including infusion therapies administered in hospitals, assuming that the infusion center provides an adequate screening protocol. we agree that in these patients the risk of causing a disease relapse when interrupting biologic therapies is probably higher than the risk of developing severe covid- because of the effects of immunomodulation; however, every effort should be made to protect them from the risk of infection. in hospitals where covid- patients are treated (like ours), the delineation of well-defined "clean" pathways is pivotal to minimize the risks of infection for non-covid- patients. if possible, medical and nonmedical personnel involved at the infusion center should be excluded from the care of covid- patients. counseling by phone, as addressed before, is of great importance to improve and secure adherence. for patients being treated using thiopurines, suspension of therapy is not recommended, even though these therapies have a prolonged effect after their suspension. combination therapy with biologics may be a situation of particular concern, but again there are currently no data available to support the suspension of one agent in this setting. in our clinical practice we discuss this issue with each patient, making decisions on a case-by-case basis, according to disease history and patient preferences. importantly, the practice of using corticosteroids for the treatment of ibd is commonplace and widely accepted. whereas a placebo-controlled randomized trial showed that corticosteroids may reduce sars coronavirus clearence, other studies have suggested a possible positive influence of these drugs on the sars outcome because of their potent anti-inflammatory properties. steroids are currently being used in our country to treat patients with moderate-to-severe covid- , to reduce the signs and symptoms of "super" (severe) inflammation in the later phases of the disease. however, in the early stages (the viral phase of infection) they should be avoided because their use may increase viral burden. the recommendation to reduce as much as possible or discontinue systemic steroids in ibd patients given by the international organization for the study of inflammatory bowel disease appears reasonable, but the decision should be made on an individual basis, discussing the benefits and risks with each patient. of course, steroid tapering (especially if accelerated) should always be conducted under strict medical surveillance, possibly with planned follow-up phone calls and, in any case, ensuring unlimited availability by phone. again, primary prevention is the most important recommendation we can give to our patients being treated using steroids. another particularly challenging issue we are facing during the covid- outbreak is conducting clinical trials. our center normally handles - ongoing clinical trials, and randomized clinical trials on novel therapeutic compounds are an important resource we use in the management of ibd patients. however, as many of us know, great efforts are commonly required to properly conduct these studies, in compliance with more and more complex protocols. as one may expect, advancing study procedures with a significant reduction of resources has become quite challenging. still, it is critical for the patients, and for the ibd center, to do whatever is necessary to guarantee continuation of the trial(s) by enrolling patients and avoiding, as much as possible, protocol deviations. even more so than routine biologic infusions, particular attention should be paid to observing clean pathways and the use of ppe to avoid potential hospital infections. in addition, the reporting of adverse events should be precise and carefully monitored to separate potential confounding factors because of the current situation. overall, although we are assuring our patients, particularly those who were already enrolled in clinical trials at the beginning of the coronavirus emergency, that our intent is to continue these studies, at present we are also carefully evaluating any new possible enrollments because of the aforementioned difficulties that may impact our ability to properly conduct these trials. not only is providing adequate follow-up for chronic diseases such as ibd complicated during the outbreak, but ensuring adequate care of patients with acute conditions is complicated as well. as already mentioned, in many hospitals gastroenterologists have had to be reassigned and are now directly involved in the care of covid- patients. even if urgent consults and endoscopic procedures are granted (a / referral service is maintained for emergent endoscopies), this reorganization adds further difficulties to the management of patients with acute gi diseases who deserve admission. as an example, acute reactivation of ibd has other unique features that may further complicate patient management. first, coronavirus infection may present with gi symptoms, such as diarrhea, weight loss, and abdominal pain, accompanied by mild to moderate fever and no (or mild) respiratory symptoms. as such, covid- may mimic ibd relapse symptoms, adding one more diagnostic challenge to this patient population. moreover, the intense pressure on the emergency system plus patients' fear of being admitted to an "infected" hospital may cause delays in the diagnosis and treatment of acute flares, consequently increasing the risk of medical treatment failure and the need for urgent surgical intervention (eg, in patients with severe ulcerative colitis). in many hospitals, medical and even surgical units have been converted to covid- units. as such, even if surgeons are still available for urgent cases, another problem to take into account is the availability of icu beds in hospitals overwhelmed with covid- patients. in fact, it is very likely that icus are completely occupied by critical covid- patients and that there is no room for "clean" patients. considering these difficulties, we preventively assigned a covid- -free hospital with ibd-dedicated gastroenterologists and surgeons, where we can promptly transfer any patients with severe ibd who have a significant risk of needing surgery and who may be admitted to our hospital during the outbreak. we strongly recommend the creation of such networks with surrounding hospitals to select dedicated hubs for the care of specific acute conditions. cooperation between ibd centers is the key to improving patient outcomes. the sars-cov- pandemic represents one of the most challenging times in the era of modern medicine. as gastroenterologists, specifically those specializing in ibd, we have to put all our efforts toward protecting our patients during these difficult days. empowering telemedicine, creating network strategies, and adequately counseling our patients are key measures to overcome this crisis and to create gold-standard strategies for improved care of ibd patients in the future. of ibd patients during covid- a new coronavirus associated with human respiratory disease in china outbreak of novel coronavirus disease (covid ): situation in italy- covid- italia-monitoraggio della situazione implications of covid- for patients with pre-existing digestive diseases evidence for gastrointestinal infection of sars-cov- advice on the use of masks in the community, during home care and in healthcare settings in the context of the novel coronavirus ( -ncov) outbreak. who. coronaviruses and immunosuppressed patients. the facts during the third epidemic hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial the cytokine release syndrome (crs) of severe covid- and interleukin- receptor (il- r) antagonist tocilizumab may be the key to reduce the mortality management of patients with crohn's disease and ulcerative colitis during the covid- pandemic: results of an international meeting effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients description and clinical treatment of an early outbreak of severe acute respiratory syndrome (sars) in guangzhou, pr china gastrointestinal manifestations of sars-cov- infection and virus load in fecal samples from the hong kong cohort and systematic review and meta-analysis key: cord- -jsqa pmu authors: samanta, jayanta; dhar, jahnvi; khaliq, abdul; kochhar, rakesh title: novel coronavirus infection: gastrointestinal manifestations date: - - journal: nan doi: . /s- - sha: doc_id: cord_uid: jsqa pmu the world is witnessing a major public health crisis in the wake of the third coronavirus strain pandemic, a novel coronavirus (severe acute respiratory syndrome coronavirus ). although initially thought to be a pure respiratory pathogen, recent reports have highlighted not only the extrapulmonary effects of the virus but also, importantly, the gastrointestinal tract (git) effects. various studies have looked into the effects of this novel coronavirus infection (coronavirus- disease [covid- ]) on git involvement with reports of more frequent involvement than previously expected. with feco-oral transmission, debate being conclusively proven with fecal samples testing positive for covid- and longer shedding time, it only underlines the importance of git involvement. moreover, the presence of other gi diseases, such as inflammatory bowel disease, with covid- infection might wreak havoc leading to poor patient outcomes. the modern world is facing a major public health crisis due to novel corona virus (severe acute respiratory syndrome coronavirus [sars-cov- ]) which has caused a pandemic involving at least countries. as we write, more than million people have already been infected and more than , deaths have been reported and this is just the fourth month since the initial cases were detected in the wuhan city of china in december . just like its close kin, severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), sars-cov- was primarily noted as a respiratory pathogen spreading via droplets and aerosols. the extrapulmonary effects and modes of transmission gained attention when the first confirmed case of sars-cov- reported from the united states had gastrointestinal (gi) complaints of nausea and vomiting followed later by diarrhea and patient's fecal specimen tested positive on day of illness. initial studies reported lower rates of gi symptoms such as diarrhea of to . %. [ ] [ ] [ ] [ ] however, with increased emphasis on reporting, this went up to as high as . % in some reports with even pure gi manifestations without respiratory symptoms. , with a flurry of data coming every day on various aspects of this novel infection, it becomes difficult to assimilate the information. moreover, multiple societies have come up with multiple guidelines regarding this, and hence can be confusing for a practicing gastroenterologist. this review aims to comprehensively outline the gi manifestations of this virus, its potential to spread via the feco-oral route and its implications and an overview of management strategies for other gi diseases, such as inflammatory bowel disease (ibd) coexisting with coronavirus- disease (covid- ) infection. as sars-cov- by the international committee on taxonomy of viruses, it was later rechristened as covid- by world health organization. this sars-cov- virus primarily transmits through fomites, air droplets, and aerosols from human to human. the receptor for attachment of this virus is angiotensinconverting enzyme (ace- ) which is present significantly in type- alveolar cells in the lungs. once attached to the spike protein (s), the viral genome enters the cells, uses human cell machinery, and produces multiple viral particles to be released to infect other cells. to contain the virus, the immune mechanism steps in and a disproportionate immune response leads to flooding of the alveoli with fluid and damaging the epithelium of the alveoli hampering oxygen exchange resulting in acute respiratory distress syndrome (ards), multiorgan failure, and death. , , , a large epidemiological data of , cases from china showed an overall case-fatality rate of . %. among them, . % affected were health care workers. covid- has already exceeded the morbidity and mortality of previous coronavirus outbreaks and looking at this rate, it would soon match the cataclysmic proportions of the spanish flu of . the main symptomatology of covid- pertains to respiratory system, with patients presenting predominantly with fever, cough, sore throat, and shortness of breath, and in serious cases, leading to ards, necessitating intensive care unit (icu) admission, and sometimes death. at the outset, respiratory signs and symptoms were being documented and established diagnosis through nasopharyngeal swabs by reverse transcription polymerase chain reaction for sars-cov- . with passing time and aggrandized focus on gi symptomatology, more and more evidences are forthcoming on the gi involvement by the virus. although liver involvement is noted too, the topic of hepatic manifestations and disease is beyond the scope of this article. gastrointestinal tract (git) involvement has been well documented by previous corona viral infections both in animals and human beings. diarrhea was seen to the tune of . % with sars and % with mers. , although earlier reports of covid- reported a low to . % - prevalence of diarrhea, these figures kept on surging as more focused gi symptomatology-related studies came to forefront. the key step of the virus entry into the host cell is via the ace- receptors, , , and ace- is an important regulator of intestinal inflammation. besides the presence of ace- receptor, successful virus entry requires transmembrane protease serine (tmprss ) which cleaves the s-protein of sars-cov- on the cell membrane and enables viral fusion. ace- and tmprss were found not only in the lung alveolar cells and esophageal epithelial cells but also in the absorptive enterocytes of ileum and colon with interestingly higher expression. using single-cell rna sequencing, liang et al demonstrated that ace- was highly expressed in the proximal and distal intestine. thus, the greater invasive nature of the virus and the abundance of its attaching receptors along the git can explain its gi manifestations. both biopsy of small intestine and rectal swabs have been detected to be positive for sars-cov- virus. the mechanisms postulated for causation of diarrhea in covid- infection are: ( ) direct virus entry through ace- receptor causing malabsorption, unbalanced intestinal secretion, and activated enteric nervous system; ( ) direct/indirect damage to the intestinal epithelium by inflammatory response ; ( ) antibiotic and/or antiviral drugs-induced intestinal dysbiosis; ( ) virus-induced disorders of the intestinal flora ; ( ) "gut-lung axis" disturbances wherein respiratory flora alteration affects the digestive system by immune regulation. the evidence of shedding of the virus through feces is another definitive proof of gi involvement and a potential route for transmission. the commonly encountered gi symptoms include diarrhea, nausea, vomiting, abdominal pain, and anorexia. different studies have reported gi symptoms with varying frequencies. a large study of , patients had nausea in % and diarrhea in . % of cases, while the latest data from wuhan reported a staggering % of the patient with gi symptoms. jin et al, in their study of patients of covid- cases with gi symptoms, interestingly pointed out that % of these cases had no respiratory symptoms. similarly, an et al reported patients presenting only with digestive symptoms at the onset. this underlines the fact that pure gi form of covid- infection, although less common, can exist and confuse the clinical scenario. this can be a red flag sign for the gastroenterologist to ensure better self-protection from possible infection. lin et al reported . % gi symptoms with . % diarrhea and . % nausea. on endoscopy in six of these cases, two severely diseased cases showed evidence of virus in esophagus, stomach, duodenum, and rectum proving that the virus can thrive all along the git. a synopsis of the various gi symptoms and their reported frequencies is given in ►table . diarrhea might sometimes be the presenting symptom. in fact, fang et al found that . % of the patients having diarrhea presented with it before the diagnosis of covid- . diarrhea usually occurred to days from the onset with a loss of taste . - mean duration of . ± . days (range: - days). the frequency has been reported to be . ± . /day. anorexia is much more commonly reported than other symptoms, but since it is difficult to objectively assess it, the most common definitive gi symptom would be diarrhea. the correlation between severity of disease and gi symptoms is dubious. some studies such as those by fang et al and guan et al found no difference in the digestive symptom rates between the critically ill and the nonsevere cases. on the contrary, wang et al recorded higher rates of gi symptoms among icu patients (anorexia . % vs. . %; abdominal pain . % vs. %) compared with the less severe cases. a recent pooled data analysis interestingly found that increased covid- severity was associated with abdominal pain but not for diarrhea. children exhibited similar rates of diarrhea ( . - %), but had higher rates of vomiting. , fecal shedding: hard evidence of gi involvement sars-cov- has exhibited gi tropism akin to its close relatives, sars-cov which had virus detected in biopsy and fecal specimens. in fact, the first case reported from the united states had fecal test positive at day of illness. in one of the earlier studies, chen et al found that of patients tested positive for sars-cov- rna in stool specimens. of them, ( . %) patients remained positive for viral rna in feces for a mean period of days even after pharyngeal swabs turned negative. overall, the proportion of patients with stool positivity has ranged between % and %. , positivity did not correlate with disease activity or digestive symptoms. the fecal test became positive around to days after the oropharyngeal swab positivity and lasted for to days. , the most ruffling data, however, is the fact that to % , , of the patients continue to show fecal positivity for a mean period of days after the respiratory specimens have turned negative. moreover, only half of these fecal positive cases actually had diarrhea. this raises a question about discharging a patient based on negative respiratory sample only. the above facts clearly depict that the git acts as a potent source of viral shedding with no correlation with clinical symptomatology, making it incalculable to label a person safe from infective potential. moreover, the virus has been shown to remain viable in aerosol form for hours and close to hours on plastic and stainless steel surfaces. in a proof of concept study, samples collected from the surface of the toilet bowl, sink bowl, and door handle of the bathroom used by a fecal positive patient turned out to be positive before cleaning, underscoring the significance of hygiene maintenance. this feco-oral or "fecomucosal" route of transmission has profound implication on the strategies needed to control the spread of the disease. the high viral load in the stool, its stability on regular surfaces, and generation of toilet fumes make the virus a formidable agent for human-to-human spread. pre-existing conditions such as ibd or other conditions may predispose the individual to more severe disease if they happen to contract covid infection. the underlying disease might flare up due to the super added infection or the covid- infection might become severe because of poor baseline immune function. hence, these groups of patients need special mention. data are not adequate yet to prove or disprove the theory of whether ibd patients are at an increased risk of infection in this covid- era. however, theoretically, ibd portends an enhanced risk of infection by two mechanisms: ( ) the virus receptor, ace- , has increased tissue concentration in the inflamed mucosa of active ulcerative colitis and crohn's disease patients. moreover, the serine protease level, the key primer to activate s-protein is times higher in patients with ibd than others. both these factors contribute to enhanced viral attachment to the host cell and thus, increased infection and ( ) ibd patients who are already on immunosuppressive medications may be at a higher risk of infection. in fact, active disease with thiopurines has been shown to increase the infection potential. of late, international organization for the study of inflammatory bowel diseases (ioibd) opined that vedolizumab and ustekinumab do not increase infection risk, while thiopurines, antitumor necrosis factor agents, and tofacitinib might pose debatable risk. another factor due to the covid- effect is the lack of the regular opd services. impaired compliance with discontinuation of medications has led to reports of disease exacerbation. this is more likely to happen in india, wherein majority of the patients hail from poor socioeconomic background with lack of proper awareness or social support. in a questionnaire survey by bai et al, among ibd cases, ( . %) had exacerbation and . % stated that covid- impacted their scheduled follow-up. probably, the advocacy of the facilities of "tele-medicine" would be able to untangle a part of this complex sociomedical issue. thus, with inadequate data of increased risk for ibd subgroup, longer clearance time for certain drugs, such as thiopurine, increased risk of disease flare on stopping ibd medications and poor hospital/clinic backup for chronic cases, it is tricky to ensure best management for active disease. real-world data on the actual outcome of patients with ibd and covid- infection are lacking. an international registry (secure-ibd) has been formulated for this purpose, and as of now, the initial data show that of the patients registered, % were treated on opd basis, while % required hospitalization and % required ventilator support with % mortality. thus, not only the infection rate but also the severity is similar for ibd cases compared with non-ibd. multiple societies have come up with guidelines for the management of ibd during this covid- pandemic. a composite synopsis of the recommendations of most of the guidelines (aga american gastroenterology association clinical practice update, ioibd, ecco european crohn's and colitis organisation task force, bsg british society guidelines expanded consensus, crohn's and colitis foundation ) is as follows: ( ) the high-risk group encompasses elderly (> years) patients, with comorbidities, pregnancy and patients on immunomodulator therapy, biologicals, and high-dose steroids (> mg/d), ( ) protective measures to be followed include the general instructions such as use of face masks (~n masks), hand hygiene, and social distancing, ( ) patients on -aminosalicylic acid ( -asa) should continue their medication, ( ) steroids to continue at the same dose; no dose reduction advised, ( ) thiopurines: to continue if already on them, ( ) biologicals to continue including infusions, provided the treating center has sars-cov- screening protocol; monotherapy preferred over combination with immunomodulators; switching not recommended, ( ) jak inhibitors to continue if already taking, ( ) for clinical trials: to continue therapies until tested positive for sars-cov- , ( ) patients tested positive for sars-cov- but asymptomatic: reduce the dose of steroids (< mg/d) or switch to budesonide; stop thiopurines, methotrexate, tofacitinib; delay dosing of monoclonal for weeks while monitoring for covid- , ( ) patients having covid- : -asa and local therapy, dietary management to continue oral budesonide may be continued; other medications to be stopped, and ( ) restart medications after weeks if the patient does not develop covid symptoms or symptoms resolve. for children, the guidelines are different. covid- and ibd behave in opposite ways in children compared with adults. while the course of ibd tends to be severe in children compared with adults, covid- infection tends to run a milder course. hence, european society for paediatric gastroenterology hepatology and nutrition guidelines clearly states that no dose reduction of any medications for ibd treatment in children is needed. in a study of cases of covid- pneumonia, % had pancreatic injury and it was associated with higher incidence of anorexia and diarrhea and more severe illness on admission. three possible explanations include: ace- receptors on pancreatic islets causing acute diabetes, cytokine storm, and drug-induced pancreatic injury. a host of drugs has been tried for the treatment of covid- including antiviral and antimalarial and antihelminthic drugs. the efficacy of these drugs is yet to be established, but these drugs can themselves cause gi side effects which the in-hospital patient might experience. a brief outline of the drugs and their commonly reported gi side effects are given in ►table . management of the gi symptoms entails routine management as for any other diseases. diarrhea is usually self-limiting and can be either viral, immune, antibiotic associated or due to dysbiosis. proper hydration is essential to maintain electrolyte balance. the use of routine antidiarrheal agents such as loperamide can be done to mitigate the symptoms. probiotics can be prescribed for dysbiosis and antispasmodics can be added for abdominal pain. abdominal pain can be disease related or maybe due to other causes and needs to be evaluated, if primary symptomatic treatment does not suffice. gi involvement with fecal shedding highlights the necessity of preventing the feco-oral route of transmission to curb its spread. in a developing country like india and as gastroenterologists, this encompasses adoption of the conventional strategies of hygiene awareness that we are so familiar with since ages. the conventional five "f" factors for feco-oral transmission are the fingers, flies, fields, fluids, and food. raising public awareness for better hygiene, safe food practices, forbidding open defecation, and ensuring clean water practices are the foundation stones for preventing the spread of this disease and need to be rigorously implemented. gi manifestations are not uncommon in patients with covid- infection and with passage of time they are more frequently being reported. in fact, a subgroup of these cases might present with pure gi symptoms. fecal shedding of the virus and its detection not only establishes the git involvement by the virus but also highlights a potential source of spread-feco-oral transmission. more data are needed to come to concrete management decisions on difficult situations such as ibd with covid infection. during this time of coronavirus pandemic, as more and more data and evidences keep pouring in, we, gastroenterologists, have to unlearn many older habits and learn a few new ones to protect ourselves and our patients. the principle should be "primum non nocere"-do no harm. catching up with the vast knowledge that pours in everyday about this virus and management protocols, we have to tread our path more carefully. who. coronavirus disease (covid- ) situation report - washington 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authors: nan title: inflammatory bowel disease date: - - journal: clinical veterinary advisor doi: . /b - - - - . - sha: doc_id: cord_uid: ncuegrq nan inflammatory bowel disease (ibd) is a poorly defined and often incorrectly used term in ferrets and other small animals for a systemic inflammatory disease primarily involving the gastrointestinal tract. clinical disease results from dysregulation of the mucosal immune response. the umbrella term ibd used in ferrets and other small animals for a variety of gastrointestinal diseases is not the same disease that is seen in humans. clinical signs, origin, endoscopic features, and histopathologic features are very different from those seen in humans. continued use of the term ibd for these diseases in small animals is a source of frustration and confusion to clinicians and pathologists alike. • crohn's disease, ulcerative colitis • incorrectly used synonyms: antibiotic responsive enteritis, eosinophilic enteritis or eosinophilic gastroenteritis, epizootic catarrhal enteritis, food allergy, gluten hypersensitivity, lymphoplasmacytic enteritis, proliferative bowel disease or colitis • ferrets have been reported incorrectly to be susceptible to ibd. • true ibd is rare in small animals. • cotton-top tamarins are natural animal models of human ibd. • ferrets are susceptible to several gastrointestinal inflammatory conditions that have erroneously been placed under the umbrella term of ibd. • identifying the cause of the ferret ibd will determine the appropriate treatment. colitis, immune suppression is the mainstay of treatment. • immunosuppressive agents that nonspecifically reduce inflammation and immunity have been the mainstay of conventional therapies for ibd. • novel protein diets for weeks or more to eliminate food intolerance or allergy (also try hydrolyzed peptide based diet); antibiotics (metronidazole - mg/kg po q - h; tylosin mg/kg po q - h; tetracycline - mg q - h) to modify intestinal microflora; steroidal antiinflammatories (prednisone mg/kg po q h initially for - weeks, then taper dose by half every weeks), or immune suppressives (azathioprine . mg/kg po q - h) are often used when a cause of ibd in ferrets cannot be determined. care should be taken to differentiate chronic ibd in ferrets from early intestinal lymphoma. • the prognosis varies depending on the cause of the ferret ibd. • crohn's disease and ulcerative colitis are lifelong systemic diseases with recurrent flare-ups. • ibd is a clinical syndrome for which it is difficult to develop a valid, objective histologic counterpart, and it should be a diagnosis of last resort, made by the clinician after alternatives such as food intolerance, motility disorders, and infectious disease have been ruled out. • the pathophysiology resulting in ibd, the basis for phenotypic variation and the mechanism for unpredictable response to treatment are not known. • the thoroughness of the clinical and laboratory investigation before endoscopic biopsy is used is influenced by the amount of time and money available to evaluate what are often elusive functional entities. endoscopic biopsies are often done early, after symptomatic medical therapy (see acute general treatment) has failed to control clinical signs. • it is not appropriate for a pathologist to issue a diagnosis of "inflammatory bowel disease." it is more appropriate to list the histologic findings, and to indicate that the changes could be "compatible with" a clinical diagnosis of that syndrome. • chronic gastrointestinal inflammatory disease in ferrets is not always cured. • emphasize that treatment is aimed at controlling clinical signs. clinical immunology and immunopathology of the canine and feline intestine alimentary and peritoneum overview of biologic therapy for crohn's disease inflammatory bowel disease-live transmission inflammatory bowel disease in veterinary medicine treatment of inflammatory bowel disease (ibd) in dogs and cats helicobacter mustelae-associated gastritis and ulcers hepatobiliary disease proliferative bowel disease salmonellosis (section vi) key: cord- - l mre authors: brenner, erica j.; ungaro, ryan c.; gearry, richard b.; kaplan, gilaad g.; kissous-hunt, michele; lewis, james d.; ng, siew c.; rahier, jean-francois; reinisch, walter; ruemmele, frank m.; steinwurz, flavio; underwood, fox e.; zhang, xian; colombel, jean-frederic; kappelman, michael d. title: corticosteroids, but not tnf antagonists, are associated with adverse covid- outcomes in patients with inflammatory bowel diseases: results from an international registry date: - - journal: gastroenterology doi: . /j.gastro. . . sha: doc_id: cord_uid: l mre background and aims the impact of coronavirus disease (covid- ) on patients with inflammatory bowel disease (ibd) is unknown. we sought to characterize the clinical course of covid- among ibd patients and evaluate the association between demographics, clinical characteristics, and immunosuppressant treatments on covid- outcomes. methods surveillance epidemiology of coronavirus under research exclusion for inflammatory bowel disease (secure-ibd) is a large, international registry created to monitor outcomes of ibd patients with confirmed covid- . we calculated age-standardized mortality ratios (smrs) and utilized multivariable logistic regression to identify factors associated with severe covid- , defined as intensive care unit admission, ventilator use, and/or death. results cases from countries were reported (median age years, % men). thirty-seven patients ( %) had severe covid- , ( %) were hospitalized, and patients died ( % case fatality rate). smrs for ibd patients were . ( % confidence interval [ci] . - . ), . ( % ci . - . ), and . ( % ci . - . ) relative to data from china, italy, and the us, respectively. risk factors for severe covid- among ibd patients included increasing age (adjusted odds ratio [aor] . , % ci . - . ), ≥ comorbidities (aor . , % ci . - . ), systemic corticosteroids (aor . , % ci . - . ), and sulfasalazine or -aminosalicylate use (aor . , % ci . - . ). tnf antagonist treatment was not associated with severe covid- (aor . , % ci . - . ). conclusions increasing age, comorbidities, and corticosteroids are associated with severe covid- among ibd patients, although a causal relationship cannot be definitively established. notably, tnf antagonists do not appear to be associated with severe covid- . inflammatory bowel disease (secure-ibd) is a large, international registry created to monitor outcomes of ibd patients with confirmed covid- . we calculated age-standardized mortality ratios (smrs) and utilized multivariable logistic regression to identify factors associated with severe covid- , defined as intensive care unit admission, ventilator use, and/or death. results: cases from countries were reported (median age years, % men). thirtyseven patients ( %) had severe covid- , ( %) were hospitalized, and patients died ( % case fatality rate). smrs for ibd patients were . ( % confidence interval [ci] . - . ), . ( % ci . - . ), and . ( % ci . - . ) relative to data from china, italy, and the us, respectively. risk factors for severe covid- among ibd patients included increasing age (adjusted odds ratio [aor] . , % ci . - . ), ≥ comorbidities (aor . , % ci . - . ) , systemic corticosteroids (aor . , % ci . - . ), and sulfasalazine or aminosalicylate use (aor . , % ci . - . ). tnf antagonist treatment was not associated with severe covid- (aor . , % ci . - . ). inflammatory bowel diseases (ibd), including crohn's disease (cd) and ulcerative colitis (uc), are chronic inflammatory conditions of the gastrointestinal tract affecting millions of people worldwide. [ ] [ ] [ ] patients with ibd and related rheumatologic, dermatologic, and neurologic autoinflammatory conditions frequently require treatment with immunosuppressant medications which can increase the risk of infection. [ ] [ ] [ ] [ ] [ ] corticosteroids, immunomodulators (thiopurines, methotrexate), biologics, and janus-kinase inhibitors, commonly used to treat chronic autoinflammatory conditions, have been associated with higher rates of serious viral and bacterial infections including influenza and pneumonia. [ ] [ ] [ ] [ ] [ ] yet, it is also possible that some forms of immune suppression may blunt the excessive immune response/cytokine storm characteristic of severe covid- infection and consequently reduce mortality, as suggested by emerging case reports of anti-il- therapy. , little is known about the impact of covid- on patients with chronic auto-inflammatory diseases such as ibd, particularly those who require systemic immunosuppressant medications. an initial report of covid- among , patients in china included only two persons with immune deficiency. a subsequent report found that cancer patients had a higher risk of severe covid- , but this conclusion was based on only patients. in italy, mazza et al reported a case of covid- pneumonia leading to death in a patient with severe acute ulcerative colitis treated with systemic corticosteroids. in order to provide better guidance to patients and their health care providers and to inform strategies for prevention of covid- and medication management, more data are urgently needed regarding the impact of ibd and treatments on covid- outcomes. in the present work, we report on the clinical course of covid- and risk factors for adverse outcomes in a large cohort of patients with ibd collected through an international registry. (table s ) . physicians and other health care providers were encouraged to voluntarily report all cases of polymerase chain reaction (pcr)-confirmed covid- occurring in ibd patients, regardless of severity. to foster international collaboration and promote transparency, we developed a project website (www.covidibd.org) to acknowledge the contributions of individual reporters and share crude, aggregate data along with an interactive web-based map displaying the geographic location of reported cases (https://covidibd.org/map/). we instructed health care providers to report cases after a minimum of days from symptom onset and sufficient time had passed to observe the disease course through resolution of acute illness or death. in the event that a patient's status changed after reporting or if there were concerns about data accuracy, we instructed reporters to re-report and contact the research team to remove their initial entry. we utilized redcap (research electronic data capture), a secure, web-based electronic data capture tool hosted at the university of north carolina at chapel hill to collect and manage study data. health care providers recorded the following information: age, country of residence, state of residence (if applicable), year of covid- diagnosis, name of center/practice/physician providing care, sex, race, ethnicity, height, weight, patient's diagnosis we removed all known duplicate or erroneous reports. we identified additional potential duplicate records based on matching age, sex, ibd disease type, country, and state (u.s. only), and reviewed these manually. reports from non-valid email addresses were flagged as potential errors and we performed a google search of reporters and practice locations to confirm legitimacy of reports. we used descriptive statistics to summarize the basic demographic and clinical characteristics of the study population. we summarized continuous variables using means and standard deviations. we expressed categorical variables as proportions. comorbidities were collapsed into the following categories: cardiovascular disease, diabetes, hypertension, stroke, lung disease, kidney disease, liver disease, and cancer. we analyzed a variety of covid- outcomes, including outpatient care only, hospitalization, icu or ventilator requirement, and death from covid- or related complications. crude data are provided for the overall study population, and stratified by a variety of demographic and clinical characteristics. to understand the impact of ibd on case fatality, we computed expected and observed deaths and age-standardized mortality ratios (smr) utilizing published agestratified covid- case fatality rates from china and italy , and publically available data from the u.s. , multivariable logistic regression estimated the independent effects of age, sex, disease (cd vs uc/ibd-u), disease activity, smoking, bmi ≥ , and number of comorbidities ( , , ≥ ) on the primary outcome of severe covid- , defined as a composite of icu admission, ventilator use, and/or death, consistent with existing covid- literature. models also included tumor necrosis factor (tnf) antagonist use (versus not) and sulfasalazine/ -aminosalicylate ( -asa) use (versus not) as these were the two most commonly reported medication classes and systemic corticosteroid use (versus not) based on increased risk of infectious complications based on prior literature and crude data. a secondary outcome was the composite of any hospitalization and/or death. we also analyzed death as a separate endpoint. we reported adjusted odds ratios (aor) and % confidence intervals (ci) for each demographic or disease characteristic. we also performed a series of exploratory sub-analyses. we compared tnf antagonist monotherapy versus combination therapy with immunomodulators ( -mercaptopurine [ mp], azathioprine, or methotrexate), controlling for the above demographic and clinical factors as well as the use of systemic corticosteroids and -asa/sulfasalazine. in addition, given the surprising association between -asa/sulfasalazine use and more severe covid outcomes in our main analyses, we performed a sub-analysis to directly compare the effects of tnf antagonists versus -asa/ sulfasalazine, controlling for the above factors as well as use of immunomodulators. the primary outcome of these exploratory analyses was the composite of any hospitalization and/or death. the number of events was too sparse to evaluate other outcomes. all data were prepared and analyzed using sas v . (sas institute, cary, north carolina). two-sided p values < . were considered statistically significant. each secure-ibd survey item met criteria for de-identified data, in accordance with the hipaa at the time of this writing, a total of cases were reported to the secure-ibd database from different countries and states within the united states (figures and ; tables s and s ). demographic, clinical, and ibd treatment related characteristics are summarized in table . the median age was years, with a range from to ≥ years, and there was a slight predominance of males ( . %). most cases were reported in whites ( . %). ethnicity was reported as hispanic/latino in . % of cases ( table ) . the majority of patients had cd ( . %), and ibd disease activity by pga was classified as remission in . % of cases. the most common class of ibd treatment was tnf antagonist therapy ( . % overall, . % monotherapy and . % combination therapy with azathioprine, mercaptopurine, or methotrexate). use of other medications is described in table . most patients ( . %) had no comorbidities other than ibd; . % had one, . % had two, and . % had three or more. four percent of the cohort reported using tobacco and/or electronic cigarettes (table ) . crude outcome data are summarized in table table . sixteen deaths ( % of reported cases) are summarized in table s . eight deaths ( %) occurred in patients ≥ years of age. no deaths occurred in patients < years of age. most deaths had comorbidities, including eight with cardiovascular disease. the age-standardized smrs for the secure-ibd population relative to china, italy, and the u.s. were . ( % confidence interval [ci] . - . ), . ( % ci . - . ), and . ( % ci . - . ) respectively (tables and ) . on multivariable analysis, increasing age (aor . , % ci . - . ), ≥ comorbidities (aor . , % ci . - . ), systemic corticosteroids (aor . , % ci . - . ), and -asa/sulfasalazine use (aor . , % ci . - . ) were positively associated with the primary endpoint after controlling for all other covariates listed in table . no significant association was seen between tnf antagonist use and the primary endpoint (aor . , % ci . - . ). similar associations were observed for our secondary outcomes, although tnf antagonist use was inversely associated with the outcome of hospitalization or death while only age and systemic corticosteroid use were positively associated with the outcome of death. in our exploratory analyses, we found that tnf antagonist combination therapy, compared to monotherapy, was positively associated with the outcome of hospitalization or death (aor . , % ci . - . ), after adjusting for clinical and demographic variables and use of systemic corticosteroids and -asa/sulfasalazine. compared to tnf antagonists, -asa/sulfasalazine was positively associated with the outcome of hospitalization or death (aor . , % ci . - . ). we report the development of an international, physician-driven, reporting system to study the was also associated with more severe covid- . reassuringly, tnf antagonist biologic therapy was not an independent risk factor for more severe covid- . in this international ibd population, we observed an age-standardized mortality ratio of approximately . to . , as compared to the general populations of china, italy, and the u.s. with confidence intervals crossing the null. we note no deaths occurred in the reported cases occurring in patients < years of age, extending the findings of an earlier case series suggesting a milder course of covid- in pediatric patients. in contrast, % of deaths occurred in patients over years of age and % of patients who died had cardiovascular comorbidities. the strong positive association between systemic corticosteroid use and our primary and secondary outcomes is consistent with extensive prior literature in ibd and other autoinflammatory conditions describing the infectious complications of corticosteroid use as well as more recent data indicating that corticosteroids are not beneficial, and may even be harmful, in the treatment of coronavirus and similar viruses (mers, sars, etc.). forty-three percent of our cohort was exposed to tnf antagonist medications. in the adjusted analysis of our primary outcome, we observed no association between tnf antagonist use and severe covid- . as tnf antagonists are the most commonly prescribed biologic therapy for patients with ibd, these initial findings should be reassuring to the large number of patients receiving tnf antagonist therapy and support their continued use during this current pandemic. in our exploratory subgroup analysis, we observed a higher risk of hospitalization and/or death with tnf antagonist combination therapy versus monotherapy, consistent with prior studies of other infectious complications. given the overall effect estimate of tnf antagonists (combination and monotherapy combined) in our primary model was . , one can hypothesize that tnf antagonist monotherapy may have a protective effect against severe covid- , as suggested in a recent commentary. we observed a higher risk of our primary outcome in patients exposed to -asa/sulfasalazine. this finding persisted after controlling for age, comorbidities, ibd disease characteristics, corticosteroid use, and other factors. furthermore, in a direct comparison, we observed that -asa/sulfasalazine treated patients fared worse than those treated with tnf inhibitors. although we cannot exclude unmeasured confounding, further exploration of biological mechanisms is warranted. conversely, although the number of reported cases exposed to other ibd treatments is currently small, it is worth noting that / ( %) patients treated with anti-il / required outpatient care only and none died. the strengths of this study include the robust, worldwide collaboration that enabled us to assemble clinical data on a large, geographically diverse sample of pediatric and adult ibd patients and rapidly define the course of covid- in this population. the reporting directly by physicians or their trained medical staff strengthens the validity of these data. although our study sample is diverse in terms of age, geography, race, and other factors, we acknowledge the possibility of reporting bias. reported cases may over-represent more severe covid- patients who come to the attention of their provider and patients in areas with readily available covid- testing. conversely, our sample may under-represent those severely ill patients who may be hospitalized at an outside hospital or die without their physician's awareness. the registry includes only confirmed cases of covid- in accordance with other reporting initiatives from national authorities and the world health organization, , , though we recognize many patients with suspected infection are never tested. although we adjusted for many factors such as age, comorbidities, and ibd disease type and severity, we acknowledge the possibility of unmeasured confounding. additional research is needed to further evaluate causality between the use of corticosteroids and other medications and covid- outcomes. finally, we computed age-standardized mortality ratios using case fatality rates reported from china, italy, and the u.s., yet our study sample arose from different countries. given the profound effects of age on covid- related mortality, we believe it was useful to standardize to existing data. that our smr estimates were roughly equivalent when standardizing to chinese, italian, or u.s. data suggests the overall validity of this approach. in summary, older age, increased number of comorbidities and systemic corticosteroid use among patients with ibd are strong risk factors for adverse covid- outcomes. maintaining remission with steroid-sparing treatments will be important in managing patients with ibd through this pandemic. it appears that tnf antagonist therapy is not associated with severe covid- , providing reassurance that patients can continue tnf antagonist therapy. turner d, huang y, author names in bold designate shared co-first authorship. table escaping pandora's box -another novel coronavirus case-fatality rate and characteristics of patients dying in relation to covid- in italy characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention preliminary estimates of the prevalence of selected underlying health conditions among patients with coronavirus disease -united states worldwide incidence and prevalence of inflammatory bowel disease in the st century: a systematic review of population-based studies crohn's disease ulcerative colitis acg clinical guideline: management of crohn's disease in adults evidence-based clinical practice guidelines for inflammatory bowel disease second european evidence-based consensus on the prevention, diagnosis and management of opportunistic infections in inflammatory bowel disease infection-related hospitalizations are associated with increased mortality in patients with inflammatory bowel diseases risk of serious and opportunistic infections associated with treatment of inflammatory bowel diseases increased risk of pneumonia among patients with inflammatory bowel disease systematic review with meta-analysis: efficacy and safety of oral janus kinase inhibitors for inflammatory bowel disease the incidence of influenza and influenza-related complications in inflammatory bowel disease patients across the united states tocilizumab, an anti-il receptor antibody, to treat covid- -related respiratory failure: a case report first case of covid- in a patient with multiple myeloma successfully treated with tocilizumab clinical characteristics of coronavirus disease in china cancer patients in sars-cov- infection: a nationwide analysis in china a fatal case of covid- pneumonia occurring in a patient with severe acute ulcerative colitis the data model concept in statistical mapping a fatal case of covid- pneumonia occurring in a patient with severe acute ulcerative colitis we acknowledge the physicians and other healthcare providers worldwide who have reported cases to the secure-ibd database and the organizations who supported or promoted the secure-ibd database (reporter names available at www.covidibd.org/reporteracknowledgment/. see table s for organization names). limitations: possibility of reporting bias and unmeasured confounding. maintaining remission with steroid-sparing treatments is important in managing ibd patients through this pandemic. tnf antagonist therapy does not appear to be a risk factor for severe covid- . we created an international registry of ibd patients who developed covid- . corticosteroids, but not tnf antagonists, were associated with adverse outcomes. other risk factors were similar to the general population. key: cord- - exaaik authors: rao, vishwas; maulik, romit; constantinescu, emil; anitescu, mihai title: a machine-learning-based importance sampling method to compute rare event probabilities date: - - journal: computational science - iccs doi: . / - - - - _ sha: doc_id: cord_uid: exaaik we develop a novel computational method for evaluating the extreme excursion probabilities arising from random initialization of nonlinear dynamical systems. the method uses excursion probability theory to formulate a sequence of bayesian inverse problems that, when solved, yields the biasing distribution. solving multiple bayesian inverse problems can be expensive; more so in higher dimensions. to alleviate the computational cost, we build machine-learning-based surrogates to solve the bayesian inverse problems that give rise to the biasing distribution. this biasing distribution can then be used in an importance sampling procedure to estimate the extreme excursion probabilities. characterizing high-impact rare and extreme events such as hurricanes, tornadoes, and cascading power failures are of great social and economic importance. many of these natural phenomena and engineering systems can be modeled by using dynamical systems. the models representing these complex phenomena are approximate and have many sources of uncertainties. for example, the exact initial and boundary conditions or the external forcings that are necessary to fully define the underlying model might be unknown. other parameters that are set based on experimental data may also be uncertain or only partially known. a probabilistic framework is generally used to formulate the problem of quantifying various uncertainties in these complex systems. by definition, the outcomes of interest that correspond to high-impact rare and extreme events reside in the tails of the probability distribution of the associated event space. fully characterizing the tails requires resolving high-dimensional integrals over irregular domains. the most commonly used method to determine the probability of rare and extreme events is monte carlo simulation (mcs). computing rareevent probabilities via mcs involves generating several samples of the random variable and calculating the fraction of the samples that produce the outcome of interest. for small probabilities, however, this process is expensive. for example, consider an event whose probability is around − and the underlying numerical model for the calculation requires ten minutes per simulation. with mcs, estimating the probability of such an event to an accuracy of % will require two years of serial computation. hence, alternative methods are needed that are computationally efficient. important examples of extreme events are rogue waves in the ocean [ ] , hurricanes, tornadoes [ ] , and power outages [ ] . the motivation for this work comes from the rising concern surrounding transient security in the presence of uncertain initial conditions identified by north american electric reliability corporation in connection with its long-term reliability assessment [ ] . the problem can be mathematically formulated as a dynamical system with uncertain initial conditions. in this paper, the aim is to compute the extreme excursion probability: the probability that the transient due to a sudden malfunction exceeds preset safety limits. typically, the target safe limit exceedance probabilities are in the range − - − . we note that the same formulation is applicable in other applications such as data assimilation, which is used extensively for medium-to long-term weather forecasting. for example, one can potentially use the formulation in this paper to determine the likelihood of temperature levels at a location exceeding certain thresholds or the likelihood of precipitation levels exceeding safe levels in a certain area. in [ ] , we presented an algorithm that uses ideas from excursion probability theory to evaluate the probability of extreme events [ ] . in particular we used rice's formula [ ] , which was developed to estimate the average number of upcrossings for a generic stochastic process. rice's formula is given by where the left-hand side denotes the expected number of upcrossings of level u, y is the derivative of the stochastic process in a mean squared sense, and ϕ t (u, y) represents the joint probability distribution of the process and its derivative. in this paper, we build on our recent algorithm [ ] , which we used to construct an importance biasing distribution (ibd) to accelerate the computation of extreme event probabilities. a key step in the algorithm presented in [ ] involves solving multiple bayesian inverse problems, which can be expensive in high dimensions. here, we propose to use machine-learning-based surrogates to obtain the inverse maps and hence alleviate the computational costs. the mathematical setup used in this paper consists of a nonlinear dynamical system that is excited by a gaussian initial state and that results in a non-gaussian stochastic process. we are interested in estimating the probability of the stochastic process exceeding a preset threshold. moreover, we wish to estimate the probabilities when the underlying event of the process exceeding the threshold is a rare event. the rare events typically lie in the tails of the underlying event distribution. to characterize the tail of the resulting stochastic process, we use ideas from theory excursion probabilities [ ] . specifically, we use rice's formula ( ) to estimate the expected number of upcrossings of a stochastic process. for a description of the mathematically rigorous settings used for the rare event problem, we refer interested readers to [ , § ] and references therein. evaluating ϕ t (u, y), the joint probability distribution of the stochastic process and its derivative, is central to evaluating the integral in rice's formula. however, ϕ t (u, y) is analytically computable only for gaussian processes. since our setup results in a non-gaussian stochastic process, we linearize the nonlinear dynamical system variation around the trajectories starting at the mean of the initial state. we thus obtain a gaussian approximation to the system trajectory distribution. in [ ] , we solve a sequence of bayesian inverse problems to determine a biasing distribution to accelerate the convergence of the probability estimates. for high-dimensional problems, however, solving multiple bayesian inverse problems can be expensive. in this work, we propose to replace multiple solutions to bayesian inverse problems with machine-learning-based surrogates to alleviate the computational burden. the rest of the paper is organized as follows. in sect. we review the existing literature for estimating rare event probabilities. in sect. we reformulate the problem of determining the ibd as a bayesian inference problem, and in sect. we develop a machine-learning-based surrogate to approximate the solution to the bayesian inference problem. in sect. we demonstrate this methodology on a simple nonlinear dynamical system excited by a gaussian distribution. in sect. we present our conclusions and potential future research directions. most of the existing methods to compute the probabilities of rare events use mcs directly or indirectly. the mcs approach was developed by metropolis and his collaborators to solve problems in mathematical physics [ ] . since then, it has been used in a variety of applications [ , ] . when evaluating rare event probabilities, the mcs method basically counts the fraction of the random samples that cause the rare event. for a small probablilty p of the underlying event, the number of samples required to obtain an accuracy of is o( − p − ). hence mcs becomes impractical for estimating rare event probabilities. a popular sampling technique that is employed to compute rare event probablities is importance sampling (is). is is a variance reduction technique developed in the s [ ] to estimate the quantity of interest by constructing estimators that have smaller variance than mcs. in mcs, simulations from most of the samples do not result in the rare event and hence do not play a part in probability calculations. is, instead, uses problem-specific information to construct an ibd; computing the rare event probability using the ibd requires fewer samples. based on this idea, several techniques for constructing ibds have been developed [ ] . for a more detailed treatment of is, we direct interested readers to [ , ] . one of the major challenges involved with importance sampling is the construction of an ibd that results in a low-variance estimator. we note that the approach may sometimes be inefficient for high-dimensional problems [ ] . a more detailed description of mcs and is in the context of rare events can be found in [ , § ] and references therein. other methods use the notion of conditional probability over a sequence of nested subsets of the probability space of interest. for example, one can start with the entire probability space and progressively shrink to the region that corresponds to the rare event. furthermore, one can use the notion of conditional probability to factorize the event of interest as a product of conditional events. subset simulation (ss) [ ] and splitting methods [ ] are ideas that use this idea. several modifications and improvements have been proposed to both ss [ , , , , ] and splitting methods [ , ] . evaluating the conditional probabilities forms a major portion of the computational load. compute the conditional probabilities for different nested subsets concurrently is nontrivial. large deviation theory (ldt) is an efficient approach for estimating rare events in cases when the event space of interest is dominated by few elements such as rogue waves of a certain height. ldt also has been used to estimate the probabilities of extreme events in dynamical systems with random components [ , ] . a sequential sampling strategy has been used to compute extreme event statistics [ , ] . most of the work in this section is a review of our approach described in [ , § ] . here we reformulate the problem of constructing an ibd as a sequence of bayesian inverse problems. consider the following dynamical system, where c is a canonical basis vector and x , the initial state of the system, is uncertain and has a probability distribution p. the problem of interest is to estimate the probability that c x(t) exceeds the level u for t ∈ [ , t ]. that is, we seek to estimate the following excursion probability, where x(t, x ) represents the solution of the dynamical system ( ) for a given initial condition x . we note that where μ is the respective measure transformation subject to ( ) and Ω(u) ⊂ Ω represents the excursion set hence, estimating Ω(u) will help us in estimating the excursion probability p t (u). in general, however, estimating the excursion set Ω(u) analytically is difficult. rice's formula, ( ) gives us insights about the excursion set and can be used to construct an approximation to the excursion set. recall that in rice's formula ( ), ϕ t (u, y) represents the joint probability density of c x and its derivative c x for an excursion level u. the right-hand side of ( ) can be interpreted as the summation of all times and slopes at which an excursion occurs. one can sample from yϕ t (u, y) to obtain a slope-time pair (y i , t i ) at which the sample paths of the stochastic process cause an excursion. now consider the map g : r d× → r that evaluates the vector c x(t) c x (t) based on the dynamical system ( ), given an initial state x and a time t. by definition of the excursion set Ω(u), there exists an element x i ∈ Ω(u) that satisfies the following relationship, where ε > . we can use this insight to construct an approximation of Ω(u) by constructing the preimages of multiple slope-time pairs. observe that the problem of finding the preimage of a sample (y i , t i ) is ill-posed since there could be multiple x i 's that map to u + ε i y i at t i via operator g. we define the set and an approximation Ω(u) to Ω(u) can be written as note that the approximation ( ) improves as we increase n . for a discussion on the choice of ε i , we refer interested readers to [ , § . ] . the underlying computational framework to approximate Ω(u) consists of the following stages: -draw samples from unnormalized yϕ t (u, y) -find the preimages of these samples to approximate Ω(u). we use mcmc to draw samples from unnormalized yϕ t (u, y) . we note that irrespective of the size of the dynamical system, yϕ t (u, y) represents an unnormalized density in two dimensions; hence, using mcmc is an effective means , draw samples from it. drawing samples from yϕ t (u, y) requires evaluating it repeatedly, and in the following section we discuss the means to do so. we note that yϕ t (u, y) can be evaluated analytically only for special cases. specifically, when ϕ t (u, y) is a gaussian process, then the joint density function yϕ t (u, y) is analytically computable. consider the dynamical system described by ( ) . when p is gaussian and f is linear, we have assuming a is invertible, x(t) can be written as where i represents an identity matrix of the appropriate size. given that x is normally distributed, it follows that x(t) is a gaussian process: where and Φ := exp(a(t − t ))Σ (exp(a(t − t ))) . we now can evaluate yϕ t (u, y) for arbitrary values of u i , y i , and t i as where Υ := c Φc c Φa c c aΦ c c aΦa c and | Υ | denotes the determinant of Υ . note that the right-hand side in ( ) is dependent on t i via Υ . when f is nonlinear, yϕ t (u, y) cannot be computed analytically-a key ingredient for our computational procedure. we approximate the nonlinear dynamics by linearizing f around the mean of the initial distribution. assuming that the initial state of the system is normally distributed as described by eq. ( ), linearizing around the mean of the initial state gives where f represents the jacobian of f at t = and x = x ; this reduces the nonlinear dynamical system to a form that is similar to eq. ( ). thus, we can now use eqs. ( ) , ( ) , and ( ) to approximate yϕ t (u, y) for nonlinear f . in [ ] we formulated the problem of determining preimages ( ) as a bayesian inverse problem. however, solving multiple bayesian inverse problems can be expensive. hence we approximated our ibd by using the solutions of a small number of bayesian inverse problems. in this section we build a simple datadriven surrogate for approximating the preimages x i described in eq. ( ) . using the surrogate, we can approximate the preimages of several y i 's obtained by sampling from yϕ t (u, y). the surrogate developed here approximates the inverse of the map defined in eq. ( ) . to that end, we wish to approximate the map where the input space corresponds to (u + ε i , y) | ti and the output lives in the domain of the state space (Ω here). this is equivalent to augmenting t i as an additional input variable and building a surrogate that maps from r → r d . we utilize a fully connected deep neural network to approximate this map. a one-layered neural network can be expressed as where f is a differentiable activation function that imparts nonlinearity to this transformation; l is the input dimension of an incoming signal; m is the number of hidden-layer neurons (in machine learning terminology); c m ∈ r m ×l are the weights of this map; m ∈ r m are the biases; and ξ j ∈ r j is the nonlinear output of this map, which may be matched to targets available from data or "fed-forward" into future maps. note that ξ j is the postactivation value of each neuron in a hidden layer of j neurons. in practice, multiple compositions of this map may be used to obtain nonlinear function approximators, called deep neural networks, that are very expressive. for nonlinear activation, we utilize for all its activation functions. in addition, we concatenate three such maps as shown in eq. to ultimately obtain an approximation for g − . two such submaps have j fixed at , and a final transformation utilizes j = . we note that the function f for the final transformation is the identity, as is common in machine learning algorithms. a schematic of this network architecture is shown in fig. . the trainable parameters (c m and m for each transformation) are optimized with the use of backpropagation [ ] , an adjoint calculation technique that obtains gradients of the loss function with respect to these parameters. a stochastic gradient optimization technique, adam, is used to update these parameters [ ] with a learning rate of . . our loss function is given by the l -distance between the prediction of the network and the targets (i.e., the mean-squared error). our network also incorporates a regularization strategy, called dropout [ ] , that randomly switches off certain units ξ j (here we utilize a dropout probability of . ) in the forward propagation of the map (i.e., from d → ). through this approach, memorization of data is avoided, while allowing for effective exploration of a complex nonconvex loss surface. our map is trained for epochs with a batch size of ; in other words, a weight update is performed after a loss is computed for samples. each epoch is completed when the losses from the entire data set are used for gradient update. during the network training, we set aside a random subset of the data for validation. losses calculated from this data set are used only to monitor the learning of the framework for unseen data. these are plotted in fig. , where one can see that both training and validation losses are reduced to an equal magnitude. figure also shows scatter plots for this validation data set where a good agreement between the true and predicted quantities can be seen. we may now use this map for approximating the ibd. the following procedure is used to construct the ibd. . obtain different realizations of the initial conditions of the dynamical system by sampling from the initial pdf p. . use g to obtain the forward maps of these realizations. . use the forward maps and the corresponding random realizations of the initial conditions to train the inverse map g − . . we now apply this trained inverse map on samples generated from yϕ t (u, y) to obtain the approximate preimages of samples y i . . use a gaussian approximation of these inverse maps is used as an ibd. assume that this gaussian approximation has pdf p ibd . . sample from the ibd, and use importance sampling to estimate the probabilities. we can now estimate p t (u) using the ibd as follows: where x , . . . , x m are sampled from the biasing distribution p ibd and i( x i ) represents the indicator function given by also, ψ( x i ) represents the importance weights. the importance weight for an arbitrary x i is given by we demonstrate the application of the procedure described in sect. and sect. for nonlinear dynamical systems excited by a gaussian distribution. we use the lotka-volterra equations as a test problem. these equations, also known as the predator-prey equations, are a pair of first-order nonlinear differential equations and are used to describe the dynamics of biological systems in which two species interact, one as a predator and the other as a prey. the populations change through time according to the following pair of equations, where x is the number of prey, x is the number of predators, and dx dt and dx dt represent the instantaneous growth rates of the two populations. we assume that the initial state of the system at time t = is a random variable that is normally distributed: and we are interested in estimating the probability of the event p (c x ≥ u), ] , and u = . the first step of our solution procedure involves sampling from yϕ t (u, y) to generate observations y i . we linearize the dynamical system about the mean of the distribution of x (eq. ( )) and express ϕ t (u, y) as a function of t and y as described by eq. ( ) . we compute yϕ t (u, y) as shown in eq. ( ) . we use the delayed rejection adaptive metropolis (dram) markov chain monte carlo (mcmc) method to generate samples from yϕ t (u, y). (for more details about dram, see [ ] .) to minimize the effect of the initial guess on the posterior inference, we use a burn-in of , samples. figure shows the contours of yϕ t (u, y) and samples drawn from it by using dram mcmc. in [ ] we then solved the bayesian inverse problem by using both mcmc and laplace approximation at map to construct a distribution that approximately maps to likelihood constructed around y i . here, we replace the solution to the bayesian inverse problem with a machine-learned inverse map described in sect. . multiple samples generated from yϕ t (u, y) can be used to construct the ibd, as described in sect. . , and the ibd can be used to estimate p t (u), as explained in sect. . . figure compares the results between the conventional mcs and machine learning based importance sampling (ml-based is) methods. note that we use an mcs estimate with million samples as a proxy for the true probabilities. the "true" probability is . × − . ml-based is gives fairly good estimate even with small number of model evaluations. when the training dataset size is large enough, the improvements are dramatic. notice that for a true probability of the order of − we obtain an estimate that has a relative error of less than %. notice that our method gives the same (or better) accuracy as the mcs with hundred times lesser computational cost. the convergence with just training samples is acceptable and these results improve dramatically for and training samples. we believe the results could be even better when we use a gaussian mixture to represent the ibd instead of a simple gaussian approximation. . comparison between conventional mcs and ml-based is. we observe even with a small amount of training data, we obtain fairly accurate estimates; and as we increase the training data, the accuracy improves dramatically. in fig. , we havent included the costs associated with generating training data, training costs, and cost for approximating the inverse map as these costs are almost negligible when compared to the overall costs. note that generating samples is approximately equivalent to model evaluations (this is because a single model evaluation can be used to generate the slope and state at different times and each of them can be used as a training sample). the training of the ml framework, for this problem, required very little compute time. each training was executed on an th-generation intel core-i machine with python . . and tensorflow . and took less than s for training samples (this is less than model evaluations). inference (for prediction points) costs were less than s, on average. in this work we developed a ml-based is to estimate rare event probabilities and we demostrated the algorithm on the prey-predator system. the method developed here builds on the approach in [ ] and replaces the expensive bayesian inference with a machine learning based surrogate. this approach yields fairly accurate estimate of the probabilities and for a given accuracy requires atleast three orders of magnitude lesser computational effort than the traditional mcs. in future, we aim to test this algorithm for larger problems and also use an active learning based approach to pick the training samples. scaling this algorithm to high dimensions (say o( )) could be challenging and to address it, we will use state-of-the-art techniques developed by machine learning and deep learning community in the future. the geometry of random fields. siam stochastic simulation: algorithms and analysis power system blackouts-literature review estimation of small failure probabilities in high dimensions by subset simulation rare-event simulation bayesian subset simulation efficient monte carlo simulation via the generalized splitting method introduction to rare event simulation hybrid subset simulation method for reliability estimation of dynamical systems subject to stochastic excitation reliability estimation for dynamical systems subject to stochastic excitation using subset simulation with splitting rogue waves and large deviations in deep sea extreme event quantification in dynamical systems with random components exploring monte carlo methods oceanic rogue waves dram: efficient adaptive mcmc estimation of particle transmission by random sampling methods of reducing sample size in monte carlo computations a two-stage subset simulation-based approach for calculating the reliability of inelastic structural systems subjected to gaussian random excitations geometric insight into the challenges of solving high-dimensional reliability problems adam: a method for stochastic optimization monte carlo strategies in scientific computing the monte carlo method a sequential sampling strategy for extreme event statistics in nonlinear dynamical systems sequential sampling strategy for extreme event statistics in nonlinear dynamical systems efficient computation of extreme excursion probabilities for dynamical systems gaussian processes for machine learning. adaptive computation and machine learning mathematical analysis of random noise monte carlo statistical methods. springer texts in statistics us department of commerece, national ocanic and atmospheric administration, national environmental satellite data and information service learning representations by backpropagating errors dropout: a simple way to prevent neural networks from overfitting the north american electricity reliability corporation bayesian post-processor and other enhancements of subset simulation for estimating failure probabilities in high dimensions key: cord- -xqj en authors: petras, robert e.; frankel, wendy l. title: large intestine (colon) date: - - journal: modern surgical pathology doi: . /b - - - - . - sha: doc_id: cord_uid: xqj en nan normal large bowel gross anatomy and microscopic anatomy - are outlined here. the large bowel extends from the ileocecal valve to the anus and measures cm to cm in adults. it can be clinically useful to divide the large bowel into regions. the cecum, the most proximal saccular part of the large bowel, lies inferior to a horizontal line defined by the ileocecal valve. the cecum is completely invested by peritoneum and contains the opening of the vermiform appendix. the right colon, cm to cm in length, extends to the hepatic flexure. the right colon lacks mesentery and lies mostly in the retroperitoneum except for its anterior and right lateral serosa. the transverse colon averages cm to cm in length, runs from the hepatic flexure to the splenic flexure, and has a mesentery. the descending colon begins at the splenic flexure, becomes retroperitoneal, and extends for cm to cm. at the distal portion of the descending colon, the large bowel once again acquires a mesentery to become the sigmoid colon, which measures approximately cm in length. the sigmoid colon arbitrarily becomes the rectum at approximately the level of the third sacral vertebra. the rectum, measuring cm to cm in length, ends at the anal canal. the upper one third of the rectum is covered by peritoneum; the lower two thirds lies in the retroperitoneum surrounded by the fatty mesorectum. beneath the mesothelium-covered serosa lies a subserosal layer of fibroadipose tissue. the muscularis externa of the large bowel is composed of an inner circular layer and an outer longitudinally running layer of smooth muscle that condenses into three longitudinally running taeniae coli, the mesocolic taenia and two antimesenteric taeniae. the taeniae unite at the base of the vermiform appendix. they flare at the rectum and incorporate into its external muscular layer. the inner and outer layers of muscularis externa are separated by the myenteric plexus of auerbach. appendages of subserosal fat typically hang from the large bowel to form the epiploic appendices. extending luminally from the muscularis externa lie the fibroadipose tissue, blood vessels, lymphatics, and nerves of the submucosa. the submucosa contains meissner's plexus, which is usually found closely juxtaposed to the muscularis mucosae. the inner surface of the large bowel is characterized by horizontally oriented folds (plicae semilunares) and fine innominate grooves of the mucosa. the proximal colon to the splenic flexure derives its blood supply from the superior mesenteric artery through the ileocolic, right colic, and middle colic branches. the remainder of the colon is supplied by the left colic and sigmoid branches of the inferior mesenteric artery. the inferior mesenteric artery and iliac vessels provide blood to the rectum. veins accompany the arteries and share their names. the large bowel venous drainage enters the portal circulation except for the distal rectum, which drains into the systemic circulation through the middle and inferior rectal veins. in portal hypertension, this area can serve as a portalsystemic shunt and can be a site of varices. the lymph node drainage is divided into those lymph nodes close to the bowel wall (e.g., pericolic, perirectal) and those that follow the blood vessels (e.g., mesenteric). the vagus nerves supply stimulatory nervous activity to the right colon and proximal transverse colon. the remainder of the large bowel is supplied by pelvic postganglionic parasympathetic nerves. the inhibitory nervous activity is derived from the superior and inferior mesenteric plexuses. the large bowel mucosa is composed of a single-cell layer of colorectal epithelium covering the lumen and lining the crypts, the supporting lamina propria and a small smooth muscle band referred to as the muscularis mucosae. the normal colorectal luminal surface of the mucosa is straight; the glands are made up of tubules (crypts) that are tightly packed, parallel, nonbranching, and closely approximating the muscularis mucosae ( fig. - ) . the appearance of the colonic tubules is similar to rows of test tubes placed in a rack. goblet cells interspersed between colorectal absorptive cells line the colonic tubules. scattered neu-roendocrine cells can be observed usually near the base of the crypt and contain basally oriented eosinophilic cytoplasmic granules. the lamina propria contains a modest amount of mixed inflammatory cells including plasma cells, lymphocytes, eosinophils, mast cells, and macrophages. intraepithelial lymphocytes are present normally but usually are fewer than per colorectal epithelial cells. the muscularis mucosae is arranged into two layers (an inner circular and outer longitudinally running layer) and is usually thin and regular. the submucosa is typically devoid of inflammatory cells. scattered mucosal and submucosal lymphoid follicles are normally encountered, especially in younger individuals. in areas of mucosal lymphoid follicles, the mucosal architecture may be distorted and the muscularis mucosae can be incomplete. flattened epithelial cells known as m cells overlie the mucosal lymphoid aggregates. the epithelium of the m-cell zone typically contains numerous intraepithelial lymphocytes. [ ] [ ] [ ] paneth cells with their basal nuclei and luminal cytoplasmic refractile red granules are seen in the base of colonic crypts but are considered normal only in the cecum and proximal right colon. , mucosal biopsy interpretation can be hampered by changes associated with bowel preparation and with the trauma of the biopsy procedure itself. changes ascribed to bowel preparation include decreased intraepithelial mucin, increased numbers of mitotic figures, surface apoptosis with karyorrhectic debris in the superficial lamina propria, and small numbers of neutrophils and eosinophils in surface or crypt epithelium. [ ] [ ] [ ] [ ] [ ] [ ] edema and recent hemorrhage into tissues not associated with other degenerative or inflammatory changes most likely represent biopsy-related trauma. muciphages (foamy macrophages containing faintly periodic acid-schiff [pas]-positive material) are often present in the lamina propria of the large bowel, especially the rectum, where they most likely represent a nonspecific response to mucosal injury (i.e., trauma). , muciphages should be distinguished from xanthelasma/xanthomatous polyp, which can also occur in the large intestine ( fig. - ). an adenoma, defined as a benign intraepithelial neoplasm composed of epithelial cells exhibiting cytologic dysplasia, is considered the precursor lesion of most colorectal carcinomas. , [ ] [ ] [ ] dysplasia is characterized by decreased intraepithelial mucin, epithelial nuclear enlargement with hyperchromasia, nuclear stratification, and increased numbers of mitotic figures. large bowel adenomas are highly prevalent in western societies. the frequency of these tumors markedly increases after age years and reaches a peak at age years. adenomas are usually asymptomatic but large ones may bleed. adenomas usually produce a raised endoscopically or grossly detectable abnormality, generally a protrusion or polyp that can often be further subclassified as sessile or pedunculated. some adenomas appear flat; some may cause mucosal depressions. adenomas occur singly or can be multiple. multiple (≥ ) adenomas may indicate a genetic syndrome such as familial adenomatous polyposis (fap), attenuated fap, or myh-associated polyposis syndrome. most adenomas are small, measuring less than mm. adenomas should be classified histologically based on the pattern of growth as tubular, villous, or tubulovillous following the world health organization (who) guidelines. , adenomas in which simple tubules make up more than % of the area are classified as tubular. adenomas with greater than % of their area showing a villiform configuration are called villous adenomas ( fig. - ) ; all others should be reported as tubulovillous adenomas. once discovered, adenomas are characteristically removed by endoscopy or surgery because they are an important precursor lesion to colorectal carcinoma. there-fore, it is not surprising that occasionally a resected polyp thought to be a benign adenoma may contain an area of carcinoma. the various nomenclatures applied to colorectal adenomas, dysplasia, and malignant polyps can be confusing. unfortunately, no unified accepted guidelines exist. [ ] [ ] [ ] most surgical pathologists use variations of the who terminology. in this system, the terms dysplasia, adenocarcinoma in situ, intramucosal adenocarcinoma, and invasive adenocarcinoma are accepted. each has a precise meaning when applied to colorectal polyps and appropriate patient care requires that the endoscopist, surgeon, and surgical pathologist understand the significance of each of these terms. all adenomas demonstrate at least low-grade epithelial dysplasia. without dysplasia, an adenoma cannot be recognized and distinguished from normal colonic mucosa. lowgrade dysplasia is characterized by a slight decrease in the amount of intracellular mucin, mild nuclear enlargement with hyperchromasia, some nuclear stratification, and an increased number of mitotic figures (fig. - ) . increasing degrees of dysplasia (low-grade to high-grade) show progressive loss of intracellular mucin, progressive increase in nuclear size with stratification, and a loss of nuclear polarity. adenocarcinoma in situ describes the next step in the dysplasia-carcinoma sequence. here, the atypical glands assume a complex cribriform or back-to-back gland configuration but the basement membrane remains intact (fig. . some experts consider adenocarcinoma in situ as part of the spectrum of high-grade glandular dysplasia and report both under the same term. when carcinoma cells infiltrate into the lamina propria or muscularis mucosae only, terms such as high-grade glandular dysplasia and adenocarcinoma in situ are technically no longer applicable because both require an intact basement membrane. there- fore, the term intramucosal adenocarcinoma is more accurate (fig. - ). , finally, when carcinoma cells have invaded the submucosa (or beyond) the lesion is labeled invasive adenocarcinoma. invasion is invariably associated with an infiltrative pattern to neoplastic glands associated with tumor desmoplasia (fig. - ) . this tumor desmoplasia is extremely helpful in recognizing invasion of at least the submucosa, especially in small biopsy specimens. the nomenclature controversy principally centers on the observation that in the colon and rectum, infiltrating carcinoma cells do not become clinically significant (i.e., able to metastasize) until they have invaded the submucosa. , , , only polyps containing invasive adenocarcinoma require a decision for additional treatment on the part of the clinician. adenoma, adenocarcinoma in situ, and even intramucosal adenocarcinoma lack metastatic capability and are considered adequately treated by polypectomy alone. , , , , as a result, some pathologists advocate modification of the nomenclature to account for clinical behavior and promulgate use of the term high-grade glandular dysplasia to encompass high-grade dysplasia, adenocarcinoma in situ, and even intramucosal adenocarcinoma. , although the who guidelines accepted and defined two (low-grade, high-grade) or three (mild, moderate, severe) grades of dysplasia, adenocarcinoma in situ, and intramucosal adenocarcinoma, the authors of those guidelines recommended a similar behavior-based modification for intramucosal carcinoma and stated that "… intramucosal adenocarcinoma of the colon has not been shown to metastasize, and for this reason 'carcinoma in situ' is more appropriate." the version of the who classification added little clarification and introduced new and even more confusing terms. the authors stated that the defining feature of colorectal adenocarcinoma is invasion through the muscularis mucosae into the submucosa. however, once defined, worrisome lesions not fulfilling this criterion become difficult to describe. for example, the who classification defines adenocarcinoma in situ and intramucosal adenocarcinoma as lesions with morphologic characteristics of "adenocarcinoma" confined to the epithelium or that "invade" the lamina propria alone and lack invasion through the muscularis mucosae. the who goes on to state that these lesions have virtually no risk of metastasis. according to the who, the term "… high-grade intraepithelial neoplasia is more appropriate than adenocarcinoma in situ and … intramucosal neoplasia is more appropriate than intramucosal adenocarcinoma." in the version, the who believes that use of these terms will help avoid overtreatment. the problems with this classification are many. the inaccurate use of the term invasion to describe lesions that are not by definition invasive carcinoma is confusing. the lesser lesion of high-grade intraepithelial neoplasia sounds worse than the term used to describe intramucosal adenocarcinoma (intramucosal neoplasia). furthermore, all adenomas, strictly speaking, are intraepithelial neoplasia. an effort to achieve consensus (largely between eastern [japanese] and western pathologists) [ ] [ ] [ ] [ ] resulted in the vienna classification of gastrointestinal (gi) neoplasia, presented in table - . problems with the vienna system include the following: ( ) inaccurate use of the word invasion; ( ) category "noninvasive" high-grade neoplasia including potentially dangerous lesions (e.g., suspicious for invasive adenocarcinoma); and ( ) category "invasive neoplasms" including intramucosal adenocarcinoma, which is widely accepted to be clinically benign in the colon and rectum. it is unlikely that this system of categories without clinical correlation will ever gain widespread acceptance. as modified from the who classification, low-grade dysplasia, high-grade dysplasia, adenocarcinoma in situ, and intramucosal adenocarcinoma exist and can be recognized by pathologists. , this nomenclature remains attractive because it can be applied throughout the gi tract. if one chooses to diagnose high-grade dysplasia, adenocarcinoma in situ, and intramucosal adenocarcinoma in colorectal biopsy specimens, specific mention in the report that these lesions lack metastatic potential is helpful to clinicians. because infiltrating carcinoma cells in a colorectal polyp do not become clinically significant (i.e., able to metastasize) until they have invaded the submucosa, , - , - only a polyp containing invasive adenocarcinoma (invasion of at least the submucosa) should be considered malignant. only invasive adenocarcinoma requires a decision regarding additional treatment. therefore, the presence or absence of invasive adenocarcinoma should be specifically mentioned in the pathology report. to comply with the american college of gastroenterology (acg), the u.s. multi-society task force on colorectal cancer, and the american cancer society guidelines, , , a villous component (villous or tubulovillous adenoma) and high-grade dysplasia should be reported because these features require more frequent surveillance. carcinoma in situ and intramucosal adenocarcinoma can be reported parenthetically as high-grade dysplasia. most mistakes that pathologists make in reporting colorectal adenomas, dysplasia, and malignant polyps occur in three major categories: ( ) the pathology report is not clear (nonspecific or noncommittal terms are used or the presence or absence of invasive adenocarcinoma is not clearly stated); ( ) mispositioned glands (pseudocarcinomatous invasion) are misinterpreted as invasive adenocarcinoma; and ( ) the margin of excision is either not identified or not discussed. a common problem concerns differentiating invasive carcinoma complicating a colorectal adenoma from pseudocarcinomatous invasion (pseudoinvasion). pseudoinvasion describes a situation in which neoplastic glands of the adenoma are mispositioned, presumably by trauma, into or beneath the muscularis mucosae. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] pseudoinvasion is relatively common, reported in % to % of resected colorectal polyps. , , distinguishing this epithelial mis- placement from invasive adenocarcinoma can be difficult. some reported series of "malignant polyps" have included and even illustrated polyps with pseudoinvasion as examples of invasive adenocarcinoma associated with adenoma. histologic features favoring pseudoinvasion include the lack of an infiltrative pattern, the lack of tumor desmoplasia, the presence of lamina propria around mispositioned glands, the lack of increased atypia in mispositioned epithelium as compared with the surface epithelium of the adenoma, and the presence of hemorrhage or hemosiderin deposits in nearby connective tissue ( fig. - ) . occasionally, the misplaced glands of pseudoinvasion can become cystic, can rupture, and can be associated with dissection of mucus into the connective tissues of the polyp. in this case, the distinction between mucinous adenocarcinoma and misplaced glands can be extremely difficult. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] table - illustrates histologic features that can aid in this differential diagnosis. examination of additional sections can help in difficult cases because almost all mucinous adenocarcinomas contain at least small foci of typical nonmucinous-type adenocarcinoma. a rational decision concerning management of a patient with an endoscopically removed malignant colorectal polyp (one containing invasive adenocarcinoma) requires weighing the chances of finding residual or metastatic cancer with a follow-up surgical excision (whom do i help?) against the risk of surgical mortality and morbidity (whom do i hurt?). some investigators have advocated surgical resection for all patients. currently, however, almost all surgeons and gastroenterologists embrace a more conservative approach and use certain gross and histologic features as indications for follow-up colectomy. , these features include sessile growth, , residual villous adenoma, a short stalk (< mm), stalk invasion, level invasion, , lymphatic or vascular permeation, lack of a residual adjacent adenoma (so-called polypoid carcinoma), poor differentiation, , , , and invasive carcinoma at or near a margin of resection. , , we and others , , , believe that two features identify patients likely to avoid an adverse outcome defined as residual or metastatic adenocarcinoma in a subsequent colectomy specimen or during clinical follow-up. patients with favorable histology (defined as well-or moderately differentiated adenocarcinoma with a -mm tumor-free margin of resection in the polypectomy specimen) experienced no adverse outcome and are considered adequately treated by polypectomy alone. similar, although not identical, therapeutic recommendations have been adopted by the acg. , these guidelines consider colonoscopic polypectomy definitive treatment for a patient with a malignant polyp if the following criteria are fulfilled: ( ) the polyp is considered completely excised at endoscopy, ( ) the specimen is properly processed by the pathology laboratory, ( ) the cancer is not poorly differentiated, ( ) no histologic evidence of vascular or lymphatic involvement exists, and ( ) the resection margin is not involved by carcinoma. lymphatic or venous invasion, proposed as an indication for follow-up colectomy, remains controversial. , , , , , , , only a few malignant polyps with these features have been reported and almost all have had positive margins, contained poorly differentiated invasive carcinoma, or both. we think that lymphatic or venous invasion is not a reliable criterion because the distinction from retraction artifact is frequently difficult. cooper and colleagues encountered significant interobserver variation in assessing this feature. furthermore, no guidelines exist that establish the extent to which a pathologist must go to diagnose lymphatic or venous invasion (e.g., number of sections or use of immunostains). although patients can be stratified into high-risk and low-risk groups based on margin status and grade of invasive adenocarcinoma, and lymphatic or and in deference to these guidelines, the presence or absence of angiolymphatic invasion should be reported. as a guide for therapy, the major studies of endoscopic polypectomy for malignant polyps have shown that the chance of finding residual or metastatic cancer in a subsequent colon resection specimen or during follow-up in the "favorable histology" group is less than %. weighing these odds against the published operative mortality rates for colectomy that range between % and %, [ ] [ ] [ ] it seems that subsequent major surgical resection should be avoided in the "favorable histology" subgroup. , if a decision for subsequent colorectal resection is made, a cancer operation is recommended rather than a more limited procedure because cancer was the indication for surgery. residual carcinoma in a follow-up resection specimen can be expected in only % of cases. these cases of residual or metastatic carcinoma that are discovered within this subset of pt lesions are overrepresented by cases containing poorly differentiated carcinoma. evaluation of the resection line is critical to proper patient management; therefore, correct handling of the polypec-tomy specimen is of utmost importance. , , , the entire polyp should be immediately placed into fixative. following adequate fixation, a polyp with a stalk should be trimmed on either side of the stalk as illustrated in figure - . the section of the polyp with stalk and margin can be embedded in a block, to maintain the correct anatomic relationship. the remainder of the polyp should be submitted in separate blocks. for polyps without stalks (sessile growths or those in which the stalk has retracted), look for the effect of cautery on the gross specimen. this will appear as a lightercolored area or a defect on the external surface of the polyp. carefully trim on either side of this defect (fig. - ) and place this tissue in a block. again, the remaining tissue should be submitted in separate blocks. routine examination of a minimum of three step-sections stained with hematoxylin-eosin (h&e) from each block is recommended. in the pathology report, the presence or absence of invasive carcinoma must clearly be stated. with malignant polyps, the grade of carcinoma must be noted, the resection line must be identified and assessed, and the status of that resection line must be clearly stated in the pathology report. a distance measurement of carcinoma-free margin should be included in the report. in deference to the acg, the presence or absence of angiolymphatic invasion should be investigated and reported. the treating physician must individualize the decision for follow-up colorectal excision by weighing the patient's wishes against the estimated cancer recurrence risk and the predicted operative morbidity and mortality. advances in laparoscopic resection of the colon and rectum could drastically reduce the morbidity and mortality of operative resection that now constitutes the major contraindication for surgery. these newer surgical techniques may require reassessment of the current management recommendations for malignant colorectal polyps. more than , new cases of colorectal carcinoma occur in the united states each year and account for approximately , deaths annually. the peak incidence occurs between the ages of and years; fewer than % of cancers occur in patients less than years of age. risk factors for carcinoma include diets rich in animal fat, sedentary lifestyle, and coexisting inflammatory bowel disease (ibd). at least five separate but overlapping molecular pathways to colorectal cancer may exist. approximately % of colorectal carcinomas occur sporadically, whereas % appear to have a genetic basis. , the genetic group includes the % of cases related to lynch's syndrome (hereditary nonpolyposis colon cancer syndrome [hnpcc] ) and the % associated with fap and its variants. the other % of cases show strong familial clustering but a specific genetic cause has yet to be found. colorectal carcinoma can also be viewed another way. approximately % of colorectal cancers are thought to originate through the chromosomal instability pathway. these tumors typically demonstrate dna aneuploidy and have abnormalities of chromosomes , , and and contain mutational changes in apc gene, k-ras proto-oncogene, dcc tumor suppressor gene, and p tumor suppressor gene. fap colorectal carcinomas arise through this pathway. approximately % of colorectal carcinoma appears to arise in the so-called mutator phenotype. these cancers tend to be dna diploid and are associated with microsatellite instability (msi). the cancers related to lynch's syndrome are associated with the mutator phenotype. dna integrity is essential for normal cell function. dna insults can result from the direct effects of chemicals or radiation and are usually corrected through the excision repair system. dna replication errors are of two types: ( ) simple mispairing of nucleotides, the most common type; and ( ) "slipping" errors, in which genes may contain too many or too few copies of repeat short dna nucleotide sequences known as microsatellites. normally, these errors are recognized, the cell cycle is arrested, and the mismatched segment is corrected. for those errors not immediately corrected by dna polymerase, the mismatch repair (mmr) system acts as a backup for additional proofreading of dna. failure to repair mismatches allows the error (mutation) to persist and to become the template for subsequent dna replication. the known mmr genes and their relative frequency in lynch's syndrome are presented in table - . msi is best viewed as an epiphenomenon found in colorectal tumor dna but not in non-neoplastic tissues. it indicates that extensive mutation exists in the nonencoding repetitive dna sequences that are particularly prone to replication error, the microsatellites. the majority of msi is linked to somatic inactivation of hmlh through hypermethylation inactivation of the promotor region, but it can also be detected in persons with germline mmr gene mutations, the definition of lynch's syndrome. msi is detected in % of colorectal cancers overall and is present in more than % of the cancers found in patients with lynch's syndrome. because patients with lynch's syndrome have a germline mutation of an mmr gene, they are at increased lifetime risk for colorectal (≈ %) and other cancers. , these cancers develop at significantly younger ages (e.g., average age for colorectal carcinoma ≤ years). other tumors related to lynch's syndrome include cancers of the endometrium, ovary, stomach, biliary tract, urinary tract, kidney, central nervous system, small bowel, and skin. patients and families with lynch's syndrome can sometimes be identified by taking a careful patient and family medical history, can be suggested from the pathologic findings of excised tumors, and can be detected by direct evaluation of the mmr system. pathologic features of colorectal cancer that suggest msi/lynch's syndrome include right-sided location, synchronous or metachronous large bowel cancers, large and bulky polypoid tumors with circumscribed pushing margins, tumors showing prominent lymphoid infiltrate, and cancers of poor differentiation (medullary or undifferentiated carcinoma) or mucinous and signet ring cell histologic pattern (figs. - and - ) . , , the diagnosis of lynch's syndrome is evolving. originally, the amsterdam criteria were used to identify hnpcc clinically including patients with lynch's syndrome. the original amsterdam criteria included ( ) three or more relatives with colorectal cancer with at least one first-degree relative, ( ) colorectal carcinoma in two generations, and ( ) one or more colorectal carcinomas occurring in a person less than years of age. to increase the sensitivity, the amsterdam criteria were modified (amsterdam ii criteria) to include ( ) three or more relatives with any carcinoma related to lynch's syndrome, ( ) colorectal carcinoma in two generations, and ( ) and one or more carcinomas related to lynch's syndrome in a person younger than years of age. detecting lynch's syndrome based on the amsterdam criteria alone poses many problems. patient histories are less useful now than in the past because of requires than just one of the following criteria be met: ( ) colorectal cancer before age years, ( ) synchronous or metachronous colorectal or other tumor related to lynch's syndrome regardless of the patient's age, ( ) colorectal cancer with msi-high pathology (msi-h) in a patient less than years old, ( ) person with colorectal cancer and a first-degree relative with colorectal adenoma or carcinoma or other lynch's syndrome-related tumor (cancer < years; adenoma < years), and ( ) colorectal cancer with two or more relatives with colorectal or other lynch's syndrome-related tumor regardless of age. the american gastroenterological association position states that genetic testing should be performed on ( ) families meeting amsterdam criteria, ( ) any affected person meeting the modified bethesda guidelines, and ( ) any firstdegree relative of those with known mutations of mmr genes. these guidelines suggest that after pretest genetic counseling and written informed consent, immunohistochemical analysis for mmr gene products or msi testing by pcr be performed on tumor tissue. the international guidelines for evaluation of msi by pcr recommend use of consensus markers: bat , bat , d s , d s , and d s . if two or more markers are abnormal, the carcinoma is considered msi-h. if one marker is abnormal, the tumor is classified as msi-low (msi-l). if no markers are abnormal, the cancer is referred to as msi-stable (mss). laboratories using more than five loci modify this classification with % to % or more abnormal defined as msi-h, less than % to % as msi-l, and none abnormal as mss. immunohistochemistry can be used to detect msi. almost all msi-h cancers can be identified if the antibody panel includes mlh , msh , pms , and msh (figs. - and - ) . , immunohistochemical analysis and msi analysis by pcr have specific advantages and limitations. pcr requires a molecular laboratory and usually requires normal tissue for comparison. immunohistochemistry is more widely available but can be limited by poor tissue fixation or poor technique rendering interpretation difficult. immunohistochemistry may be superior because the findings can direct gene sequencing and msi is not always seen in lynch's syndrome kindred with msh germline mutation. patients with msi-h cancer should undergo additional genetic testing including gene sequencing. mss and msi-l tumors require no further testing. additional genetic evaluation may be considered if the clinical history is compelling. the clinical significance of identifying lynch's syndrome is that affected individuals and at-risk persons are recognized and can be screened and treated with correct surgical procedures. subtotal colectomy is usually recommended to treat colon cancer related to lynch's syndrome because of the high likelihood of synchronous or metachronous cancers. partial colectomy with colonoscopy every to years is a reasonable alternative. furthermore, clinicians can institute proper screening such as colonoscopy at a young age (beginning at age or years younger than the youngest cancer history in the family), periodic endometrial sampling (every to years starting at age years), pelvic ultrasound, ca serum testing, and urine cytology or molecular testing for urinary tract carcinoma. many experts screen all resected colorectal cancers for msi initially by pcr. immunohistochemical analysis is a useful alternative and some prefer this as the initial test because an abnormality in protein expression correlates almost invariably with msi-h by pcr. in cases showing normal mmr proteins or equivocal staining by immunohistochemical analysis, msi testing by pcr should be done in clinically suspicious cases to exclude a germline mutation that can yield an antigenic protein that is biologically inactive. msi testing in sporadic colorectal carcinoma is a subject of considerable contemporary interest and debate. much like their counterparts in patients with lynch's syndrome, sporadic msi-h carcinomas have a predilection for the right colon, mucinous histologic features, and a prominent lymphoid infiltrate. strong arguments exist for routine testing for msi in all resected colorectal carcinomas including the lower mortality rate independent of tumor stage. sporadic msi-h cancer can also be associated with an increased rate of metachronous tumors with subsequent clinical implications for cancer surgery, surveillance, and follow-up. msi status may also have implications for chemotherapy. improved survival is reported in patients with mss and msi-l stage ii and stage iii cancers who are treated with fluorouracil-based regimens. , finally, routine msi testing could increase the detection of lynch's syndrome because % of probands were more than years old and up to % of patients with lynch's syndrome did not fulfill amsterdam or bethesda guidelines. colorectal serrated polyps and the serrated pathway to colorectal cancer hyperplastic polyps are the most common benign polyps of the large intestine. , these polyps are usually small (< mm), sessile, and are often about the same color as the surrounding colonic mucosa. histologically, evenly distributed absorptive and goblet cells line crypts that are elongate and dilated (fig. - ). inhibition of normal apoptosis is thought to be the underlying mechanism for polyp formation and because more epithelial cells are present per unit area than normal, the cells must pseudostratify, thus imparting a serrated or micropapillary appearance. characteristically, the basement membrane under the surface epithelium is thickened and hyalinized. regenerative epithelial changes, mitotic figures, and active inflammation can be quite prom- c d inent at the crypt bases. this regenerative area can occasionally cause diagnostic confusion with dysplasia and carcinoma, especially in a variant referred to as inverted hyperplastic polyp. , in this inverted variety, the regenerative epithelium of the crypt base is misplaced into or beneath the muscularis mucosae. most examples of inverted hyperplastic polyp are now probably best classified as sessile serrated polyp (see later) and are easily recognized if one is cognizant of its existence and also notes the overall architectural and cytologic similarity to hyperplastic polyp/sessile serrated polyp. the entity is distinguished from invasive adenocarcinoma by the lack of infiltration and tumor desmoplasia. the differential diagnosis between hyperplastic polyp and tubular adenoma can be difficult, especially in a diminutive polyp that has been treated by hot biopsy (so-called thermal polyp). useful features in the differential diagnosis are found in table - . when the choice between hyperplastic polyp and tubular adenoma is difficult, as long as an adenoma diagnosis is not going to result in surgical resection (e.g., right colonic adenoma incompletely excised), we err on the side of adenoma to ensure that the patient will receive more frequent surveillance. mixtures of hyperplastic polyp, sessile serrated polyp, and adenoma occur. , mixed polyps and serrated adenomas are considered in more detail later. rare examples of patients with colons carpeted by hyperplastic-like polyps (hyperplastic polyposis) have been described ( fig. - ). the who defines hyperplastic polyposis as ( ) or more hyperplastic polyps proximal to the sigmoid colon of which are larger than cm, ( ) any number of hyperplastic polyps proximal to the sigmoid colon if the person has a first-degree relative with hyperplastic polyposis, and ( ) more than hyperplastic polyps of any size and any molecular events involved in the serrated polyp family are now recognized. methylation-induced inactivation of mmr genes occurs in both hyperplastic polyps and carcinoma. as shown in table - , methylation inactivation of genes and certain gene mutations (especially braf) appear to be involved in the serrated pathway to carcinoma. , these molecular events have been verified. [ ] [ ] [ ] [ ] [ ] [ ] hyperplastic polyps associated with carcinoma have been unusually large and right-sided lesions. they have been reported using numerous synonyms, including giant hyperplastic polyp, sessile serrated adenoma, sessile serrated polyp, inverted hyperplastic polyp, and polyp with epithelial serrated proliferation. it is becoming clear that several different pathologic entities have been called hyperplastic polyps in the past. this serrated polyp family includes conventional hyperplastic polyp, mixed hyperplastic/sessile serrated polyp/adenoma ( fig. - ), serrated adenoma (epithelial dysplasia defined, usually pedunculated and left sided, having eosinophilic cytoplasm and showing gastric foveolar change and often referred to as the traditionally defined serrated adenoma) ( fig. - ) , and hyperplastic-like polyps with unusual features that have been referred to as sessile serrated polyps or sessile serrated adenomas. , , [ ] [ ] [ ] [ ] sessile serrated polyps appear related to serrated adenomas and mixed polyps and could be the specific precursor lesion to sporadic msi-h carcinoma. transitions from sessile serrated polyps to areas of dysplasia and carcinoma with loss of hmlh protein expression have been described . , sessile serrated polyps as the name implies are sessile, large (frequently ≥ cm), right sided, and often show poor endoscopic circumscription. numerous cytologic and architectural abnormalities have been reported in sessile serrated polyps, especially those associated with carcinoma. , , , the abnormal proliferation or dysmaturation features include persisting nuclear atypia with large nuclei and nucleoli high (upper third) in the crypts, high (upper third of the crypt) mitotic figures, and irregular distribution of dystrophic goblet cells. architectural abnormalities include basal crypt dilatation, horizontally oriented crypts, crypt branching, an increased epithelial-to-stromal ratio (> %), inverted crypts, prominent serration, increased surface villosity or papillations, and the lack of a surface basement membrane thickening typical of conventional hyperplastic polyps. some authors suggest that a diagnosis of sessile serrated polyp requires the presence of at least four of the architectural and abnormal proliferation features mentioned earlier (figs. - and - ) . once recognized, the sessile serrated polyp creates a patient management dilemma. calling them "sessile serrated adenomas" may not be an appropriate default. first and foremost, these lesions do not show the cytologic dysplasia that should be definitional for adenoma. sessile serrated adenoma is often confused by gastroenterologists and surgeons with serrated adenoma or villous adenoma. it is unknown whether colonic resection (which is typically done for incompletely excised adenomas) should be recommended for sessile serrated polyps that are incompletely excised at endoscopy. furthermore, endoscopic follow-up for serrated adenoma would typically be in years (if the clinician considers serrated adenoma or sessile serrated adenoma a variant villous adenoma) or in years. in a cohort of patients with sessile serrated polyps preceding msi-h carcinomas, predated the carcinomas by less than years. we think that these lesions should be diagnosed as sessile serrated polyps and that they should be treated by complete endoscopic excision if possible. until more is known, a shorter surveillance interval (e.g., to years) rather than resection seems prudent for these types of polyps that are incompletely excised or associated with additional, endoscopically similar polyps that have remained unsampled. [ ] [ ] [ ] [ ] colorectal carcinoma occurs more often in men (male-tofemale ratio, : ), with a median age of years. most patients present with rectal bleeding, anemia, change in bowel habits, bowel obstruction, or less often perforation. patients with right-sided colon carcinoma are more likely to present with anemia and fatigue, whereas left-sided car-cinoma is more likely to produce melena, constipation, and change in bowel habits. approximately half of all large bowel carcinomas occur in the rectum, % occur in the sigmoid colon, and the rest are evenly distributed throughout the remainder of the colon. that said, with increased use of colonoscopy with removal of adenomas, clinicians have seen a right-sided migration of carcinomas since the late s. carcinomas of the right colon tend to produce large, exophytic tumors (see fig. - ) . carcinomas of the descending colon are more likely to be stenotic and produce the so-called "napkin ring" tumor. carcinomas anywhere can be fungating, ulcerated, or necrotic masses; the most common macroscopic appearance is an ulcer with raised, indurated edges (fig. the following features adversely affect prognosis: advanced stage, extensive local spread, lymph node involvement, aggressive histologic type, high histologic grade, extramural venous invasion, and free mesothelial surface invasion. , , [ ] [ ] [ ] although useful information is gleaned through these classic grading and staging exercises, the process is not without problems and controversy. no general agreement exists on staging or grading and all current schemes have shortcomings. [ ] [ ] [ ] using current systems, most fall into a moderate-stage, moderate-grade category in which the probability of survival is roughly / . the cap considered and commented on the multitude of reputed prognostic factors in a consensus statement and concluded that certain factors have been definitively proved to be of prognostic import, including local extent of tumor (pt), regional lymph node metastases (pn), blood or lymphatic vessel invasion, and residual tumor following surgery with curative intent. other factors that have repeatedly been shown to be of prognostic importance include tumor grade, radial margin status, and residual tumor in specimens following neoadjuvant therapy. the cap recommends that these additional features should also be included in pathology reports. although customarily included in pathology reports, parameters such as tumor size and gross configuration have been well studied and are of no prognostic significance. that still leaves an incredibly large group of factors that may be considered prognostic but have not yet been sufficiently studied. this discussion focuses on controversial areas in classic staging and grading including methods of lymph node dissection and assessment of histologic grade and type. additionally, the role of flow cytometric analysis, markers of proliferative activity, and other ancillary testing will be examined. the single most important factor related to patient prognosis is the presence or absence of lymph node metastases. no doubt exists that searching for lymph nodes in a resection specimen is tedious. the lymph node yield per case is directly proportional to the dissector's enthusiasm and skill. as a general rule, a standard resection specimen for carcinoma of the sigmoid colon or rectum should contain to lymph nodes, although we all have had cases in which the dissector found far fewer. minimum numbers of lymph nodes harvested is increasingly considered a measure of quality. , therefore, the routine use of clearance techniques for lymph node dissection has been debated. certainly, clearance techniques have advantages. one is likely to find more lymph nodes in a specimen and the lymph node yield will no longer depend solely on the dissector's ability and enthusiasm. however, the clearance process is time consuming and it may delay reporting. , clearing is relatively expensive because of the large volumes of clearing agents used and the prolonged technologists' or pathologists' time. common clearing agents are often flammable and toxic. cawthorn and colleagues showed that clearance techniques increase the yield of lymph nodes per specimen when compared with routine dissection. however, the proportion of stage , , and cases did not change. the number of positive lymph nodes found was similar between cleared and noncleared groups. this finding was later confirmed. clearance techniques are considered unnecessary for routine cases. , additional controversy is added by consideration of nontraditional methods of lymph node examination such as the following: immunohistochemical analysis for carcinoembryonic antigen (cea), cytokeratins, and epithelial membrane antigen; pcr testing looking for tumor dna or rna; and sentinel lymph node examination. the biologic significance of these nontraditional methods lacks validation, , [ ] [ ] [ ] [ ] [ ] and "positive" nodes found by these techniques may have no effect on prognosis. currently, the cap recommends that all grossly identified lymph nodes be sectioned (without multiple levels) in a routine fashion. , pathologists should find as many lymph nodes as possible and should recognize that the rules of representative sampling and probability apply. as a general rule, negative lymph nodes usually correlate with true pn status. , , , extramural tumor nodules of any size with smooth con- tours are counted as replaced regional lymph nodes. sentinel lymph node examination does not accurately predict either conventionally defined nodal metastasis or micrometastasis and is not considered useful in the study of patients with colorectal carcinoma. pathologists admit that grading is more art than science. grading is subjective and prone to interobserver and intraobserver variation. one multicenter trial noted % welldifferentiated adenocarcinomas from one institution, whereas another hospital reported % well-differentiated cases. marked heterogeneity exists within a given tumor. some observers grade on the average, whereas others assign a grade corresponding to the least-differentiated area. many grading systems are used for colorectal carcinoma. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] all employ slightly different criteria that are poorly defined. some use three grades and others four. some exclude mucinous carcinoma altogether and others include it as grade iv or grade iii. criteria for mucinous carcinoma are hardly ever defined. we follow the guidelines of dukes and bussey and use a three-grade system. well-differentiated adenocarcinoma (grade i), which should account for % to % of cases, shows tubular differentiation; the nuclear polarity is easily discerned, and nuclei are generally uniform in size . approximately % of adenocarcinomas are moderately differentiated (grade ii) exhibiting a more complex and irregular tubular pattern, and the polarity of nuclei is lost or is only barely discernible ( fig. - ). the remaining poorly differentiated adenocarcinomas (grade iii) consist of highly irregular glands or may show an absence of glandular architecture. nuclear polarity is lost ( fig. - ). when variability exists within a given tumor, the grade is determined by the worst area no matter how small. mucinous carcinoma and signet ring cell carcinoma are considered poorly differentiated or grade iii. jass and colleagues investigated grading using a cox regression analysis model in resection specimens. the only grading parameters associated with prognosis were the amount of tubule configuration, the pattern of growth (expanding versus infiltrative), and the degree of lymphocytic infiltration. when stage-related parameters were added into the cox regression model, only three factors emerged as significant: ( ) lymph node involvement, ( ) local spread (i.e., the components of stage), and ( ) the amount of lymphocytic infiltration in the neoplasm (i.e., a reflection of msi status). this study provided the scientific verification of the original observation by dukes and bussey that grade was subservient to stage in prognosis and re-emphasizes the need for careful specimen dissection and examination to determine the amount of local spread and lymph node status. many clinicians believe that mucinous carcinoma and signet ring cell carcinoma are associated with significantly worse prognosis than nonmucinous adenocarcinoma. unfortu- nately, the definitions of mucinous and signet ring cell carcinoma vary. , , [ ] [ ] [ ] [ ] [ ] work by sasaki and colleagues and umpleby and associates verified that mucinous and signet ring cell carcinomas are associated with a worse prognosis, but these tumors manifest at high stage and are associated with extensive local spread. sasaki and colleagues scrutinized a large cohort of mucinous, signet ring cell, and nonmucinous carcinomas using a cox multiple regression model. according to this study, the only significant adverse prognosis-related independent variables were the presence of lymph node metastases and the extent of local spread (i.e., the components of stage), along with an infiltrative growth pattern and minimal lymphocytic infiltration. sasaki and colleagues and umpleby and associates concluded that mucinous carcinoma (> % to % by volume) and signet ring cell carcinoma (> % cells with signet ring morphology) ( fig. - ) are more aggressive. these histologic features were not, however, significantly associated with poor prognosis when they were controlled for stage. flow cytometry for examination of dna content in human tumors involves cells or isolated nuclei stained in suspension with a fluorescent dye that binds stoichiometrically with double-stranded dna. these stained cells or nuclei are then passed one by one through an excitor light source (laser). the amount of fluorescence produced by the bound dye is detectable by a photoelectric cell, and the information is stored electronically. with this technique, thousands of measurements can be made in seconds and displayed on a histogram. the position of peaks on the x-axis is proportional to the amount of dna per cell, and the height of the peaks on the y-axis is proportional to the number of cells demonstrating a particular dna content. using this method, diploid cell populations can be distinguished from nondiploid (including dna aneuploid) cell populations. studies of paraffin-embedded and fresh colorectal carcinoma specimens have demonstrated an inconsistent association between dna aneuploidy and survival. , in at least one of these studies, stage was retained as a strong independent variable associated with prognosis after mul-tiple regression analysis. other investigators showed no independent association between dna aneuploidy and prognosis in a large group of patients with colorectal carcinoma, with stage as the only independent variable associated with prognosis. dna content analysis by flow cytometry is of no proved clinical value. the technology and methods lack standardization, and the results among groups generally are not comparable. most published studies have employed paraffin-embedded material. this may not be optimal because dna fragments and partial nuclei tend to stick together, thus leading to increased yields of pseudoaneuploid histograms. the proportion of cases showing aneuploid peaks is lower when fresh intact cells are used. in terms of interpretation, control histograms are easy to read but tumor histograms are less clean and interpretations are subject to interobserver variation. in a cohort of patients with colorectal carcinoma prospectively studied, results of flow cytometric analysis showed no correlation between dna aneuploidy and any standard staging or grading parameter and had no independent association with prognosis. , the cap believes that dna analysis has not been adequately studied for determination of prognostic value and the data are insufficient to recommend a specific technologic method. various proliferation markers have been studied in colorectal carcinoma. for example, a cohort of patients with colorectal carcinoma were studied using an antibody that recognizes ki- , a nuclear antigen expressed in all phases of the cell cycle except g . no correlation was found between ki- scores and stage, grade, or prognosis. stage, growth pattern, and lymphocytic infiltration were the only factors independently associated with prognosis. the cap believes that data are insufficient to recommend inclusion of proliferation indices in pathology reports for prognostic information. early nonpolypoid colorectal carcinoma early invasive colorectal carcinoma (pt -invasive carcinoma limited to the submucosa) warrants special mention given the advent of endoscopic techniques allowing gastroenterologists and surgeons to resect some carcinomas locally either surgically or through the endoscope (e.g., endoscopic mucosal resection [emr]). because lymph node status is the strongest prognostic factor in colorectal carcinoma, the question asked, particularly by surgeons, is whether local excision or emr is enough or should definitive surgical resection be performed for pt lesions. the issue is further complicated by the low rate of lymph node metastases in pt colorectal carcinoma, estimated at % to %. , [ ] [ ] [ ] [ ] this dilemma prompted evaluation of histologic parameters and molecular markers that correlate with positive lymph node status in excised pt colorectal carcinoma. features that consistently correlate with positive lymph node status in pt colorectal carcinoma include angiolymphatic invasion, poor differentiation, tumor budding (fig. [ ] [ ] [ ] [ ] [ ] [ ] , and sm invasion (invasion of the deepest third of the submucosa). [ ] [ ] [ ] [ ] [ ] although various immunostains and molecular markers have not been significantly associated with lymph node status, gene expression profiling could improve the prediction of patients likely to have positive lymph nodes and could improve outcomes. cetuximab is a chimeric monoclonal antibody that binds to epidermal growth factor receptor (egfr). it has clinical significant activity when given alone or in combination with irinotecan in patients with advanced irinotecan-refractory colorectal carcinoma. [ ] [ ] [ ] approximately % of colorectal cancers express egfr on immunohistochemical analysis but that expression does not correlate with gene amplification. immunohistochemical analysis for egfr is sometimes used as a selection criterion for cetuximab. the threshold for positive staining results has been extraordinarily low ( + staining in > % of cancer cells) and neither the proportion of positive tumor cells nor the intensity of staining has correlated with clinical response. some patients who tested negative for egfr responded to cetuximab and many patients who tested positive did not. consequently, the national comprehensive cancer network guidelines for colorectal cancer management recommend against using egfr expression based on immunohistochemical results to select patients for cetuximab therapy. , patients with colorectal cancer bearing mutated k-ras do not benefit from cetuximab. a bevacizumab is a monoclonal antibody directed against vascular endothelial growth factor (vegf), which is critical in the regulation of angiogenesis. this monoclonal antibody added to fluorouracil-based chemotherapy regimens resulted in significant improvement in patients with advanced colorectal carcinoma. , vegf is expressed in approximately % of colorectal carcinomas. neither microvessel density determination, which is prone to significant methodologic variation, nor vegf determinations by immunohistochemical analysis were considered a selection criterion. before , fluorouracil, a thymidylate synthase inhibitor, was the only effective treatment for advanced colorectal cancer and because leucovorin (folinic acid) enhances the effect by stabilizing the bond between fluorouracil and thymidylate synthase, both agents are often given together. , , other cytotoxic drugs such as irinotecan, an inhibitor of topoisomerase i, and oxaliplatin, which distorts dna by cross-linking into adducts, are now approved for treatment for advanced colorectal carcinoma. , , irinotecan has efficacy as a first-line treatment for advanced colorectal cancer but lacks efficacy as adjunct therapy. irinotecan is hydrolyzed into an active metabolite (sn- ) by hepatic carboxyl esterase. sn- is converted into an inactive form by uridine diphosphate glucuronosyltransferase isoform a (ugt a ). in patients with polymorphisms of ugt a , the toxicities of irinotecan (diarrhea, nausea, vomiting, myelosuppression, alopecia) are more severe. although results indicate that ugt a * polymorphisms have some relevance to toxicity, especially hematologic toxicity with the first cycle of chemotherapy, determination of the polymorphism seems to have marginal clinical implications. the observed toxicities can be managed clinically, other ugt a enzymes may play a role as well, and data do not support dose reduction based on a molecular test. cisplatin and its analogues (oxaliplatin) are particularly toxic and molecular markers to identify patients likely to respond have been investigated. oxaliplatin adducts are repaired by the nucleotide excision repair complex. ercc (excision repair cross-complementation group ) is of genes that encode proteins of this complex. polymorphisms that reduce levels of ercc correlate with clinical sensitivity to oxaliplatin , and could be used for patient selection. the medullary variant of colorectal carcinoma occurs predominantly in women and usually occurs in the cecum and right colon. , histologically, it is composed of uniform polygonal cells arranged in a nesting or trabecular pattern with minimal gland formation. immunohistochemistry is often employed to rule out neuroendocrine carcinoma or melanoma. other characteristic features of medullary carcinoma include a prominent lymphoid component that can either be peritumoral, often described as crohn's diseaselike, or intratumoral with tumor-infiltrating lymphocytes (more than five per high-power field; see figs. - and - ) . medullary carcinoma is seen with increased frequency in msi-h colorectal carcinoma whether sporadic or in association with lynch's syndrome. adenosquamous carcinoma, defined by having a malignant glandular and squamous component, occurs rarely as a primary carcinoma in the colon and rectum (fig. - ). the possibility of metastasis must always be considered. adjacent adenoma can help to confirm a primary tumor. adenosquamous carcinoma has been described in patients with ulcerative colitis (uc), fap, schistosomiasis, and endometriosis. occasionally, squamous differentiation can be found in adenomas (fig. - ). pure squamous colorectal carcinoma outside of the anal canal is extremely rare. the possible diagnosis of metastasis must be excluded. squamous carcinoma has been reported in fistulas, in association with radiation, and in patients with ibd, tuberculosis, and schistosomiasis. , , occasional case reports of microglandular goblet cell adenocarcinoma (goblet cell carcinoid) identical to that seen in the vermiform appendix have been described in the large bowel. these tumors are composed of trabeculae and nests of well-differentiated adenocarcinoma cells showing differentiation toward goblet cells. scattered or no endocrine differentiation is characteristically seen by immunohistochemical analysis for chromogranin and synaptophysin. although cytologically bland, these carcinomas are often aggressive in the colon and rectum. carcinosarcoma carcinosarcoma is often referred to as spindle cell carcinoma or metaplastic carcinoma and can occur rarely in the large bowel. frequently, a high-grade squamous or glandular component is detected. the mesenchymal component can be undifferentiated or can show striated or smooth muscle differentiation or areas of cartilage or bone. these tumors are associated with a poor prognosis. , , giant cell carcinoma and choriocarcinoma can occur purely or as focal components of an otherwise typical high-grade adenocarcinoma or carcinosarcoma. in choriocarcinoma, the giant cells express beta human chorionic gonadotropin (hcg). carcinomas with giant cells that fail to stain for beta hcg are referred to as giant cell carcinomas. , , endocrine tumors involving the colon and rectum can be classified into three types based on morphology: ( ) carcinoid tumor (well-differentiated neuroendocrine tumor), ( ) intermediate-grade neuroendocrine carcinoma, and ( ) high-grade neuroendocrine carcinoma, which can be further subdivided into small cell and large cell types. , rightsided endocrine tumors are usually large, whereas rectal endocrine tumors manifest as small polyps, most being solitary and less than . cm in greatest cross dimension. rectal carcinoids are quite common. they most often exhibit hindgut patterns of growth with trabeculae, small acini, and tubular structures that may contain luminal mucin and that must not be confused with goblet cell carcinoid (microglandular goblet cell adenocarcinoma) . mitoses are rare. the cytoplasm of the carcinoid tumor cells may be clear or eosinophilic and can sometimes appear granular. nuclei are regular and ovoid and contain granular chromatin. neuroendocrine differentiation can be proved with immunohistochemical analysis for chromogranin and synaptophysin. hormonally inactive carcinoids that are less than . cm in greatest cross dimension without angiolymphatic invasion act in a clinically benign fashion. , intermediate-grade neuroendocrine carcinoma acts more aggressively and is often characterized by angiolymphatic invasion, more frequent mitotic figures, nuclear pleomorphism with open nuclear chromatin, increased apoptotic bodies, and areas of necrosis ( fig. - ). transitions from low-grade to intermediate-grade and high-grade neuroendocrine carcinoma can be observed in rare cases. high-grade neuroendocrine carcinoma is important to recognize because of its particularly poor prognosis and because of chemotherapeutic considerations. , the small cell variant is composed of sheets of densely packed small to intermediate-size cells with dark hyperchromatic nuclei, inconspicuous nucleoli, frequent mitoses, and very little cytoplasm with molding ( fig. - ). foci of squamous and glandular differentiation can be seen in high-grade neuroendocrine carcinoma. large cell neuroendocrine carci-noma shows an endocrinoid growth pattern but contains larger cells that are round or polygonal and contain more abundant cytoplasm. the nuclei show a coarse chromatin pattern and in contrast to small cell carcinoma are often nucleolated ( fig. - ). large cell carcinoma and small cell carcinoma of the colon and rectum have a poor prognosis. virtually any subtype of lymphoma or leukemia can involve the colon and rectum. the reader is directed to the section covering the hematolymphoid system for more detailed discussions of these entities. use of the who classification for tumors of the hematopoietic and lymphoid tissues is recommended. this discussion focuses on the most common lymphoproliferative disorders affecting the colon and rectum. approximately one half of extranodal lymphomas involve the gi tract. diffuse large b-cell lymphoma makes up the majority of these cases and is usually easily identified by the surgical pathologist as malignant. b-cell lineage should be confirmed with cd immunostaining. an immunohistochemical panel to rule out undifferentiated carcinoma, melanoma, and high-grade neuroendocrine carcinoma is helpful in practice. differentiation of germinal center type (better prognosis) from nongerminal center type of diffuse large b-cell lymphoma should be made using immunostains for cd , bcl- , and mum- . burkitt's lymphoma and burkitt-like lymphoma can involve the colon and rectum. , , these lymphomas are composed of relatively uniform medium-sized cells with coarse chromatin and prominent nucleoli. b-cell lineage should be confirmed with cd immunostain and, as described earlier, other high-grade neoplasms should be excluded. most investigators recommend the workup of burkitt's and burkitt-like lymphoma with mib- immuno- staining, which should highlight at least % of cells. burkitt's lymphoma cells also characteristically stain positive for cd and bcl- . lymphoid hyperplasia must be distinguished from follicular lymphoma involving the colon and rectum. this is especially true in the setting of low rectal or anorectal lymphoid hyperplasia (so-called anorectal tonsil) and in defunctioned bowel seen in the setting of primary ibd. localized lymphoid hyperplasia of the anorectal area can manifest with bleeding, prolapse, or anorectal discomfort. the endoscopic appearance can vary from a sessile polyp, which can measure up to . cm in greatest dimension, to mere thickening of the mucosa resembling cobblestoning. histologically, one sees lymphoid hyperplasia with germinal center formation and scattered interfollicular collections of pale-staining histiocytes. distinction from follicular lymphoma can usually be made with standard h&e-stained sections but immunostaining for bcl- , which is overexpressed in follicular lymphoma because of the t( ; ) translocation, can be an extremely helpful adjunct. positive bcl- staining is seen in neoplastic follicles but not in reactive germinal centers. up to % of cases of multiple lymphomatous polyposis are caused by follicular lymphoma (fig. - ). mantle cell lymphoma usually manifests with widespread lymphadenopathy and frequent bone marrow involvement. overt gi tract involvement occurs in % to % of patients and clinically occult disease can affect up to % of patients. colonic involvement can manifest as a mass, a polyp, diffuse mucosal thickening, or multiple lymphomatous polyposis. approximately one third of patients with lymphomatous polyposis have mantle cell lymphoma. the histologic pattern of mantle cell lymphoma typically shows mucosa and submucosal infiltrates by small atypical lymphocytes surrounding germinal centers with effacement. the lymphoma cells express the b-cell marker cd and often coexpress cd . the t( ; ) translocation causes overexpression of cyclin d- , which is now considered defining. , extranodal marginal zone b-cell lymphoma of the mucosa-associated lymphoid tissue (malt) type can involve the colon and rectum and is responsible for up to % of cases manifesting as multiple lymphomatous polyposis. , similar to its appearance in the stomach, colorectal marginal zone b-cell lymphoma of malt type creates destructive lymphoepithelial lesions and produces an expansive proliferation of marginal zone lymphocytes. an appropriate immunohistochemical profile includes positive staining for pan b-cell markers and negative staining for cd , cd , cd , cd , and cyclin d- . gene rearrangement studies by pcr can be helpful in difficult cases. , , mesenchymal tumors involving the colon and rectum are unusual and include gi stromal tumors (gists), true smooth muscle tumors, neural tumors, inflammatory fibroid polyp, fatty tumors, fibrohistiocytic tumors, tumors of skeletal muscle, and vascular lesions (see later). primary mesenchymal tumors of the colon and rectum must be distinguished from mesenchymal differentiation within a high-grade carcinoma. the reader is directed to the section on soft tissues for more detailed discussions of these entities. gists of the colon and rectum are rare and usually show high-grade features using the national institutes of health consensus approach for classification. approximately % of colon and rectum gists have shown aggressive clinical behavior. overexpression of c-kit using cd immunostaining must be ascertained, preferably as part of a panel of immunostains that includes cd , smooth muscle actin, desmin, cytokeratin, s- protein, and melan a to verify gist and to exclude other differential diagnostic possibilities. schwannoma in the colon and rectum is rare and is usually encountered in the absence of a predisposing condition. schwannomas are rarely seen in patients who have neurofibromatosis syndrome or multiple endocrine neoplasia type iib (men b) and must be distinguished from gists in that setting. , , neurofibromas can manifest as an isolated mass lesion or nodule but are more commonly seen as a diffuse infiltration of the bowel wall in the setting of neurofibromatosis. care must be taken to distinguish neurofibroma from schwannoma and gist, which also occur in neurofibromatosis. , , granular cell tumor can manifest as a polyp or nodule frequently in the distal rectum and anal canal where it can mimic a hemorrhoid. the tumor is composed of masses of histiocytic-like cells with granular eosinophilic or amphophilic cytoplasm, which can sometimes be spindled . the cytoplasmic granules stain positive on pas and stain intensely for s- protein. , true smooth muscle tumors are rare but leiomyomas of the muscularis mucosae are commonly encountered in biopsy specimens. these tumors are usually small, less than mm to mm in greatest cross dimension, and are composed of smooth muscle cells. atypical mitoses and necrosis typical of leiomyosarcoma are not seen and the reported behavior has been benign. primary glomus tumors of the colon and rectum are exceptionally rare , and can manifest as a polyp or mucosal thickening. microscopically, these tumors are composed of lobules of small round cells intimately related to vascular spaces and usually show positive immunoreactivity for smooth muscle actin and vimentin. the cases reported so far have acted in a benign fashion. lipohyperplasia of the ileocecal valve, which can be accentuated in obese persons, is the most common fatty tumor of the colon. , occasionally, the fat may produce a more localized tumor. patients are usually asymptomatic and the degree of lipohyperplasia generally correlates with body weight. lipomas are frequently encountered during colonoscopy. these tumors are composed of benign adipose tissue. most are small and asymptomatic but larger ones can bleed, can be subject to trauma or prolapse, cause abdominal pain, and may lead to resection ( fig. - ). lipomas are usually recognized at colonoscopy by their yellow color and pliability (pillow sign). atypical lipomas and even liposarcoma have rarely been described. rare examples of malignant fibrous histiocytoma, rhabdoid tumor, and rhabdomyosarcoma have been reported in the large bowel. numerous classifications for intestinal vascular lesions have been proposed. , we find it useful to divide the vascular lesions into two groups, those isolated to the gi tract and those associated with recognized clinical syndromes (tables - and - ). vascular ectasia of the right colon has numerous synonyms, including angiodysplasia, arteriovenous malformation, and the type i lesion of moore and colleagues. , , , vascular ectasia of the right colon is a well-defined clinicopathologic entity with a distinct type of lesion and pathogenesis. patients are typically elderly and present with episodes of recurrent low-grade lower gi bleeding. approximately % of patients present with massive gi hemorrhage. the lesions nearly always manifest in the cecum or right colon and are often multiple. vascular ectasia of the right colon is usually not associated with other conditions. reports linking vascular ectasia of the right colon to aortic valvular disease have been criticized. , however, bleeding vascular ectasias may be associated with aortic stenosis and associated abnormalities in von willebrand factor (deficiency of the largest multimers). colonoscopic and radiographic examination will show almost all lesions. endoscopically, vascular ectasias appear as erythematous mucosal macules or papules measuring . mm to mm in diameter ( fig. - ). they may be round, oval, or stellate. a "feeding" vessel may be visible. angiographic signs of vascular ectasia include the densely opacified, slowly emptying vein, an opacified vascular tuft, early opacification (filling) of a draining vein, or, rarely, extravasation of contrast media into the lumen. vascular ectasia is usually treated primarily by the endoscopist (heat probe or other endoscopic ablation technique) or by the radiologist (vasopressin infusion or transcatheter embolization) at the time of diagnosis. , the typical curative surgery, right hemicolectomy, is rarely performed and is often reserved for those cases in which endoscopy or radiography could not be performed or treatment was not successful or for perforative complications of endoscopic or angiographic treatment. vascular ectasia is notoriously difficult to identify by standard pathologic techniques in resection specimens. demonstration of the lesion usually requires some form of vascular injection. boley and brandt's technique employs injection of the fresh specimen vasculature with a silicon rubber compound. the specimen is then fixed in formalin, dehydrated in graded alcohols, and cleared with methylsalicylate. this technique produces a transparent specimen in which the vasculature can be examined by epi-illumination and transillumination under a dissecting microscope. using this technique, the vascular ectasias appear as "coral reef-like" arrangements measuring . cm to . cm in diameter contrasted against the honeycombed pattern of the normal colonic mucosal vasculature. histologic examination reveals abnormal thin-walled ectatic veins, venules, and capillaries localized to the mucosa and submucosa. the lesions are thought to result from repetitive partial obstruction to venous drainage caused by contraction of the muscularis propria. wall tension is directly proportional to lumen diameter, a finding that may explain the lesion's predilection for the right colon, where the diameter is the greatest. obviously, vascular injection techniques require preparation in terms of materials. if caught unprepared, one may still demonstrate larger ectasias by carefully and gently washing off the mucosal surface of the specimen with water. sometimes, a blood clot adheres to the bleeding point and this may direct the histologic sectioning. we have been disappointed with injection of contrast media and specimen radiography but have had success demonstrating right-sided vascular ectasia by injecting brightly colored ink into the ileocolic artery. some investigators have reported success by bluntly dissecting the mucosa from the muscular wall of the formalin-fixed bowel followed by dehydration and transillumination. hemangiomas many isolated hemangiomas may be classified on the basis of the predominant vessel type as capillary, cavernous, venous, arteriovenous, or mixed. capillary hemangiomas are typically small, rarely multiple, and composed of small, closely packed capillaries. these lesions may be found in the colon but are usually encountered in the small intestine, appendix, and perianal skin. some lesions reported as capillary hemangiomas may, in fact, be vascular ectasias (see earlier). capillary hemangiomas rarely cause clinical symptoms. cavernous, venous, arteriovenous, and other mixture hemangiomas occur in localized or diffuse forms [ ] [ ] [ ] [ ] and have the same clinical significance regardless of vessel type . cavernous hemangiomas are composed of blood-filled, sinus-like spaces supported by connective tissue. , the stroma occasionally contains smooth muscle. cavernous hemangiomas can occur in the small bowel but have a predilection for the colon and rectum. patients may present with symptoms of a mass or with rectal bleeding. localized cavernous hemangiomas can be treated by excision, fulguration, or sometimes radiation therapy, whereas diffuse forms of hemangiomas may be unresectable and treatment must be directed toward controlling complications. a lesion composed of abnormal proliferating veins and arteries with evidence of arteriovenous shunting is referred to as an arteriovenous hemangioma. venous hemangiomas are characterized by large, thick-walled vessels that resemble normal veins but occasionally have more disorganized smooth muscle in their walls. arteriovenous and venous hemangiomas have the same distribution and significance as cavernous hemangioma. rare reports have noted tumors composed of admixtures of dilated vascular chan- nels and lymphatics. these tumors can be referred to as hemangiolymphangiomas, lymphaticovenous malformations, or even generically as vascular malformations. , these lesions have the same clinical significance as cavernous hemangiomas. pure lymphangioma of the large bowel is extremely rare. depending on size and location, these lesions may be asymptomatic or they may cause pain, diarrhea, or bleeding. reported size has varied to up to cm. microscopically the lesions are composed of thin-walled, anastomosing endothelial-lined channels that are bloodless but usually contain eosinophilic material. the term phlebectasia defines a non-neoplastic venous varicosity. these lesions may occasionally cause severe gi bleeding. the most frequent site of bleeding caused by phlebectasias associated with portal hypertension is the lower esophagus. other sites have been reported, especially the rectosigmoid colon and rarely the small intestine. we have also seen cases of idiopathic colonic varices in children. because phlebectasias are encountered rarely, if ever, in surgical specimens, they are not considered further. hereditary hemorrhagic telangiectasia (hht) is inherited as an autosomal dominant trait. , two disease-causing genes have been identified, endoglin and alk- . vascular lesions occur in the skin, mucous membranes, and internal organs, including liver, genitourinary tract, meninges, eyes, and spinal cord. the mucocutaneous lesions appear in the second and third decades of life. many patients present with recurrent epistaxis in childhood. approximately % of patients with hht, usually older patients, have gi bleeding. the histopathologic pattern of the colorectal lesion is simple dilatation of normal vascular structures (i.e., telangiectasia) and in general resembles the lesions of right-sided vascular ectasia ( fig. - ). progressive systemic sclerosis can be associated with gi tract telangiectasias. , a predilection exists for the stomach, rectum, and colon. intestinal bleeding develops only rarely. the blue rubber bleb nevus syndrome can occur as an autosomal dominant trait or in a sporadic form. the characteristic histologic lesion is a cavernous hemangioma that can involve the skin, gi tract, and other viscera. [ ] [ ] [ ] the skin lesion is typically blue and tender and ranges in size from . cm to . cm. the lesion empties on digital pressure to leave a wrinkled blue sac that in time refills with blood. the preferred site of involvement in the gi tract is the small intestine but colorectal lesions have been reported. turner's syndrome is characterized by numerous somatic abnormalities associated with a xo karyotype. telangiectasias of the small and large intestine have been described in this disorder and may cause gi bleeding. klippel-trenaunay-weber syndrome occurs sporadically. patients demonstrate unilateral soft tissue and bony hypertrophy associated with a port-wine hemangioma. gi cavernous hemangiomas have been described in these patients. , pseudoxanthoma elasticum shows marked clinical and genetic variability. clinically, "plucked-chicken" skin and angiomatoid streaks in the retina have been described. bleeding in the gi tract may occur from ruptured blood vessels caused by the abnormalities in elastic structure. patients usually bleed from the stomach. arteriovenous hemangiomas have also been described. , the term ehlers-danlos syndrome describes more than subclasses of metabolic disorders associated with abnormal collagen synthesis. most patients demonstrate hyperelasticity of skin and joints (i.e., the circus rubber man). vascular ectasias or proliferative vascular lesions have not been described, but spontaneous arterial or intestinal rupture has been reported in some forms (especially type iv [vascular ehlers-danlos syndrome with mutations of col a gene ]) of ehlers-danlos syndrome. [ ] [ ] [ ] [ ] [ ] [ ] [ ] kaposi's sarcoma characteristically cause pigmented skin nodules predominantly on the lower extremities. currently, an aggressive variant of kaposi's sarcoma in patients with acquired immunodeficiency syndrome (aids) is most commonly seen [ ] [ ] [ ] and has been linked to infection with human herpesvirus . the gi tract of patients with aids is frequently involved by kaposi's sarcoma. investigators have estimated that about half of the patients with cutaneous or lymph node kaposi's sarcoma have concomitant gi tract involvement. [ ] [ ] [ ] rarely, the gi tract can be involved without skin lesions. gi tract kaposi's sarcoma is typically asymptomatic but occasional gi kaposi's sarcoma can cause bleeding, obstruction, perforation, or protein-losing enteropathy. , gi kaposi's sarcoma has numerous appearances grossly and endoscopically. although red macules have been described, kaposi's sarcoma more characteristically causes a red or purple nodule or plaque measuring mm or more. some patients with gi kaposi's sarcoma have large tumors. despite the vascular nature of the neoplasm, lesions can be sampled for biopsy and in most instances bleed surprisingly little. the diagnostic yield with biopsy has been notoriously low ; in one report, only % of upper gi specimens and % of sigmoidoscopic specimens tested positive. usually, the low yield is attributed to the preferential submucosal location of kaposi's sarcoma that may not be sampled in an endoscopic biopsy specimen. that said, examples of kaposi's sarcoma are easily overlooked by even experienced pathologists, because the sarcoma is subtle and can be dismissed as granulation tissue. in positive biopsy specimens, kaposi's sarcoma typically causes an expansion of lamina propria by a spindle cell proliferation that may not be very atypical. the spindle cells are usually not extremely mitotically active and at first glance may suggest granulation tissue (although macrophages and mononuclear inflammatory cells are typically absent) or the fibromuscular obliteration of the lamina propria associated with the solitary rectal ulcer syndrome (prolapse or trauma-related change). generally, gi kaposi's sarcoma is histologically similar to kaposi's sarcoma seen elsewhere. the histologic pattern shows spindle cells, vascular clefts, extravasated red blood cells frequently associated with hemosiderin deposits, and occasionally tumor necrosis ( fig. - ). kaposi's sarcoma cells usually stain with antibodies directed against factor viii-related antigen but more consistently stain positive with endothelial markers such as cd and cd . as mentioned earlier, the changes are subtle and diagnosis can be difficult. important clues to the proper diagnosis include ( ) knowledge of the clinical situation, ( ) knowledge of the endoscopic appearance, ( ) recognition of crowding and hyperchromasia in the spindle cell nuclei, ( ) recognition of slits and spaces containing red blood cells, and ( ) recognition of the tendency for kaposi's sarcoma cells to overrun or obliterate the muscularis mucosae. aggressive antiretroviral therapy is now considered the first-line treatment for gi kaposi's sarcoma. , other forms of treatment are reserved for symptomatic tumors. surgical procedures are performed for obstruction or severe bleeding. chemotherapy approaches have improved but the treatment is risky in an already immunocompromised patient. these therapies offer palliation at best. treatment with zidovudine (formerly azidothymidine [azt]) does not have an effect on gi kaposi's sarcoma. , immunomodulation has been tested but is not used. , , the addition of taxanes (paclitaxel and docetaxel) to aggressive antiretroviral therapy appears promising. , angiosarcoma angiosarcomas are only rarely encountered in the colon and rectum. the varied histologic spectrum ranges from wellformed vascular structures to sheets of malignant polygonal or spindle cells. grossly and endoscopically, angiosarcomas form red-gray polypoid masses. an immunohistochemical panel including cd and factor viii-antigen can be useful in diagnosis. the term dieulafoy's lesion refers to an inappropriately large artery located directly beneath the mucosa where it can erode and cause profuse bleeding. although typically described in the stomach, similar lesions are found in the colon and rectum. metastatic neoplasia to the colon and rectum come principally from the lung, breast, ovary, and skin (melanoma). advanced prostate carcinoma can cause confusion with primary rectal carcinoma or with neuroendocrine carcinoma. sometimes perirectal lymph node metastases in resections performed for colorectal carcinoma contain metastatic deposits from prostate carcinoma. fap is inherited as an autosomal dominant trait. bussey recognized that or more colorectal adenomas (recognized grossly) phenotypically identified patients with fap and distinguished them from patients with multiple adenomas in whom inheritance was not seen. , in typical fap, hundreds to thousands of adenomas develop within the colon ( fig. - ). the adenomas begin to appear in the second or third decades of life and are surprisingly asymptomatic considering their usually large numbers. symptom-atic patients present with signs and symptoms of increased bowel motility and the passage of blood or mucus, or both, which often heralds the onset of carcinoma. the average age of patients with colon cancer and fap is years. two thirds of patients with these so-called propositus cases present with carcinoma and nearly one half of them have more than one carcinoma in the colon. this high risk of invasive cancer in symptomatic patients forms the basis for polyposis registries and the extensive screening of asymptomatic kindred at risk for fap. screening recommendations have evolved with increased genetic information. genetic testing should be considered for fap, attenuated fap, and muty homologue (myh)-associated polyposis (map) when or more colorectal adenomas are found in a patient during a single examination or over time. screening of first-degree relatives of affected individuals should begin at the age of years. in the absence of genetic testing, endoscopic screening is still useful to detect fap. all affected patients have adenomas within the range of a flexible sigmoidoscope. it is therefore recommended that screening sigmoidoscopy begin at age years with re-examination every years. the diagnosis of fap must be confirmed with biopsy because lymphoid polyposis and hyperplastic polyposis can mimic fap grossly and endoscopically. once a diagnosis of fap has been established, prophylactic proctocolectomy is recommended. most investigators recommend sigmoidoscopy for mutation-negative kindred at age years just in case the genetic test is erroneous. thyroid examination for associated thyroid lesions (usually papillary carcinoma with cribriform pattern ) and determination of serum alpha fetoprotein (to screen for hepatoblastoma) are recommended. regular upper endoscopy should also be performed. gastric and duodenal polyps develop in % to % of patients with fap. the gastric lesions are usually fundic gland polyposis, whereas the duodenal polyps are usually adenomas. the fundic gland polyps can develop a peculiar surface epithelial atypia called foveolar dysplasia, but progression to carcinoma is extremely rare. the incidence of duodenal adenomas in fap increases with increasing age. duodenal adenomas have a propensity to develop in the periampullary region. adenomas anywhere in the gi tract can proceed through the dysplasia-carcinoma sequence. the relative risk of duodenal or periampullary carcinoma in patients with fap is approximately to times that seen in the general population, and duodenal or periampullary carcinoma has become the major cause of morbidity and mortality in patients with fap in the post-prophylactic colectomy era. the gene responsible for fap (apc gene) has been localized to the long arm of chromosome ( q -q ) and has been cloned. [ ] [ ] [ ] [ ] [ ] some apc gene mutation-negative cases may be caused by mutation of myh (see later). mutation in most patients with fap creates a stop codon resulting in a truncated protein product. the apc gene is a tumor suppressor gene and the apc protein is part of the wntsignaling pathway , involved in cell growth control. when the apc gene is mutated, beta-catenin accumulates, thus altering expression of certain genes affecting proliferation, differentiation, migration, and apoptosis. most patients are now diagnosed by dna sequencing, which has largely replaced the assay to detect the truncated apc protein (ptt). , monoallelic mutation analysis (mama) examines the two apc alleles independently and can detect more than % of patients with fap. another test, multiplex ligation-dependent probe amplification (mlpa), is useful in detecting large deletions. more than disease-causing apc gene mutations have been reported. localization of mutations within the apc gene locus correlates with phenotype. for example, germline mutations between codon and codon are associated with very large numbers of colonic adenomas, whereas mutations elsewhere, especially near the ′ end or the ′ end of the apc gene and an area of exon , yield lesser numbers of colonic adenomas (see the later discussion of attenuated fap). , [ ] [ ] [ ] in gardner's variant, in addition to colonic adenomas and upper gi polyps, patients can exhibit extraintestinal manifestations such as osteomas, epidermal inclusion cysts and other benign skin tumors, desmoid tumors of the abdomen or abdominal wall, fibrosis of mesentery, dental abnormalities, carcinoma of the periampullary region or duodenum, and carcinoma of the thyroid. patients with gardner's syndrome have apc gene mutations; however, no particular apc mutation distinguishes fap from gardner's variant. even within a "gardner's family," gardner's stigmata can be variably expressed and can skip generations. therefore, some unknown disease-modifying factors are required for phenotypic expression of the extraintestinal manifestations. turcot's syndrome has been the subject of some controversy. in many investigators' zeal to publish, the phenotypic spectrum has been unduly broad with colonic manifestations ranging from a single adenoma to a virtual carpeting of the colonic mucosa with polyps. furthermore, the brain tumors have comprised almost every histologic type. molecular studies done on families with turcot's syndrome have clarified the situation. families with turcot's syndrome and germline mutations of the apc gene have a typical fap colonic phenotype and develop medulloblastomas. other patients originally thought to have turcot's syndrome have mutations in the dna mmr genes that are characteristic of lynch's syndrome. the brain tumors in this group have varied, with many reported as glioblastoma. mutations of the apc gene near the ′ end and ′ end and in a particular region of exon result in fewer adenomas (< ; average, ), a tendency for the adenomas to be macroscopically flat, and a propensity for these adenomas to cluster in the right colon. originally reported as hereditary flat adenoma syndrome, this form is now more accurately referred to as attenuated fap (afap). , as in typical fap, these patients can develop fundic gland polyposis, duodenal adenomas, and periampullary carcinoma. the risk of colorectal carcinoma is increased in these patients albeit to a lesser degree than in the other form of fap and the cancers tend to occur later in life (average age, years). inherited variants of a base-excision repair gene myh have been associated with colorectal polyposis with an autosomal recessive mode of inheritance. , , some cases phenotypically resemble fap or attenuated fap and are referred to as myh polyposis or map. of those patients with a phenotype typical of fap or suspected afap in whom an apc gene mutation is not found, % to % will have mutation of the myh gene. approximately % of affected persons have one of two specific myh mutations (y c or g d). if one is found, then sequencing is done to find the mutation on the other allele because map is biallelic. these patients should be treated and followed similarly to patients with fap. juvenile polyps can occur in a sporadic form or can be part of juvenile polyposis syndrome. in the sporadic form, juvenile polyps have their peak prevalence in children between and years of age. some evidence indicates that juvenile polyps once formed can regress; they can certainly be seen in adults. sporadic juvenile polyps typically occur singly but patients can have up to five, usually located in the rectum. juvenile polyps typically range in size up to cm and can be associated with overt prolapse (fig. - ). because these polyps are often attached by a small pedicle, they are prone to autoamputation. histologically, typical juvenile polyps consist of a hamartomatous overgrowth of the lamina propria accompanied by elongation and cystic dilatation of colonic crypts lined by nondysplastic colonic epithelium ( fig. - ). osseous and cartilaginous stromal metaplasia can occur ( fig. - ). the inflammatory component of juvenile polyps can be quite prominent with neutrophils and lymphoid follicles within the lamina propria. frequently, the distinction between juvenile polyps and inflammatory polyps of primary ibd cannot be made on histologic grounds alone and requires clinical correlation. nonsyndromatic juvenile polyps appear to have no malignant potential. juvenile polyposis syndrome can be familial or nonfamilial and usually becomes clinically apparent within the first decade of life with painless rectal bleeding, prolapse, iron deficiency anemia, or passage of an autoamputated polyp. patients are considered to have juvenile polyposis syndrome if they have six or more juvenile polyps in the colon and rectum, have juvenile polyps throughout the gi tract, or have any number of juvenile polyps in association with a positive family history. , in the nonfamilial forms of juvenile polyposis syndrome (≈ % of the total), patients frequently have associated abnormalities, such as cardiac defects, hydrocephalus, gut malrotation, undescended testes, and skull abnormalities. patients with the familial forms usually lack these extraintestinal manifestations. inheritance has varied although almost all are considered autosomal dominant with variable penetrance. familial forms of juvenile polyposis syndrome appear to be associated with an increased risk of colorectal carcinoma. prophylactic colectomy may be prudent in juvenile polyposis syndrome. patients may also have an increased risk of gastric, small intestinal, and pancreatic carcinoma. juvenile polyposis syndrome coexisting with hht (osler-weber-rendu syndrome) is rarely reported. the number of polyps in juvenile polyposis syndrome typically ranges from a few dozen to several hundred ( fig. - ) . phenotypically, juvenile polyposis syndrome appears to occur in three varieties: ( ) polyps limited to the colon, ( ) polyps limited to the stomach, and ( ) polyps throughout the entire gi tract. [ ] [ ] [ ] the mucosal polyps found in the context of juvenile polyposis syndromes are often unusual histologically. in addition to the typical juvenile polyps described earlier, one can find juvenile polyps with atypical features such as more epithelium than lamina propria. in addition, mixture polyps (juvenile polyps with areas of adenoma or dysplasia) are quite frequent. , [ ] [ ] [ ] seen in % of cases. one should consider genetic testing for juvenile polyposis syndrome when three or more juvenile polyps have occurred in one individual or when juvenile polyps are found outside the colon. madh and bmpr a are both components of the signaling pathway for transforming growth factor-b and the bone morphogenetic proteins. patients with madh gene mutation are more likely to have gastric juvenile polyposis. , , juvenile polyps can be found in patients with other hamartomatous syndromes of the colon, such as intestinal ganglioneuromatosis or ganglioneurofibromatosis (see later), [ ] [ ] [ ] although some of these syndromes are now best classified as pten (phosphatase and tensin homologue) syndrome (see later). patients can sometimes be managed with endoscopy and polypectomy (every to years); however, colectomy must be considered for patients with large numbers of polyps or polyps with dysplasia or for patients with complications (e.g., bleeding, obstruction). screening colonoscopy every years should commence with symptoms or in the early teenage years in an asymptomatic patient. , upper endoscopy is also recommended in patients with juvenile polyposis syndrome. esophagogastroduodenoscopy and small bowel examination (every years) should begin at age years. , ruvalcaba-myhre-smith syndrome (bannayan-zonana syndrome, ruvalcaba-myhre-smith syndrome consists of macrocephaly, mental deficiency, unusual craniofacial appearance, pseudopapilledema, pigmented macules on the penis, and hamartomatous polyps in the gi tract. the syndrome appears to be passed on as an autosomal dominant condition. the gi polyps have been indistinguishable from juvenile polyps and in rare instances intestinal ganglioneuromatosis has also been described. the syndrome has been linked to mutations or deletions in the pten gene ( q . ) , , and with cowden's syndrome and can be considered as one of the pten polyposis syndromes. peutz-jeghers polyps can be found throughout the gi tract, either sporadically or as part of the peutz-jeghers syndrome. , , the polyp itself is characterized by fairly normal epithelium and lamina propria lining an abnormal arborizing network of smooth muscle that represents hamartomatous overgrowth of the muscularis mucosae ( fig. - ). , , peutz-jeghers syndrome, usually inherited as an autosomal dominant trait, is the combination of skin hyperpigmentation and peutz-jeghers polyps in the gi tract. the diagnosis of peutz-jeghers syndrome is considered definitive if the patient has a peutz-jeghers polyp and at least two of the following criteria: ( ) family history, ( ) mucocutaneous hyperpigmentation, and ( ) small bowel polyposis. , the pigmentation consists of clusters of brown or black freckles about the lips, buccal mucosa, and perianal and genital region. pigmented areas can occasionally be seen on the fingers and toes. the spots appear in the first year of life and tend to fade toward middle age. the polyps usually number only in the dozens and can be found throughout the gi tract. these polyps have a propensity to form in the small intestine where they often cause intussusception. in rare kindred, peutz-jeghers polyps have been limited to the large bowel. cases of complicating gi carcinoma have been reported. , approximately % of female patients with peutz-jeghers syndrome have a peculiar ovarian tumor, sex cord tumor with annular tubules (sctat). the rate of detection may increase if the ovaries are carefully examined, , and some tumors may be associated with sexual precocity. male patients with peutz-jeghers syndrome occasionally have unilateral or bilateral sertoli cell tumors of the testes. , adenoma malignum and pancreaticobiliary tract carcinomas are reported to occur at increased rates. the peutz-jeghers syndrome has been linked to the stk (serine/threonine-protein kinase , also known as lkb ) gene on chromosome p . [ ] [ ] [ ] [ ] and can be demonstrated in % of cases. this is a tumor suppressor gene involved in transduction of intracellular growth signals. investigators have suggested that genetic testing be considered for peutz-jeghers syndrome when any peutz-jeghers polyps or typical perioral pigmentations are found. meta-analysis of cancer risk in an evaluation of patients with known mutations of the stk gene have shown increased lifetime risk for cancer of the esophagus, stomach, small bowel, colon, pancreas, and breast. , putting this into perspective, the risk for breast cancer in peutz-jeghers syndrome is similar to the risk seen in individuals with germline mutations of brca and brca and peutz-jeghers syndrome is the strongest known risk factor for pancreatic carcinoma except for hereditary pancreatitis. screening at-risk individuals (first-degree relatives of a patient with peutz-jeghers syndrome) should begin at birth with an annual history and physical examination to look specifically for melanotic spots, precocious puberty, and testicular tumors. asymptomatic at-risk individuals without stigmata by age years should be tested for stk /lkb gene mutations. if mutation is not found in the family, small intestinal contrast radiography every years until age years is recommended. other investigators suggest that upper and lower endoscopy with small bowel series should be done at ages , , and years. esophagogastroduodenoscopy and upper gi radiographic series with small bowel follow through are recommended in patients with peutz-jeghers syndrome commencing at age years and repeated every years thereafter. , colonoscopy every years is recommended starting with symptoms or by age years if symptoms have not occurred. , testicular examination, pelvic examination by age years, mammographic examination by age years, and endoscopic ultrasound examination of the pancreas by age to years have been recommended. , annual transvaginal ultrasound examination and serum ca- determination are also recommended commencing at age years. intestinal ganglioneuromatosis is defined as proliferation of ganglion cells, neurites, and supporting cells that can affect any layer of the gi wall ( fig. - ). these proliferations often manifest as mucosal polyps in the colon. although these lesions most often occur as an isolated phenomenon, the importance of intestinal polypoid ganglioneuromatosis is in recognizing the other settings in which it can occur, such as von recklinghausen's disease (nf gene mutation), men b (ret gene mutation), cowden's syndrome (pten mutation), ruvalcaba-myhre-smith syndrome (pten mutation), and tuberous sclerosis (tsc [ q ] or tsc [ p ] mutation). [ ] [ ] [ ] [ ] [ ] intestinal ganglioneuromatosis can coexist with juvenile polyps although these patients may be better classified as having pten polyposis. [ ] [ ] [ ] cowden's syndrome describes an autosomal dominant multiple hamartoma syndrome in which patients have multiple orocutaneous hamartomas (e.g., facial trichilemmomas, mucosal papillomas, acral keratosis, subcutaneous lipomas), fibrocystic disease of the breast, an increased risk of breast carcinoma, thyroid abnormalities, and hamartomatous polyps in the stomach, small intestine, and colon. polyps of the gi tract, when described, often have shown an abnormal proliferation of the smooth muscle in the lamina propria and generally have resembled the polypoid variant of solitary rectal ulcer syndrome. some juvenile polyp-like proliferations have been described. intestinal ganglioneuromatosis has also been reported. other associated abnormalities include macrocephaly, high arched palate, hypoplastic mandible and maxilla, microstomia, supernumerary nipples, pectus excavatum, hemangiomas, ovarian cysts, and uterine leiomyomas. , the gene (pten) for cowden's disease has been mapped to chromosome ( q - ). , , cowden's syndrome and ruvalcaba-myhre-smith syndrome are sometimes referred to as the pten polyposis syndromes. genetic testing is suggested when features of this syndrome are present. esophagogastroduodenoscopy and small bowel examination every years beginning at age years is recommended. cronkhite-canada syndrome is an acquired nonfamilial syndrome characterized by intestinal polyposis, dystrophic changes of the fingernails, alopecia, and cutaneous hyperpigmentation. , patients first present with diarrhea, abdominal pain, and anorexia that progresses to weight loss and protein-losing enteropathy. many patients complain of loss of taste (hypogeusia) and loss of smell. as a rule, the ectodermal changes occur weeks to months after the other symptoms. the nail dystrophy consists of thinning, splitting, and separation from the nail bed (onycholysis). onychomadesis (complete loss of the nail) can also occur. the hair loss is rapid and may be seen in the scalp, eyebrow, face, axilla, or pubic region. the cutaneous hyperpigmentation ranges from small macules to confluent areas of hyperpigmentation that can be cm or more. histologically, the pigmented macules result from increased melanin in the basal layer. cronkhite-canada polyps are found throughout the gi tract but are most commonly seen in the stomach and large bowel. grossly, they are sessile; a few are pedunculated. the polyps tend to occur on a background of diffuse mucosal thickening ( fig. - ) . histologically, the polyps themselves are identical to juvenile polyps. however, the mucosa between polyps is abnormal and shows edema, congestion, and inflammation (chronic inflammation often with prominent eosinophils) of the lamina propria coupled with glandular ectasia (fig. - ) . carcinomas of the colon and stomach have been described rarely in patients with cronkhite-canada syndrome. the malabsorption in this syndrome is usually progressive and with no specific therapy available, the prognosis is generally poor. death results from anemia, septic shock, bleeding, or postoperative complications. treatment consists of supportive therapy, antibiotics, corticosteroids, and surgery. within the stomach, cronkhite-canada syndrome closely mimics ménétrier's disease. ménétrier's disease, however, is confined to the stomach and has no associated ectodermal changes. heterotopic gastric, pancreatic, sebaceous, and salivary gland tissues have been described in the colon and rectum. these ectopic tissues can be found throughout the gi tract but are most often seen in the rectum where they can cause a plaque, polyp, or mass ( fig. - ). [ ] [ ] [ ] inflammatory fibroid polyp is most commonly found in the stomach but can be encountered throughout the gi tract including the colon and rectum. , - symptoms include abdominal pain and bleeding. the polyp is usually solitary. it can be sessile or pedunculated and typically has a solid pale tan cut surface. microscopically one sees a loose myxoid fibrous tissue background containing regularly distributed blood vessels, some of which show hyalin change in their walls. the fibrous tissue can layer in a whorl-like fashion around these vessels in an onion-skin pattern. most lesions are rich in inflammatory cells including plasma cells and eosinophils ( fig. - ) . scattered macrophages and touton-type giant cells can also be seen. the stroma in most lesions is positive for cd but negative for cd . mutations in plateletderived growth factor receptor alpha gene have been described. a the mucosa overlying these typically submucosal tumors can be ulcerated, presumably by trauma, and can show areas of inflamed granulation tissue. the ulcerated surface can contain bizarre stromal cells, which are also seen in a variety of inflammatory polyps with chronic ulceration (e.g., ibd, trauma or prolapse, and radiation injury). the inflammatory fibroid polyp is benign and typically does not recur. malakoplakia, an abnormal immune response to gramnegative bacteria, can cause a tumor or polyp in any site of the gi tract, including the large bowel. histologically, it is characterized by xanthogranulomatous inflammation accompanied by the pathognomonic michaelis-gutmann body (fig. - ). the partially digested bacteria accumulate in macrophages and lead to deposition of calcium and iron on the residual bacterial glycolipids. an association between colorectal malakoplakia and colorectal neoplasia may exist. defined as the presence of endometrial glands or stroma usually with hemorrhage and hemosiderin deposits in an extrauterine location, endometriosis tends to affect sites closest to the female genital tract such as the sigmoid colon and rectum. symptoms include episodic abdominal pain. hematochezia can occur with mucosal involvement. endometriosis usually involves the serosa and muscularis externa and can cause smooth muscle proliferation and stricture. mucosal and submucosal involvement can cause mucosal polyps. endometriosis must be distinguished from müllerian adenosarcoma and endometrial stromal sarcoma. the glandular component can be confused with colitis cystica profunda and adenocarcinoma. immunohistochemical analysis for cd , which highlights endometrial stromal cells, can be helpful in the differential diagnosis as can recognition of ciliated epithelial cells. differential cytokeratin immunostaining can also help because endometriosis commonly stains positive for ck , whereas colorectal epithelium usually expresses ck . examples of malignant transformation (mostly endometrioid carcinoma and clear cell carcinoma) in endometriosis have been reported. injection of materials containing lipid bases into the lower rectum and anus can cause a mass or polyp referred to as an oleogranuloma. the lesion is composed of lipidcontaining cysts surrounded by a foreign body giant cell reaction. benign fibroblastic polyps and perineurioma have been described in the colon and rectum where they may represent the same or a similar lesion. - these mucosal polyps are usually solitary but can be multiple and have been reported throughout the gi tract, most commonly in the colon and rectum. histologically, these polyps contain proliferations of small tightly packed spindle cells within the lamina propria that often orient themselves parallel to the muscularis mucosae. this lesion frequently coexists with hyperplastic polyp-like epithelial proliferations, and indeed the polyp could represent a trauma-related change seen in the otherwise typical hyperplastic polyp or sessile serrated polyp ( fig. - ) . the spindle cells test negative for immunoreactive s- protein and other neuromarkers; they can express immunoreactive epithelial membrane antigen by immunohistochemical analysis. areas of increased elastin fibers in the submucosa and muscularis mucosae are referred to as elastosis or elastofibromatous change and can cause polyps in the colon and rectum. histologically, the elastosis appears as finely granular or fibrillar amphophilic material usually with a fibrous component and is often centered around prominent blood vessels (fig. - ) . the change could also be a manifestation of mucosal trauma or prolapse. elastosis can be confused with amyloid deposits but results of congo red stains have been negative. benign spindle cell proliferations that express immunoreactive s- protein can manifest as mucosal polyps in the colon and rectum and are usually termed mucosal neuromas or schwann cell hamartomas. care must be taken not to overlook ganglion cells, which would indicate a ganglioneuroma. these lesions can be seen in patients with neurofibromatosis but most occur sporadically and are unassociated with syndromes. with increased availability of total colonoscopy and flexible sigmoidoscopy, pathologists can expect an ever-increasing number of colorectal biopsy specimens. the pathologist plays a critical role in the diagnosis and management of patients with colitis and suspected colitis. when evaluating a colorectal biopsy specimen, it is useful to scan the tissue at low magnification and ask several questions. are abnormalities present? if present, are the changes diffuse or focal? is the luminal border of the specimen straight or irregular? is intraepithelial mucin (the goblet cell population) preserved or depleted? if intracellular mucin is depleted, is this change focal or diffuse? are inflammatory cells increased in the lamina propria or epithelium? is this increase diffuse or focal? what kind of inflammatory cells are they? is the lamina propria obliterated by fibrous or fibromuscular tissue? are crypt abscesses present? are colonic tubules straight? do the colonic tubules reach the muscularis mucosae or are they short, branched, or budded? with careful consideration of the features outlined earlier, one can often recognize a pattern of abnormality that when coupled with clinical and endoscopic information can lead to a fairly specific diagnosis in a large number of patients. the following patterns of inflammation can be identified in mucosal biopsy specimens: chronic colitis, diffuse active colitis, focal active colitis, ischemic-type injury, traumarelated change, apoptotic colopathy, and intraepithelial lymphocytosis. identification of an inflammation pattern can be helpful in assessing patients by creating a clinically relevant differential diagnosis. quiescent uc best typifies the chronic colitis pattern of injury. the predominant features are mucosal atrophy and architectural distortion (fig. - ) . [ ] [ ] [ ] [ ] the luminal border is often irregular and the number of crypts decreased. the remaining crypts typically appear short (i.e., they do not touch the muscularis mucosae), they lose their parallel arrangement, and they become branched and budded. the goblet cell population is usually preserved. chronic inflammatory cells including plasma cells are increased in the lamina propria. paneth cells may be present. the muscularis mucosae is usually hypertrophied. the foregoing changes, although consistent with a diagnosis of chronic uc, must be interpreted in light of the clinical, radiologic, and endoscopic findings because similar changes can be seen in focal, healed, or healing areas of other chronic colitides such as crohn's disease, ischemia, chronic irradiation injury, tuberculosis, and schistosomiasis. care must be taken when interpreting biopsy specimens obtained from the normal mucosa adjacent to lymphoid follicles, from normal mucosa containing the innominate groove, and from the lower portion of the rectum near the transition zone. these areas normally show some loss of crypt parallelism and should not be misinterpreted as evidence of chronic colitis. , conversely, histologically normal biopsy specimens must not be reported as showing "chronic nonspecific inflammation" consistent with uc. unless one or more of the features discussed in the preceding para- graph are also present, it is a good rule not to diagnose primary ibd based only on an evaluation of inflammatory cells within the lamina propria. the term active colitis describes inflammatory conditions in which neutrophils are present within the lamina propria, within epithelial cells (cryptitis), or within the lumen of crypts (crypt abscesses). included under this heading are uc in an active phase, most examples of crohn's disease, and infectious colitis or acute self-limited colitis. , uc in an active phase represents the prototype diffuse active colitis. biopsy specimens usually demonstrate diffuse abnormalities, meaning that changes are of approximately the same intensity in all areas of the tissue submitted from a particular region of the colon. the luminal border of the mucosa is irregular. [ ] [ ] [ ] [ ] [ ] [ ] [ ] increased numbers of chronic inflammatory cells are present in the lamina propria and may occasionally spill over into the superficial portion of the submucosa. intracellular mucin in goblet cells is diffusely depleted. cryptitis and crypt abscess formation are often prominent (fig. - ). it is surprising that even in uc of extremely short overt clinical duration, atrophy, branching, and budding of crypts are already apparent in many specimens. , [ ] [ ] [ ] [ ] [ ] [ ] this crypt distortion coupled with basal plasmacytosis, which is defined as increased plasma cells in the lower fifth of the mucosa, has been proposed as the most useful histologic criterion to differentiate primary ibd from infectious colitis or acute self-limited colitis. , [ ] [ ] [ ] remember, the most a pathologist can conclude from a biopsy specimen showing this pattern of injury is that the changes are consistent with uc in an active phase. the diffuse active colitis pattern of injury can also be seen in some examples of crohn's disease , and in some cases of documented infectious colitis, although we think it is likely that these cases represent an infectious exacerbation of an underlying, albeit clinically latent, primary ibd. the diffuse active colitis pattern of injury has been reported in a form of colitis associated with diverticular disease. however, this entity can be distinguished from classic uc by virtue of its rectal sparing and the presence of inflammation only in areas of diverticula. the focal active colitis pattern of injury refers to the patchy distribution of architectural or acute inflammatory change in mucosal biopsy specimens. chronic colitis showing diffuse chronic changes as described earlier, coupled with patchy acute inflammation, is not considered focal active colitis but is classified as chronic colitis showing mild activity. the focal active colitis pattern consists of limited areas of increased inflammatory cells sometimes coupled with focal minimal architectural distortion (fig. - ) . characteristically, some areas of the biopsy specimen from a region of the colon must maintain an essentially normal appearance. the focal active colitis pattern of injury is usually not seen with uc and when present suggests crohn's disease, , , injury related to nonsteroidal anti-inflammatory drugs (nsaids), or infectious colitis or acute self-limited colitis (see later). , , , , , the focal active colitis pattern of injury, however, can be seen in resolving uc under active medical management. , areas previously involved in the colon and rectum in uc can return to an almost normal histologic appearance with therapy. granulomas typically found in crohn's disease should be sought in all biopsy specimens, but especially those showing the focal active pattern. although serial sectioning of biopsy specimens is advocated by some investigators for the detection of granulomas, in our experience, granulomas are rarely missed. that said, germinal centers, tangential cuts of blood vessels, tangential cuts of the pericryptal fibroblastic sheath, and an inflammatory reaction to extravasated mucin (so-called mucin granulomas) are often misinterpreted as the granulomas of crohn's disease. in the absence of true granulomas, biopsy specimens from patients with crohn's disease often show the focal active colitis pattern of injury without neutrophils in the lamina propria. some examples of crohn's colitis can be indistinguishable from resolving mucosal uc in biopsy specimens. , a fibrinopurulent exudate in the specimen overlying but separate from the mucosa is always abnormal. the clinician should be informed that ulcers are likely to be present in a more proximal location in the bowel. on a practical note, all inflammatory exudates should be examined under high magnification for trophozoites of entamoeba histolytica, because this is their preferred location and these organisms are easy to overlook. the definitive classification of colonic inflammation depends on clinical pathologic correlation. in our opinion, the task of the pathologist is to convey the histologic pattern of injury to the clinician who then collates that information with the clinical history and data obtained from endoscopic, radiologic, and laboratory examination. antineutrophil cytoplasmic antibodies (ancas) have been identified in patients with ibd. [ ] [ ] [ ] the immunofluorescence pattern (perinuclear) differs from the diffuse cytoplasmic reaction detected in patients with wegener's granulomatosis. although initially thought to be specific for uc, ancas more likely reflect inflammatory conditions involving the colon. in one study, up to % of patients with presumed uc had anca positivity with a % prevalence in patients with colonic crohn's disease and a % prevalence in patients with infectious-type colitis. another antibody, anti-saccharomyces cerevisiae antibody (asca), has been closely linked to crohn's disease. ascas can be identified in up to % to % of patients with crohn's disease, as opposed to % to % in uc and % of controls. , some investigators have proposed the use of anca and asca together to increase specificity. these tests clinically have demonstrated low sensitivity (≈ % to %) but have been reported to be highly disease specific. these tests may have value in classification of indeterminate colitis but this remains to be proved. the following histologic appearances have been described in culture-proved or toxin-proved infectious colitis: normal colon, nonspecific increases in chronic inflammatory cells, the diffuse active colitis pattern of injury (although these likely represent infectious exacerbations of an underlying primary ibd), ischemic-like changes, , and the focal active colitis pattern of injury. although colonic mucosal biopsy appearance in infectious colitis can vary greatly, many specimens have the focal active colitis pattern. in general, invasive organisms cause greater changes in morphology than those producing their effects by toxins. histologic evaluation, although helpful in suggesting infection and ruling out primary ibd, can only rarely suggest a specific cause. the definitive diagnosis of infectious colitis requires laboratory identification of the serologic features of the offending organism. even after extensive microbiologic workup, a subset of patients presumed clinically to have infectious colitis will experience spontaneous recovery in less than months and will have biopsy specimens that demonstrate the focal active colitis pattern of injury without an identifiable infectious cause. the term acute self-limited colitis has been used to describe such patients. [ ] [ ] [ ] we prefer the term infectious-type colitis to acute self-limited colitis because some examples of acute self-limited colitis may not be self-limited. other investigators prefer the term nonrelapsing colitis. , the ischemic pattern the characteristic pattern of acute ischemic-type injury consists of hemorrhage into the lamina propria associated with superficial epithelial coagulative necrosis, with relative sparing of the deep portions of the crypts (fig. - ) . , , these changes can occasionally be associated with more extensive necrosis of epithelium with inflammatory pseudomembrane formation. surprisingly, acute and chronic inflammatory cells, especially plasma cells, are typically scant in ischemic-type damage and this feature can help differentiate ischemic-type damage from primary ibd. the differential diagnosis of ischemic-type damage is wide and includes all causes of true ischemia such as inadequate perfusion, narrowing of blood vessels, obstructing lesions of the bowel, and bowel distention. ischemictype change is also associated with a variety of drugs, including vasopressors, oral contraceptives, nsaids, cocaine, and glutaraldehyde, which is sometimes used to clean endoscopes. [ ] [ ] [ ] [ ] some infectious agents typically cause ischemic-type damage, including cytomegalovirus (cmv), clostridium difficile, clostridium septicum, and the enterohemorrhagic escherichia coli (ehec). trauma-type histologic changes frequently coexist clinically with mucosal ulcers or erosions. the characteristic traumatype histologic pattern is found in the mucosa adjacent to ulcers or in polypoid mucosa areas and consists of fibro- muscular proliferation within the lamina propria associated with mucosal architectural distortion and intramucosal capillary ectasia (fig. - ). [ ] [ ] [ ] [ ] the trauma-type histologic features can be seen in the solitary rectal ulcer syndrome, localized colitis cystica profunda, inflammatory cloacogenic polyp, the mucosa adjacent to orifices of colonic diverticula, and inflammatory cap polyposis and are frequent findings adjacent to neoplasia and in the vicinity of the ileocecal valve. surface colonic epithelial apoptosis and karyorrhectic debris within the superficial lamina propria are commonly seen in mucosal biopsy specimens and are widely attributed to bowel preparation. apoptotic bodies in the deep crypt are only rarely seen (< per crypts) outside of pathologic conditions. increased deep apoptotic bodies can be seen in ischemic-type damage, cmv infection, chemotherapy or radiation, and in association with mycophenolate mofetil (cellcept) (fig. - ). although associated with a variety of injurious agents, apoptosis is the characteristic form of cell death in cell-mediated immune cytotoxicity as demonstrated in graft-versus-host disease (gvhd), other immune deficiency syndromes , and thymic neoplasia. many patients with aids with diarrhea demonstrate deep apoptosis in the absence of pathogens, a finding suggesting that primary immune-mediated apoptotic colopathy may cause some of the diarrhea and wasting seen in aids. , intraepithelial lymphocytosis the term collagenous colitis, first coined by lindström in , describes a distinct clinicopathologic syndrome causing watery diarrhea, predominantly in middle-aged or older (mean age, years) women (male-to-female ratio, : . ). , results of colonoscopic and barium enema examination in these patients are usually normal. therefore, diagnosis depends on recognition of characteristic changes in biopsy specimens. the primary histologic change of collagenous colitis consists of a patchy increase in the thickness of the subepithelial collagen plate (fig. - ) . , the normal colonic epithelial collagen layer measures approximately µm in thickness, but in collagenous colitis it may increase to µm or more. we use µm as a morphologic cutoff point for collagenous colitis. a mild to moderate increase in chronic inflammatory cells including plasma cells and eosinophils expands the lamina propria. patchy injury to the surface epithelium characterized by increased numbers of intraepithelial lymphocytes, epithelial degeneration, and sloughing also occur. atrophy, mucosal architectural distortion, and acute inflammation are usually not present or are minimal. , , read and colleagues introduced the term microscopic colitis to describe patients with chronic watery diarrhea of unknown origin occurring in middle-aged (mean, years) patients. as in collagenous colitis, the colonoscopic appearance and the barium enema were usually described as normal. biopsy specimens demonstrated increased inflammatory cells in a pattern not specific for any established entity. since this initial report, investigators have refined the clinical and especially the histologic diagnostic criteria of lymphocytic colitis. , the histologic changes include increased chronic inflammatory cells in the lamina propria and surface epithelium, degenerative changes of the surface epithelium, minimal architectural change, and minimal acute inflammation (fig. - ) . the changes of lymphocytic colitis in general resemble the surface epithelial and lamina propria changes of collagenous colitis, and indeed changes identical to lymphocytic colitis are seen in biopsy specimens from patients with collagenous colitis when areas where the collagen plate is not well developed are sampled. the similarities between lymphocytic colitis and collagenous colitis are striking. they share similar symptoms and endoscopic findings. the histologic similarities include increased intraepithelial lymphocytes, surface epithelial damage, and increased chronic inflammatory cells within the lamina propria. , , both collagenous colitis and lymphocytic colitis can be associated with other autoimmune phenomena such as thyroid disease, enteropathic arthritis, rheumatoid arthritis, myasthenia gravis, and celiac sprue. , some cases of lymphocytic colitis demonstrate thickening of the subepithelial collagen plate, and distinguishing lymphocytic colitis from collagenous colitis may not be so clear-cut in all cases. it appears likely that lymphocytic colitis and collagenous colitis are either the same or very similar conditions, perhaps representing different morphologic phases of one disease process. , some minor differences between lymphocytic colitis and collagenous colitis deserve mention, however. collagenous colitis has been reported predominantly in women, whereas lymphocytic colitis affects men and women equally. , differences in human leukocyte antigen (hla) haplotypes between collagenous colitis and lymphocytic colitis have been described. finally, collagenous colitis and lymphocytic colitis differ histologically with the presence of the thickened collagen plate. lymphocytic colitis and collagenous colitis are associated with net fluid secretion in the colon that is responsible for the diarrhea. several investigators could find no association between the thickness and extent of the collagen plate and the amount of diarrhea. therefore, most investigators conclude the damage to the surface epithelial cells and the inflammation, rather than the collagen deposits, cause diarrhea. the presence and thickness of the subepithelial collagen plate appear unrelated to the patient's age or the duration of disease. some patients have also demonstrated evidence of small bowel dysfunction such as salt wasting, fatty acid malabsorption, small bowel net secretion, and rarely a small bowel villous lesion that resembles celiac sprue (see later). spontaneous resolution of collagenous and lymphocytic colitis has occurred, thus rendering evaluation of therapeutic regimens difficult. a few patients with these colitides have presented with relatively mild diarrhea and have achieved medical control with dietary restriction (elimination of caffeine and lactose-containing foods), bulking agents, and antimotility drugs (loperamide hydrochloride, diphenoxylate hydrochloride, atropine). a trial of bismuth compounds is worthwhile. many times, symptomatic therapy has failed, thus necessitating the addition of antiinflammatory agents. approximately % to % of patients eventually respond to therapy with sulfasalazine, prednisone, or budesonide. , , etiology and pathogenesis nsaids and ticlopidine have been linked to some occurrences of collagenous colitis, , and lymphocytic colitis has been reported in patients receiving ruscus extract (cyclo fort), ranitidine, ticlopidine, and flutamide. however, most cases cannot be linked to drug ingestion and the bulk of the evidence suggests that lymphocytic and collagenous colitis share common immune-mediated etiology and pathogenesis. both conditions have a striking histologic similarity to celiac sprue, a condition known to be autoimmune and possibly having a viral or infectious trigger. in addition, both lymphocytic and collagenous colitis have been linked to other conditions thought to have autoimmune pathogenesis such as crohn's disease, hypothyroidism, hyperthyroidism, inflammatory arthropathies, pernicious anemia, small bowel villous atrophy, iritis, diabetes mellitus, and myasthenia gravis. , , both conditions respond dramatically to anti-inflammatory agents. , the finding of "lymphocytic colitis-like" histologic features reported in an epidemic outbreak of brainerd diarrhea linked to a water tank aboard a cruise ship supports an infectious trigger for some cases of lymphocytic colitis (see later). the association between lymphocytic colitis or collagenous colitis and celiac sprue and spruelike lesions deserves special attention. in the experience of dubois and associates and wolber and colleagues, approximately % of patients with "celiac sprue" who had colonic biopsies also showed changes of lymphocytic colitis. colonic microscopic abnormalities in patients with celiac sprue occur after experimental exposure to wheat or gliadin enemas, a finding suggesting that the entire intestinal tract may be susceptible to gluten-induced injury. it is possible that in some patients with true celiac sprue (responsive to gluten withdrawal), occult dietary gluten reaches the colon and induces the histologic changes of lymphocytic colitis. however, approximately one half of the patients with spruelike small bowel lesions and lymphocytic colitis have not responded to gluten withdrawal. the term lymphocytic enterocolitis has been coined to describe this refractory spruelike condition associated with colonic intraepithelial lymphocytosis. as more cases are investigated, it is not unusual to see variations in histologic pattern commingled with the classic changes of lymphocytic and collagenous colitis. these variations include architectural change, cryptitis, paneth cell metaplasia, ulcers, and inflammatory membranes. [ ] [ ] [ ] [ ] approximately one third of patients demonstrate cryptitis. rarely, one encounters inflammatory pseudomembranes. neither histologic pattern correlates strongly with infection. approximately % of patients have ulcers, and architectural abnormalities have been reported in % of patients. paneth cell metaplasia is seen in % to % of patients, most often in collagenous colitis in which it may correlate with increased severity of diarrhea. acute inflammation is seen with increased frequency in patients taking antibiotics. ulcers have been linked with concomitant nsaid usage. some investigators have suggested that the "stiff" colon seen in collagenous colitis could be at increased risk for the development of ulcers and perforation during colonoscopy. other variants with subepithelial giant cells have been described (fig. - ). histologic features, aberrant or otherwise, do not seem to correlate with symptoms, results of medical treatment, or outcome. no patient with unusual histologic features, including architectural change and paneth cell metaplasia, has yet to develop primary ibd (e.g., crohn's disease or uc). the term brainerd diarrhea has been applied to outbreaks of diarrhea of unknown origin characterized by acute onset and prolonged duration. the disease was named after brainerd, minnesota, where in residents developed watery diarrhea after drinking unpasteurized milk. a second outbreak occurred in henderson county, illinois in , when people developed watery diarrhea associated with drinking contaminated well water at a roadside restaurant. in each outbreak, patients underwent extensive diagnostic evaluations including comprehensive microbiologic studies of their stool and the implicated exposure site. despite the workup, no causative agent was identified. the clinical and epidemiologic characteristics were typical of point-source epidemic infectious diarrhea. however, unlike in typical infectious diarrhea, these patients developed a chronic watery diarrhea syndrome with symptoms lasting longer than months and often lasting for years. in general, the small bowel biopsy specimens from patients with brainerd diarrhea were histologically normal. colonic biopsy specimens revealed surface epithelial lymphocytosis without distortion of mucosal architecture, surface degenerative changes, or thickened subepithelial collagen plate. the degree of surface epithelial lymphocytosis was similar to that seen in cases of collagenous and lymphocytic colitis. , , many experts believe that patients with the clinical syndrome of chronic watery diarrhea of unknown origin and patients reported as having lymphocytic colitis represent a heterogeneous group that may contain persons with unrecognized brainerd diarrhea. with long-term follow-up, cases of brainerd diarrhea appear to be self-limited, and patients recover in less than years. from a practical standpoint, surface epithelial lymphocyte counts should be performed on all colonic biopsy specimens from patients with chronic diarrhea, especially chronic watery diarrhea. currently, brainerd diarrhea cannot be recognized outside the setting of an epidemic. the solitary rectal ulcer syndrome, localized colitis cystica profunda, inflammatory cloacogenic polyp, prolapsing mucosal folds in areas of diverticular disease, and inflammatory cap polyposis are closely allied conditions that have been linked to large bowel mucosal prolapse and trauma. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] affected patients with solitary rectal ulcer, localized colitis cystica profunda, and inflammatory cloacogenic polyp often demonstrate abnormal function of the anal and pelvic floor musculature during defecation , , that leads to rectal mucosal prolapse or even intussusception. the resulting trauma is thought to cause the clinical symptoms and the pathologic changes. the term solitary rectal ulcer syndrome is quite a misnomer because the ulcers are often multiple, a preulcer polypoid phase is reported, and similar lesions occur in the anal canal and proximal colon. , , , the terms colitis cystica profunda and inflammatory cloacogenic polyp are also misnomers. because all three conditions share a common histologic appearance, clinical presentation, clinical course, and pathogenesis, we prefer to consider them together under the heading mucosal prolapse syndromes. patients with mucosal prolapse syndrome range in age from to years with the majority presenting in the third and fourth decades of life. the condition occurs more commonly in women. all patients report difficulties in defecation. other common symptoms include rectal bleeding, passage of mucus, anal and abdominal pain, and intermittent bowel habits in which periods of constipation interrupt bouts of loose stools. furthermore, surprising numbers of patients admit to the use of digital manipulation, spoons, or other instruments to assist in rectal evacuation. the diagnosis of mucosal prolapse syndromes can be difficult because the history often suggests primary inflammatory or ischemic bowel disease. in addition, mucosal prolapse is often covert requiring special techniques such as evacuation cine proctography and defecography to demonstrate abnormalities in these patients. , , , ulcers, when present, typically occur on the anterior or anterolateral wall of the rectum, are irregular in shape, and often appear well demarcated. in patients without ulceration, the mucosa appears polypoid, roughened, or erythematous. inflammatory cloacogenic polyp , involves the lower rectum and anal transition zone. the clinical impression in patients with mucosal prolapse syndromes is often incorrect and includes ulcer, crohn's disease, nonspecific proctitis, carcinoma, and villous adenoma. the characteristic histopathologic changes are found in the mucosa adjacent to the ulcers or in the polypoid areas and consist of fibromuscular obliteration of the lamina propria associated with mucosal architectural distortion often with a hyperplastic or villiform appearance (see fig. - ). [ ] [ ] [ ] [ ] inflammation is typically mild or absent and mucosal capillaries can be ectatic. superficial erosions occasionally occur and can be associated with acute inflammation and the formation of inflammatory pseudomembranes. on occasion, colonic glands may be misplaced into the muscularis mucosae or submucosa, a histologic pattern referred to as localized colitis cystica profunda. submucosal vessels may also be ectatic and hyalinized. the histologic features of specimens obtained from ulcerated areas appear nonspecific and usually show fibrinopurulent debris, fibrosis, and granulation tissue. differential diagnostic considerations include mucinous adenocarcinoma, chronic uc, cowden's disease, and ulcers resulting from ergotamine suppositories. the misplaced glands of mucosal prolapse syndrome (colitis cystica profunda) simulate pseudoinvasion seen in adenomas; these glands can be associated with dissecting mucous pools and easily can be mistaken for invasive mucinous adenocarcinoma. table - illustrates histologic features that aid in this differential diagnosis. the mucosal abnormalities of mucosal prolapse syndrome also closely mimic chronic uc. knowledge of the clinical picture and recognition of the characteristic fibromuscular obliteration of the lamina propria (not usually present in ibd) are helpful in this distinction. the histologic appearance of colorectal polyps from patients with cowden's disease appears identical to the histologic features seen in mucosal prolapse syndrome. mucosal prolapse syndrome and cowden's disease must be separated on clin-ical grounds. clinical history is also required to separate the rectal ulcers associated with the use of ergotamine suppositories from mucosal prolapse. these lesions can be grossly and microscopically identical to those seen in mucosal prolapse syndrome. the few studies with long-term follow-up report a chronic, stable appearance to the lesions. , , most patients with mucosal prolapse syndrome tolerate their symptoms following reassurance that they do not have cancer, the addition of fiber to the diet, and instruction to reduce straining and to avoid digital manipulation. the rare patient with severe bleeding or obstruction requires excisional therapy. medical remedies such as sulfasalazine, local or systemic corticosteroids, and antibiotics are useless. , , unusual patients with severe, incapacitating symptoms have been treated by resection, diverting colostomy, or rectal prolapse repair. results of these operations have varied and because approximately different operations have been described for mucosal prolapse syndrome, comparisons are difficult. inflammatory conditions of the colon can be caused by specific agents or conditions, such as bacteria or ischemia, or can be nonspecific. after specific causes of colitis have been ruled out, one is left with a group of diseases referred to as nonspecific or primary ibd. these include uc, crohn's disease, and ibd type indeterminate (unclassified colitis). in most cases, careful examination of gross and microscopic features will allow categorization of these nonspecific ibds into either uc or crohn's disease. approximately % of cases will cause significant differential diagnostic problems by illustrating ambiguous features. these cases are best classified as ibd type indeterminate (see later). evolving surgical techniques have changed the pathologist's role in the analysis of ibd. patients with uc have several surgical options that either create continence through ileostomy (kock ileostomy) or preserve anal sphincter function and restore continuity to the bowel (ileal pouch-anal anastomosis). [ ] [ ] [ ] these operations have in common the creation of a pouch or reservoir formed by interconnecting loops of terminal ilium. in general, these pouch procedures are contraindicated in patients with crohn's disease because of increased morbidity such as fistula and abscess. furthermore, complications requiring pouch removal can result in loss of considerable lengths of small bowel, sometimes enough to cause short-bowel syndrome. in our experience, there is nothing quite like a pouch to bring out the crohn's disease in someone. these cases highlight the limitations of the current pathologic classification especially in the setting of fulminant clinical disease. accurate pathologic diagnosis of colonic ibd contributes greatly to patient care. in patients who have had subtotal colectomy, relaxed and thorough pathologic examination of the colectomy specimen to rule out crohn's disease is pos-sible. a staged resection of the large bowel is still the safest and most advisable approach for patients with severe or fulminant colitis, even though it requires an additional operation to remove the rectum and a period with a temporary ileostomy. although pathologic assessment and interpretation are more difficult in fulminant disease, the two-stage procedure still allows for a complete examination of the colectomy specimen before any contemplated pouch construction with ileal-anal anastomosis. some surgeons do not consider two-stage proctocolectomy necessary for selected patients with colonic ibd. the one-stage operative approach therefore requires accurate preoperative diagnosis and emphasizes the importance of clinical evaluation, radiologic examination, endoscopic analysis, and proper interpretation of mucosal biopsy specimens (see earlier). pouch complications include inflammation, fistula, obstruction, incontinence, and anastomotic leaks. , although many complications result from surgical and mechanical difficulties, and others relate to the development of "primary" inflammation in the pouch (see "pouchitis," later), some of these complications represent pouch recurrence of initially unrecognized crohn's disease. these cases illustrate the inability to differentiate uc from crohn's disease reliably in severe colitis, even after thorough examination of the colectomy specimen. all reports of surgical experience with ileal pouch-anal anastomosis for presumed uc contain approximately % to % of patients in whom the final diagnosis proves to be crohn's disease. , [ ] [ ] [ ] [ ] we recommend use of a three-tiered classification system for primary ibd in colon resection specimens: uc, crohn's disease, and ibd of indeterminate type. the definitive diagnosis of uc requires all the following features: diffuse disease limited to the large intestine, involvement of the rectum, more proximal colonic disease occurring in continuity with an involved rectum (i.e., no gross or histologic skip lesions), the absence of deep fissural ulcers, the absence of mural sinuses, and the absence of transmural lymphoid aggregates or granulomas (fig. - ) . , , the definitive diagnosis of crohn's disease requires histologic verification with the demonstration of transmural lymphoid aggregates in an area not deeply ulcerated or the presence of non-necrotizing granulomas (figs. - and - ). , [ ] [ ] [ ] [ ] when the gross and clinical features suggest crohn's disease by the presence of skip lesions, linear ulcers, cobblestoning, fat wrapping, or terminal ileal inflammation, extensive histologic sampling to find the definitive histologic features of crohn's disease is suggested (fig. - ) . the term primary ibd of an indeterminate type is used for cases of idiopathic colonic ibd that have ambiguous pathologic features in the resection specimen and are inconclusive for a diagnosis of uc or crohn's disease. , , [ ] [ ] [ ] [ ] [ ] [ ] the classification system has prognostic significance. the pouch failure rate in indeterminate colitis (≈ %) is intermediate between that seen with crohn's disease (≈ %) and that reported in uc (≈ %). even so, patients without clinical, endoscopic, or radiologic evidence suggestive of crohn's disease in whom the final pathologic results are indeterminate generally are considered suitable for ileal pouch-anal anastomosis. , , [ ] [ ] [ ] [ ] [ ] [ ] colon and rectum surgically placed out of circuit acquire histologic changes associated with defunctioning alone, regardless of the original reason for diversion. [ ] [ ] [ ] [ ] the changes reflect a physiologic response to stasis and the loss of trophic factors in the feces, most notably short-chain fatty acids. most patients are asymptomatic. the mucosa of the diverted segment appears erythematous, granular, and friable ( fig. - ). histologic changes include marked lymphoid hyperplasia with germinal center formation, usually accompanied by mild colitis with crypt abscess formation (fig. - ) . these changes may be indistinguishable from follicular proctitis (ulcerative proctitis or localized uc). with time, the muscularis mucosae hypertrophies, the submucosa shows fatty and fibrous tissue infiltration, the muscularis propria thickens, and the lumen becomes progressively smaller. the mucosal lymphoid hyperplasia may be accompanied by lymphoid aggregates scattered in the deep submucosa and muscularis externa and may occur in diverted segments in patients without ibd. care must be taken not to base a diagnosis of primary ibd, especially crohn's disease, solely on the histologic changes seen in diverted specimens. in many patients, the rectum is placed out of circuit during an operation for primary ibd. in these instances, the rectum can show changes of both ibd and diversion. the histologic changes in a defunctioned rectum do not correlate with the original diagnosis or clinical outcome. a late complication of kock pouch and ileal pouch-anal anastomosis is the development of "primary" inflammation in the pouch with its associated clinical syndrome termed pouchitis. , the reported incidence ranges from % to %. this wide range may be the result of the lack of an accepted case definition of pouchitis. patients generally experience nausea, vomiting, malaise, fever, and abdominal cramping. the increased effluent/stool from the pouch may be watery, foul smelling, or grossly bloody. patients frequently become incontinent. pouch bacterial ecology is often altered and many patients respond to antibiotics such as metronidazole and ciprofloxacin, a finding that suggests a bacterial cause in some cases. some patients have required -aminosalicylates, sulfasalazine, corticosteroids, immunomodulators, or even pouch excision to manage symptoms. pouch biopsy may be performed to confirm the presence of inflammation or to evaluate the possibility of crohn's disease. pouch biopsy specimens from nondysfunctional pouches can show mild villus shortening and increased chronic inflammation with augmented crypt proliferation. most specimens from pouches that are functioning well resemble normal terminal ileum. a few neutrophils within surface epithelium and within the lamina propria are commonly seen in pouches that are functioning well. [ ] [ ] [ ] [ ] [ ] in contrast, pouches with classic pouchitis show decreased epithelial mucin and decreased or absent lymphoid follicles. the most consistent finding in this classic form of pouchitis has been ulcers with granulation tissue and patchy accumulations of neutrophils within crypt epithelium and within the lamina propria with deep crypt abscess formation. , , , , to define pouchitis more precisely, sandborn proposed the pouchitis disease activity index. , in this system, up to points are awarded for various clinical and endoscopic findings. the amount of neutrophilic infiltration and the degree of ulceration are graded on a scale of to points each. histologic findings are weighted in this system because pouchitis is diagnosed in patients who have or more points. many investigators report an inconsistent relationship between endoscopic and histologic changes in the pouch and patients' symptoms. therefore, many clinicians diagnose pouchitis on clinical criteria and reserve endoscopic examination with biopsy for those patients with refractory pouchitis or possible crohn's disease. , , , unfortunately, no reliable endoscopic or histologic criteria are available to differentiate most examples of pouchitis from a new onset or recurrence of crohn's disease in the pouch. confusion over pouchitis is easily understood when one considers that the clinical syndrome of pouchitis probably represents at least six different conditions (table - ) . , , the two variants of antibiotic-responsive pouchitis syndromes are classic pouchitis and jejunal bacterial overgrowth. , , classic pouchitis is associated with endoscopic and histologic findings that support the pouch as the source of clinical symptoms. [ ] [ ] [ ] [ ] , patients with classic pouchitis usually respond to antibiotics such as metronidazole and ciprofloxacin. occasional patients with clinical symptoms of pouchitis have had endoscopically and histologically negative pouches but have responded to antibiotics. , although rare, some of these patients have had proximal jejunal bacterial overgrowth, probably as a result of pouch distention causing an ileal brake phenomenon that decreases motility in the proximal intestine. , this decreased motility may predispose some individuals to proximal small bowel bacterial overgrowth. pouchitis syndromes refractory to antibiotic therapy include irritable pouch syndrome, short-strip pouchitis ("cuffitis"), crohn's disease, and chronic primary refractory pouchitis. , patients with irritable pouch syndrome may have severe clinical symptoms but demonstrate normal pouch endoscopy and histology. some of these patients respond to dietary fiber supplements and antidepressant therapy. to obtain a better-functioning pouch, many surgeons have abandoned rectal mucosectomy in the ileal pouch-anal anastomosis procedure. , in short-strip pouch itis, clinical symptoms may be caused by exacerbation of uc in the small retained rectal segment. , many patients with this form of pouchitis have responded to topical corticosteroids or mesalamine. although debated, missed crohn's disease is likely to manifest as a late pouch fistula or abscess rather than as refractory pouchitis. in occasional reported cases of refractory pouchitis, pouch biopsy specimens contained granulomas or the excised pouch showed histologic criteria for crohn's disease. , , invariably, the original colectomy showed either missed crohn's disease or colitis of an indeterminate type. these cases are best classified as crohn's disease. , , patients with chronic pouchitis should be investigated with endoscopy and biopsy to rule out crohn's disease or cmv infection, which has been linked to some cases of refractory pouchitis. , routine biopsy specimens of the pouch should be examined as well as biopsy specimens of the afferent limb. afferent limb ulcers tend to correlate with crohn's disease and, in patients who do not have crohn's disease, with the use of nsaids. we have seen cases of refractory pouchitis requiring surgical removal of the pouch. after careful pathologic evaluation, no specific infection and no criteria for crohn's disease can be found in either the excised pouch or the original colectomy specimen. , , these cases are best categorized as primary refractory pouchitis. the causes of classic pouchitis and primary refractory pouchitis are unknown but are probably related to a combination of stasis, bacterial overgrowth, the abnormal immune response of patients with ibd, or perhaps associated colonic-type metaplasia, which is reported to occur in a minority of pouches (see later). , , some investigators have identified histologic patterns of mucosal adaptation in pouches. [ ] [ ] [ ] approximately % to % of patients exhibit what has been called type a mucosa with normal small bowel biopsy histology or only mild mucosal atrophy with no or minimal inflammation. type b mucosa, characterized by transient atrophy with temporary moderate to severe inflammation followed by normalization of intestinal mucosa, is seen in % to % of patients. type c mucosa with permanent persistent atrophy and severe inflammation occurs in up to % of pouches. colonic-type features have been reported at least focally in pouches of all types by routine morphology, mucin histochemistry, immunohistochemistry, lectin binding, or electron microscopy. this colonic-type change is best developed in type c mucosa but is not complete. all pouches seem to retain mostly small bowel properties regardless of mucosal type or the duration of the pouch. with long-term follow-up, it appears that epithelial dysplasia can rarely develop in the pouch. this extremely rare complication seems to be limited to the subgroup of patients (< %) in whom refractory pouchitis and colonic-type metaplasia develop (type c mucosa). [ ] [ ] [ ] most investigators suggest yearly pouch surveillance once type c mucosa is established. however, until further information on cancer risk is available, it would seem prudent to survey types a and b mucosa as well, perhaps every other year. , dysplasia can also develop in the small retained rectal segment (often incorrectly referred to as the anal transition zone) in patients who have had restorative proctocolectomy and stapled ileal pouch-anal anastomosis. annual endoscopic surveillance with biopsy for these retained rectal segments seems prudent. the incidence of dysplasia is rare (< % of patients), correlates with the original colectomy specimen containing dysplasia or cancer, and can be safely treated by completion mucosectomy. [ ] [ ] [ ] colostomy changes specimens from colostomy revisions usually show changes associated with trauma and mucosal prolapse such as erosions, ulcers, hemorrhage, acute and chronic inflammation, and fibromuscular obliteration of the lamina propria. one often encounters peri-intestinal fibrosis and suture granulomas. inflammatory polyps can be seen as well. infiltration of the large intestine by large numbers of eosinophils can be seen in eosinophilic gastroenteritis. these patients usually show peripheral blood eosinophilia and have a clinical history of atopy. [ ] [ ] [ ] [ ] the entity that has been termed allergic proctitis is in our opinion a form of uc. whenever large numbers of eosinophils are encountered in a colorectal biopsy specimen, this finding should prompt thorough search for parasites, especially strongyloides species. the most common form of allergic proctitis or colitis occurs in infants as a result of dietary protein-related allergy. these children present typically with rectal bleeding, sometimes with diarrhea. , colorectal biopsy specimens show increased numbers of eosinophils within the lamina propria, occasionally with focal active colitis. biopsy specimens that contain more than eosinophils per high-power fields are suggestive of this disease. allogeneic bone marrow transplantation can be complicated by acute or chronic gvhd. , [ ] [ ] [ ] [ ] the changes of acute gvhd (apoptotic colopathy) in mucosal biopsy have been described and various grading systems for acute gvhd are used. , , grade acute gvhd correlates with increased apoptosis alone. grade changes include crypt abscesses as well as apoptotic bodies (see fig. - ) . total necrosis of crypts is classified as grade , whereas denudation of areas of bowel is considered grade . biopsy specimens should be carefully examined for cmv infection. cmv may also cause apoptotic change that can mimic gvhd and occasionally both conditions can coexist. apoptosis is seen with damage associated with chemotherapy and irradiation. patients receiving bone marrow transplantation for malignant conditions are prepared with chemotherapy and irradiation; therefore, distinguishing the effects of therapy from gvhd without a clinical history is impossible. histologic changes associated with chemotherapy and irradiation typically resolve in to days. , apoptotic colopathy identical to grade gvhd can be seen in immunodeficiency syndromes, in patients receiving mycophenolate mofetil, and in primary aids-related colitis. grade gvhd cannot be distinguished from ischemic damage without a clinical history. chronic gvhd usually spares the colon but a few examples have been described. the pathologic changes include submucosal fibrosis, mucosal calcification, and fibrosis of the lamina propria. , , chronic colitis-like changes have been seen in a subset of patients with gvhd; the relationship between this condition and chronic gvhd requires more study. a localized form of uc with a better prognosis has been described as mucosal proctosigmoiditis, follicular proctitis, or ulcerative proctitis. [ ] [ ] [ ] the gross and endoscopic mucosal appearance resembles that seen with more extensive forms of uc. mucosal biopsy specimens typically show changes of uc but some have shown prominent mucosal lymphoid aggregates (follicular proctitis) (fig. - ) . most patients respond to the local administration of corticosteroids or -aminosalicylates; approximately % of patients develop more extensive colitis. , some evidence suggests that patients with prominent lymphoid follicles are less likely to respond to treatment. inflammatory changes are relatively common in patients with diverticular disease and are often related to trauma or prolapse. ibd and diverticular disease are both common and occasionally coexist. uc-like inflammation in association with diverticular disease has been described. crohn's disease and diverticular disease share similar features such as focal mucosal inflammation, stricture, and fistula. frequently, the pathologist will ascribe all the changes to one disease (usually diverticular disease) and overlook the concurrent ibd (usually crohn's disease). features that suggest crohn's disease in this setting are the presence of fissural ulcers, patchy active mucosal inflammation outside diverticula, granulomatous inflammation, and internal fistulas other than colovesical or colovaginal fistula. approximately % to % of patients develop other bowel lesions of ibd on follow-up. [ ] [ ] [ ] [ ] [ ] [ ] systemic mastocytosis is a rare disorder characterized by the infiltration of mast cells and eosinophils in the skin, bones, lymph nodes, and other organs such as the gi tract. [ ] [ ] [ ] one group of investigators suggested that colorectal biopsy specimens showing more than mast cells per high-magnification field as highlighted by cd or mast cell tryptase immunostain should be diagnosed as mastocytic enterocolitis, a disease best treated with antihistamines or mast cell stabilizers. this controversial study did not control for thickness of immunohistochemically stained sections and lacked a treatment control group, thus making such conclusions premature. mast cells coexpressing cd usually indicate neoplastic mast cells. patients with long-standing uc are at increased risk for colorectal carcinoma. the prevalence of colorectal cancer in patients with uc is estimated to be . % overall and . % for those with pancolitis. those at greatest risk are patients with extensive colitis , who have been afflicted for more than to years. the cumulative risk of cancer in a patient with uc is approximately % at years, % at years, and % at years. , early age at onset of colitis, family history of colorectal cancer, histologic evidence of active inflammation, and sclerosing cholangitis may also increase the risk. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] colitis-associated carcinoma makes up only a small proportion of total colorectal cancer cases, but these tumors are particularly bothersome to the medical community. they frequently occur in young patients and often develop insidiously even while the patient is under active medical care. many times the carcinomas are not clinically apparent until distant spread has occurred. , colitis-associated carcinomas are often flat and infiltrative and difficult to visualize with standard radiographic and endoscopic techniques. because the symptoms associated with carcinoma mimic those of colitis, diagnosis is often delayed. the estimated risk of carcinoma complicating uc varies considerably. however, epidemiologic data suggest that patients with uc have to times the risk of developing carcinoma when compared with the general population, and looking at the subgroup of patients with uc who have extensive colitis, the risk is -to -fold. , this increased risk poses a management dilemma for physicians caring for patients not sick enough to require colectomy. several management options are available. first, the physician can ignore the risk. many physicians prefer this approach when dealing with an older patient or a patient who is not otherwise a surgical candidate, especially if the actual cancer risk is believed to be low. a second management approach is prophylactic colectomy in patients with colitis for longer than to years. this may be the best approach when dealing with a young patient because of the expected long duration of cancer risk and the possibility that surveillance can fail. furthermore, the results of ileal pouch-anal anastomosis are better in young patients. although prophylactic colectomy theoretically eliminates the cancer risk, several factors have made this approach generally unacceptable. patients with extensive colitis for more than to years are often asymptomatic or have only mild disease. such patients often find it difficult to accept the risks associated with major surgery, the possible morbidity associated with mobilizing the rectum, or the social implications of a permanent ileostomy (in the event of a pelvic pouch failure). besides, most studies suggest that prophylactic colectomy would have been unnecessary in the majority of patients because they would not have developed carcinoma. , a third option is colonoscopic surveillance with biopsy. the strategy of such surveillance is to identify a marker that signals a subgroup of patients with colitis who are at increased risk for carcinoma or the detection of carcinoma in an early, curable phase. , , currently, recognition of dysplasia in surveillance biopsy specimens is used as such a marker. dysplasia, the presumed precancerous epithelial change, has been regularly recognized in colectomy specimens both adjacent to and distant from colitis-associated carcinomas. , circumstantial evidence suggests that dysplasia may not only be the marker for carcinoma but also may itself be the carcinoma in a preinvasive phase. , dysplastic epithelium can occur in grossly flat mucosa, in mucosa with a villous or plaquelike configuration, or in a nodular growth resembling adenoma (fig. - ). dysplasia is recognized by histologic examination of biopsy specimens using well-defined cytologic criteria that include nuclear enlargement with hyperchromasia, increased mitotic figures, and decreased intracellular mucin. , most colitisassociated dysplasia resembles adenomas seen in patients without colitis. the pathologist should use the term dysplasia only as a synonym for intraepithelial neoplasia and not in reference to reactive or reparative changes seen with active inflammation. the distinction between repair and dysplasia can be difficult, requires experience, and in certain instances can be impossible. in general, cytologic abnormalities seen in the presence of active inflammation must be interpreted with caution. , similar features can be seen in repair and dysplasia and no one histologic feature is absolute in making the distinction. both conditions are associated with nuclear enlargement and hyperchromasia, increased mitotic figures, and decreased intracellular mucin. some features favor repair over dysplasia. although cells undergoing repair demonstrate nuclear enlargement with hyperchromasia, these nuclei are often evenly spaced and not crowded, round to oval with a smooth external nuclear contour, contain granular chromatin with a single or multiple chromocenter or nucleoli, and are remarkably similar to one another in size and appearance. in contrast to dysplasia, the nuclear-tocytoplasmic size ratio of regenerative cells is often actually decreased, especially in cells adjacent to ulcerated areas. during this phase, the cell cytoplasm is often eosinophilic. nearby crypt abscesses or neutrophils within epithelium (cryptitis) help to confirm the diagnosis of repair. as a general rule, reparative epithelial change is limited to or accentuated in the crypt base and will not extend onto the surface of the crypt. features that favor dysplasia over repair are variable nuclear hyperchromasia, nuclear pleomorphism, irregular external nuclear contours, nuclear stratification with irregular nuclear crowding and overlap, and a loss of nuclear polarity. the changes of dysplasia usually extend onto the surface of the crypt. major distortions of mucosal architecture also favor dysplasia. the inflammatory bowel disease-dysplasia morphology study group proposed a three-tiered classification for biopsy interpretation in ibd: positive, negative, and indefinite for dysplasia. our experience has shown that this classification is useful and reasonably reproducible. , biopsy specimens negative for dysplasia include normal colon and those biopsy specimens showing changes of active or quiescent uc. positive biopsy specimens are reported as showing high-grade dysplasia or low-grade dysplasia. most dysplasias histologically resemble adenomas seen in patients without colitis, although some do not. dysplasias in this latter group are easy to miss. in low-grade dysplasia, nuclear changes such as crowding, pleomorphism, hyperchromasia, and increased mitotic figures are present, but in general, the nuclei are limited to the basal half of the cell (fig. - ) . usually, intracellular mucin is decreased, but on occasion intracellular mucin can be increased. dystrophic or signet ring-type goblet cells are common in dysplasia although not pathognomonic. the general mucosal architecture should not be greatly disturbed. the distinction between low-grade dysplasia and highgrade dysplasia is made largely on the degree of cytologic and architectural change. in high-grade dysplasia, hyperchromasia and pleomorphism are marked (fig. - ) . stratification with loss of nuclear polarity is often present. nuclei may be found in the luminal portions of the cells. high-grade dysplasia is often associated with a distortion of mucosal architecture, most often taking the form of a villous growth similar to villous adenomas seen in patients without colitis. biopsy specimens are classified as showing changes indefinite for dysplasia when cytologic abnormalities are seen but these changes are of insufficient degree to warrant a diagnosis of true dysplasia. indefinite changes are usually encountered in a background of active inflammation in which atypical epithelial changes could represent repair or regeneration rather than dysplasia. the indefinite category also includes odd mucosal patterns of growth that have not yet been observed to give rise to carcinoma (e.g., sessile serrated polyp-like change [ fig. - ], epithelial changes that resemble gastric foveolar epithelium). the category, indefinite for dysplasia, is a legitimate diagnosis alerting the treating physician that worrisome changes are present that may place a patient in a higher risk category that requires more frequent surveillance. the inflammatory bowel disease-dysplasia morphology group originally subdivided indefinite dysplasia into three groups: probably inflamma-tory, probably dysplastic, and unknown. obviously, in a category that is already uncertain, subcategorization is cumbersome, subjective, and associated with marked interobserver and intraobserver variation. therefore, subdivision of biopsy specimens in the indefinite category is not recommended. the use of -aminosalicylate compounds decreases the risk of cancer by % to %, , as does seeing the physician regularly and having more than one colonoscopy. most patients and physicians opt for lifelong annual or biannual colonoscopic surveillance. , current practice guidelines recommend two to four biopsy specimens obtained every cm throughout the diseased bowel in addition to special sampling of macroscopic lesions. compliance with this recommendation has been inadequate. [ ] [ ] [ ] [ ] patients whose biopsy specimens remain negative for dysplasia can probably safely continue regular surveillance. most authorities recommend annual total colonoscopic examination for patients with extensive uc who have had their disease for more than to years. , , , the surveillance interval can be increased to to years for patients with negative initial colonoscopy because in only % to % of these patients has disease been shown to progress. patients demonstrating epithelial changes indefinite for dysplasia should have shorter follow-up (e.g., months to year). , they must not be ignored because % of patients in whom the initial biopsy results were indefinite progressed to high-grade dysplasia and % progressed to cancer. management recommendations for patients with low-grade dysplasia in a flat mucosa are difficult because of the paucity of long-term follow-up information. some clinicians consider it safe to continue short-term follow-up (e. g., to months) for patients with low-grade glandular dysplasia; , if dysplasia persists , , , or is associated with any suspicious gross lesion or stricture (dysplasiaassociated lesion or mass [dalm]), colectomy should be considered. , , , forty-three percent of reported patients with dalm already had carcinoma in immediate colectomy specimens. , increasing numbers of experts have advo- cated immediate colectomy for low-grade dysplasia. [ ] [ ] [ ] these experts have stated that % of patients with lowgrade dysplasia already had complicating carcinomas, and they also have cited a very high rate of progression to highgrade dysplasia or carcinoma with follow-up ( %, st. marks, london; %, mt. sinai, new york; %, mayo clinic, rochester, minn). these data are questioned because of case selection bias. if high-grade dysplasia is encountered, then colectomy should be recommended because the risk of concurrent carcinoma is estimated to be as high as %. , experience with surveillance biopsy interpretation has shown that truly negative results are rarely interpreted as dysplasia, and dysplasia, especially high-grade dysplasia, is rarely missed. , that said, variations in interpretation do occur, , , , - and in general confirmation of a biopsy diagnosis of dysplasia is desirable before colectomy. , any one or more of the following may be considered adequate confirmation: finding of dysplasia in a repeat biopsy from the same site, finding of dysplasia in one or more additional sites during the same endoscopic examination, or review and confirmation of the original dysplasia interpretation by another pathologist experienced with the classification system. , , surveillance endoscopy with biopsy has limitations. no compelling evidence proves that surveillance benefits the patient. , , , , participation in a surveillance program does not guarantee that lethal cancer will not develop. , , , , , , dysplasia is unusual and it is difficult for any one pathologist to acquire extensive experience. therefore, errors in histologic interpretation can be expected. dysplasia can be focal and is subject to tremendous sampling error. , , because most authorities consider dysplasia a neoplastic change, it is unlikely that it ever resolves spontaneously. thus, a clinician should not be lulled into a false sense of security by negative follow-up biopsy results once true dysplasia has been identified. although not proved scientifically, it seems likely that patients benefit from surveillance programs because many carcinomas are detected in a curable phase. , , , , , in addition, the incidence of carcinoma in patients whose biopsy specimen results have remained negative is low. , , it is unclear whether this benefit justifies the large cost of surveillance endoscopy. , finally, surveillance programs are pointless unless the patient complies with regular surveillance and agrees to colectomy when the end point (dysplasia) has been reached. many groups have searched for other markers for precancer in uc. sialomucins predominate in cancer and dysplasia in uc but the role of mucin histochemistry findings is debated. results of lectin binding, cea immunohistochemical study, and immunohistochemical analysis for products of c-myc and ras oncogenes have produced variable results and could not reliably differentiate dysplasia from repair. several investigators described a correlation between dna aneuploidy and dysplasia or carcinoma in uc. [ ] [ ] [ ] however, only % to % of invasive carcinomas and % to % of dysplasia demonstrated dna aneuploidy. this finding indicates that dna analysis by flow cytometry could not be used alone in a cancer surveillance program. many specimens (≥ %) interpreted as negative or indefinite for dysplasia showed dna aneuploidy. , this finding can be interpreted in several ways. it could indicate technical problems linked to false-positive results including failure to disaggregate nuclear clumps, prolonged exposure of the sample to higher temperatures, and debris in the sample. , alternatively, this test could identify a subgroup of patients different from the group identified by dysplasia that is showing objective chromosomal abnormalities in the absence of recognizable dysplasia. it is tempting to speculate that this dna aneuploidy could be used as a marker of some carcinomas complicating uc, although other investigators concluded that the detection of dna aneuploidy was not useful as a predictor of the presence of concurrent carcinoma in uc. [ ] [ ] [ ] until large prospective studies determine the usefulness of dna aneuploidy in uc, histologic dysplasia is still the only reliable marker for cancer in a surveillance program. , , dna analysis could benefit some patients. patients with diploid dna content and no signs of dysplasia probably can be examined at longer surveillance intervals. , approximately % of patients would fall into this category and a longer interval surveillance could save considerable amounts of money and time. a special problem concerns the occurrence of adenoma in patients with colitis. colorectal adenomas and ibd are both relatively common. therefore, there is probably no reason that both conditions could not coexist. because most ibdassociated dysplasias resemble adenomas, the distinction in practice is often impossible. management of patients with uc and adenoma has been controversial. does the lesion represent a harmless sporadic adenoma or is it a dangerous dalm? in general, a pathologic diagnosis of "adenoma" in a patient with ibd must be carefully evaluated by the treating physician and should at least prompt further consultation with the pathologist because the lesion could represent ibd-associated dysplasia, an ominous change that implies a substantial risk for the development of carcinoma or for coexisting cancer. , adenoma versus dysplasia-associated lesion or mass dysplasia in ibd can be broadly classified endoscopically as flat or raised. raised lesions can be further subdivided into adenoma-like (i.e., discrete, well-circumscribed sessile or pedunculated lesions that resemble sporadic adenomas [see fig. - ] ) or non-adenoma-like (i.e., irregular plaque or masslike) lesions (figs. - and - ). , most experts recommend colectomy for flat high-grade dysplasia and endoscopically raised non-adenoma-like dysplasia lesions. correct management of patients with uc who have endoscopically adenoma-like dysplasia is not clear. the differential diagnosis between conventional adenoma and dysplastic lesions in ibd has been the focus of several publications. distinction based on pathomorphologic features has been proposed. , although lesions can be classified histologically using this system, to date this approach has not been clinically validated. the study of torres and colleagues suggested that an admixture of normal and dysplastic glands at the surface of the lesion could be used to distinguish ibd-associated dysplasia from conventional adenoma. several drawbacks to this study limit its clinical usefulness. an association with carcinoma or flat dysplasia was definitional for ibd-associated dysplasia in this study. furthermore, the study had limited clinical follow-up to determine whether the distinction based on this feature (admixture of normal and dysplasia at the surface) was clinically relevant in patients not yet known to have other areas of dysplasia or carcinoma. many authorities believe that admixtures of normal and dysplastic glands are so highly prevalent in sporadic adenomas that this criterion is not helpful in practice. molecular analysis has been applied in an attempt to resolve this diagnostic problem. odze and associates reported a similar prevalence of p, apc, and p muta-tions in adenoma-like dysplasia lesions in uc and sporadic adenomas. however, non-adenoma-like dysplasia in uc showed significantly higher proportions of p and p mutation, a finding indicating different timing of molecular events in these lesions. although promising, these molecular approaches are technically difficult, are not generally available, and remain to be validated clinically. immunohistochemistry has also been used in an attempt to discriminate between conventional adenoma and ibdassociated dysplasia. , p overexpression by immunohistochemical analysis that generally correlates with mutation of the corresponding tumor suppressor gene appears to be the most promising. p gene mutations reportedly occur earlier in ibd-associated dysplasia than in conventional adenoma or carcinoma. therefore, strong p overexpression tends to correlate with ibd-associated dysplasia. unfortunately, only % of ibd-associated dysplasias overexpress p (versus % of sporadic adenomas). , this p testing is probably not sensitive enough for routine practice. combining p immunoreactivity with betacatenin expression (which exploits the relatively low prevalence of apc gene mutation in ibd-associated dysplasia) or bcl- immunohistochemistry (which is more often overexpressed in sporadic adenomas) does not appear to improve the sensitivity or specificity of this approach. , immunohistochemistry is clearly technically easier and more widely available. however, as with the molecular approaches outlined earlier, diagnoses based on immunohistochemistry have not been clinically validated. practical guide to patient management pathomorphologic, molecular, or immunohistochemical testing could eventually separate harmless sporadic adenomas from the more ominous dalms in patients with ibd but this remains to be proved. in the meantime, follow-up information is sufficient to guide a careful physician facing this dilemma. the most important criterion for stratification is the topographic relation of the adenoma-like lesion to areas of colitis. adenoma-like lesions occurring in areas not affected by colitis histologically or endoscopically have not been associated with a high risk of concurrent or subsequent carcinoma. these lesions should be considered conventional adenomas and treated by polypectomy alone. , , , , management of adenoma-like lesions occurring in areas involved by colitis is more challenging. as many as two thirds of patients have had concurrent or subsequent invasive carcinoma at follow-up. , , , from a practical standpoint, we believe that it is prudent to consider all adenoma-like lesions occurring in areas involved by colitis as potentially ibd-associated dysplasia lesions. these lesions must be excised endoscopically and not merely sampled with biopsy. the diagnosis of ibd-associated dysplasia alone in this unique setting (adenoma-like dysplasia in a macroscopic polyp) may not necessarily be an indication for immediate colectomy. , , , [ ] [ ] [ ] , endoscopic polypectomy alone may be adequate treatment provided that careful patient selection criteria are applied, including the following: ( ) the patient is in an adenoma age group (> years of age), ( ) the adenoma-like lesion is discretely defined macroscopically and can be excised in its entirety, ( ) excision of the lesion appears complete to the endosco- pist, ( ) no flat dysplasia is identified in the colon, and ( ) the colon is easy to survey (i.e., compliant patient without inflammatory polyposis). current data suggest that the majority of patients managed in this fashion will follow a clinically benign course. , , , [ ] [ ] [ ] however, these patients must receive careful short-term surveillance. a -to month initial surveillance interval can be increased to months to year following negative results of colonoscopy. colectomy should be recommended for patients not fulfilling these selection criteria. increasing epidemiologic and pathologic evidence suggests that patients with crohn's disease are also at increased risk for the development of carcinoma. , - those patients who are younger than years of age at the onset of crohn's disease may be at even higher risk. colonic carcinomas in patients with crohn's disease have in general developed after about years of disease. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the diagnosis is usually made clinically because a gross intraluminal lesion can often be visualized (fig. - ) . the colonic carcinomas have been better differentiated than their small bowel counterparts in crohn's disease, with an increased prevalence of mucinous histologic features. , , , reports using current histologic criteria almost invariably note dysplasia in the epithelium adjacent to small bowel and colonic carcinomas in crohn's disease. , [ ] [ ] [ ] dysplasia distant from carcinomas has also been encountered in specimens exhibiting both colonic carcinoma and crohn's disease. these features suggest that the dysplasiacarcinoma sequence similar to that proposed for uc also occurs in crohn's disease. , , a practice-based surveillance study of patients with crohn's colitis affecting at least one third of the colon for more than years found that % of patients developed dysplasia or cancer at years. opinions vary regarding cancer surveillance in patients with long-standing crohn's disease. , [ ] [ ] [ ] , , it is possible that surveillance could benefit an occasional patient with colonic crohn's disease by detecting dysplasia or early carcinoma, but the safety, necessity, and benefit of regular cancer surveillance are questioned. colonoscopy may be technically impossible or dangerous in some patients with crohn's disease. retained rectal stumps are often strictured and inaccessible. the absolute number of colon carcinomas reported in crohn's disease is still low, so again the expected yield of positive cases in a surveillance program would be quite small. some experts advocate a uc-like surveillance strategy for patients with at least years of crohn's colitis involving at least one third of the colon. rather than ignoring the risk in crohn's disease, options are available for a cautious clinician. , , a patient with recrudescence of colitis-like symptoms and a background of long-standing inactive crohn's colitis should be thoroughly investigated for carcinoma. yearly surveillance of an out-of-circuit rectum seems reasonable considering that approximately % of the reported cases of large bowel cancer in crohn's disease have occurred in such segments. , it may, however, be better to advise removal of the defunctioned rectum, especially if reanastomosis is not planned, not possible, or contraindicated. the presence of dysplasia in a biopsy specimen from a patient with crohn's disease must alert the physician to the possibility of coexistent invasive carcinoma. in this situation, management approaches similar to those proposed for dysplasia in uc are recommended. , , clinicians should also recognize that chronic fistula or anal stricture in crohn's disease may be complicated by squamous carcinoma or adenocarcinoma. , any abrupt change in the volume or nature of a fistula discharge or the development of an area of induration or mass near a fistula should be investigated with biopsy. so-called left-sided colonic diverticular disease commonly affects middle-aged and older individuals and shows a characteristic muscular abnormality of the bowel wall. the abnormality is characterized by marked thickening of the inner circular layer of the muscularis externa that results in a narrowed lumen and a mucosa thrown up into prominent accordion-like folds. the mucosal abnormality and associated diverticula are most prominent within the sigmoid colon but they may extend proximally for a variable distance and may even involve the entire colon. diverticula are not seen in the rectum. the diverticula are usually still enveloped by an intact longitudinal muscle layer. diverticula usually manifest in two longitudinally running rows situated between the mesenteric and two anti-mesenteric taeniae. in severe cases of diverticular disease, diverticula can also be found between the antimesenteric taeniae. similar clinical signs and symptoms occur regardless of whether objective inflammatory changes are present; therefore, the term diverticular disease is preferred rather than routinely making the distinction between diverticulosis and diverticulitis. diverticula are sometimes difficult to demonstrate in freshly resected specimens because of lost muscle tone and intraluminal pressure. as a result the diverticula often evert and are mistaken for polyps. the best way to demonstrate diverticula is to fix an intact specimen with formalin under pressure for at least day before dissection (fig. - ) . complications of diverticular disease include inflammation within the diverticula (diverticulitis), perforation of diverticula, adhesion, fistula formation, pericolonic abscess with inflammatory mass, hemorrhage, and obstruction. although colovesical and colovaginal fistula can occur in diverticular disease, other fistula combinations must raise the suspicion of coexisting crohn's disease. any granularity or ulceration of the luminal mucosa that is accompanied by microscopic findings of crypt abscesses or deep lymphoid aggregates away from inflamed diverticula must also raise the possibility of coexisting crohn's disease (see earlier). likewise, features of colitis distal to the zone of diverticula (e.g., at the distal resection margin) must also raise the question of coexisting crohn's disease. right-sided diverticular disease is often isolated and usually unassociated with diverticula in the rest of the colon. right-sided diverticula occur in younger patients and are frequently seen in asians. [ ] [ ] [ ] [ ] clinically, right-sided diverticular disease manifests with symptoms that mimic acute appendicitis. some authors classify these diverticula as congenital if the outpouching contains all layers of the bowel wall or acquired if the diverticula resemble those seen in the left-sided diverticular disease. complications include inflammation, hemorrhage, pericolonic abscess, and peritonitis. these diverticula have been implicated in some solitary nonspecific ulcers of the cecum and right colon. the clinical diagnosis of ischemic bowel disease can be difficult. patients usually present with nonspecific symptoms and routine radiographic examinations such as plain abdominal films or barium enemas are rarely of use. even arteriography may not be helpful because of the poor correlation between angiographic findings and the ultimate diagnosis or clinical outcome. angiographic demonstration of patent mesenteric blood vessels does not exclude ischemia. conversely, stenosis or occlusion of mesenteric arteries (even two of the three major vessels) does not establish a diagnosis of ischemia because these are frequent findings in asymptomatic patients. therefore, the diagnosis of ischemic bowel disease requires a high index of clinical suspicion complemented by continued monitoring of clinical, laboratory, and radiographic information. the pathologist can play an important role in the diagnosis and management of ischemic bowel disease by recognizing the patterns of injury that suggest ischemia in biopsy and resection specimens. at first glance, intestinal ischemia seems simple, but in reality the topic is almost impossibly complex; the ultimate outcome of the patient depends on complicated interrelationships among temporal, anatomic, and physiologic factors. for example, gradual occlusion of large vessels rarely causes ischemic damage because adequate collateral circulation (e.g., arc of riolan, marginal artery of drummond) will develop. in contrast, acute arterial occlusion is likely to result in infarct. considering anatomic factors, occlusion of a large vessel does not often result in ischemic damage because collateral circulation is usually adequate to protect the bowel. however, occlusion of smaller end arteries frequently results in segmental ischemic damage and infarct. finally, the mere existence of an entity referred to as nonocclusive intestinal ischemia emphasizes the importance of physiologic considerations. systemic hypotension and bowel distention lead to marked decreases in splanchnic blood flow caused by vasoconstriction and arteriovenous shunting in the bowel wall. , this phenomenon can result in widespread bowel infarct even in the presence of patent blood vessels. ischemic injury to the gi tract occurs whenever the oxygen or vascular supply cannot meet the metabolic demands of the tissue. gi ischemia has many causes, including inadequate perfusion, narrowing of blood vessels from any cause, bowel obstruction and distention, drug effects, and infections that can mimic ischemic damage. the list of differential diagnostic possibilities often overwhelms the pathologist and clinician. keeping the diagnostic considerations in their proper perspective, almost all cases of mesenteric ischemia result from systemic hypotension or occlusion of a major blood vessel. knowledge of these relative frequencies can be invaluable in decreasing the pathologist's anxiety and frustration when dealing with these types of specimens and when interacting with the concerned clinician, patient, and family. most ischemic episodes result from nonocclusive ischemic bowel disease (low-flow states) and in these cases no vascular lesion or specific cause for ischemia can be demonstrated on pathologic examination. superior mesenteric artery embolism or thrombosis is usually diagnosed by radiography or at operation. almost all significant superior mesenteric artery occlusions occur in the proximal cm to cm of the artery, an area not likely to be present in a resection specimen; such occlusive plaques, thrombi, or emboli are either bypassed surgically or removed and submitted separately to the pathology laboratory. mesenteric venous thrombosis is also diagnosed and identified at laparotomy and treated with thrombectomy. low-flow states, superior mesenteric artery occlusion, and mesenteric venous thrombosis account for more than % of major ischemic events. ischemia can result in a wide range of pathologic changes depending on the cause, severity, and duration of the hypoxia. thus, ischemia can cause little or no change, mucosal plaques or hemorrhages with erosion, discrete or serpiginous ulcers that mimic crohn's disease, strictures, or at the severe end of the spectrum, hemorrhagic infarct with perforation. any part or length of bowel can be affected depending on the cause and duration of hypoxia and the state of the collateral circulation. in general, those cases of ischemic bowel disease associated with narrowing of a blood vessel show pathologic changes in the bowel in the corresponding distribution. in contrast, nonocclusive intestinal ischemia causes patchy and irregular distribution of damage not necessarily corresponding to an area supplied by a specific blood vessel (fig. - ) . ischemic damage associated with reduced blood flow shows a propensity to affect "watershed zones" such as the splenic flexure, which is the boundary between the superior mesenteric and inferior mesenteric artery distributions. , acute, organizing, and healed phases of ischemia can be recognized histologically. acute ischemic damage can be focal or diffuse. the characteristic pattern of acute injury consists of hemorrhage into the lamina propria associated with superficial epithelial coagulative necrosis, often with sparing of the deep portion of the intestinal crypt (see fig. - ) . , the mucosal distribution of these changes can be explained by the existence of a countercurrent exchange mechanism that has been suggested for intestinal mucosa. in the mucosa, a central arteriole carries blood luminally in a direction parallel to but opposite to the direction of blood flow in the draining mucosal venule. this situation causes a gradient in oxygen tension to form, with the luminal aspect of the bowel relatively hypoxic when compared with the crypt base. , the changes of acute ischemia can occasionally be associated with more extensive necrosis of the epithelium, often associated with an attached inflammatory pseudomembrane. surprisingly, acute and chronic inflammatory cells are typically minimal in acute ischemic damage. this finding helps to differentiate ischemic damage from changes seen in primary ibd. depending on the severity and duration of the hypoxia, the acute change can be reversible or can proceed to mucosal ulcers, at which point the histologic features become relatively nonspecific. with increasing severity and duration, ischemia can lead to full-thickness infarct with hemorrhage and coagulative necrosis involving all bowel layers. granulation tissue and fibrosis with plump fibroblasts predominate in the organizing phase of ischemic damage. the histologic change becomes nonspecific. ischemia should be suspected when an ulcer or area of organizing inflammation in the bowel appears quite bland and is not associated with a marked increase in chronic inflammatory cells with plasma cells and lymphoid aggregates so characteristic of primary ibd. often, the mucosa of the ulcer edge shows changes similar to those seen in acute ischemic damage, and extensive samplings of these areas can help in diagnostic problem cases. the presence of hemorrhage and hemosiderin deposits in the connective tissues, although not pathognomonic, strongly suggests an ischemic origin. during the healing phase, the colonic mucosa often assumes an architectural appearance similar (if not identical) to chronic uc with atrophy, shortened crypts, and branched and budded glands. , these changes usually have a patchy distribution in healed ischemic damage but are diffuse in uc. ischemia usually does not show the basal plasmocytosis of primary ibd. healed phases of ischemia are often complicated by fibrous stricture in which fibrosis can affect all the layers of the bowel wall including the muscularis externa. fibrosis of the muscularis externa is distinctly unusual in primary ibd and its presence should raise the suspicion of ischemic bowel disease. again, hemosiderin deposits can be a helpful clue in recognizing the ischemic nature of the injury. most ischemic bowel lesions result from a combination of atherosclerosis and low-flow states and are referred to as nonocclusive ischemic bowel disease. specimens showing ischemic damage or infarct are usually resected on an emergency basis and include relatively short segments of mes- entery. large segments of infarct or ischemic damage are usually caused by an occlusive lesion in a proximal segment of a large artery or vein. in other words, the offending vessel is not likely to be present in the resection specimen. although one is unlikely to see specific vascular abnormality in the resection specimen, the blood vessels should be carefully dissected and many cross sections examined microscopically to detect vascular lesions such as atherosclerosis, embolus, thrombosis, or inflammation (vasculitis). the significance of vascular inflammation in areas of active ulcer and infarct is questionable because this "vasculitis" may be secondary. however, acute inflammation in vascular walls associated with fibrinoid necrosis found in apparently viable areas separated from an ulcerated and necrotic area by a margin of normal tissue may indicate systemic vasculitis. polyarteritis nodosa is a multisystem disease characterized by random necrotizing inflammation involving small and medium-sized arteries. because the distribution and severity of the vascular lesions are haphazard, polyarteritis nodosa produces protean clinical manifestations without pathognomonic signs or symptoms. abdominal pain, fever, leukocytosis, hypertension, and neuropathy are common in patients with polyarteritis nodosa. according to the american college of rheumatology, the presence of of the following criteria are considered diagnostic of the disease : weight loss, livedo reticularis, testicular pain or tenderness, myalgia or myopathy, neuropathy, hypertension, renal impairment, hepatitis b infection, abnormal arteriogram, and positive biopsy results. the diagnostic histopathologic change of polyarteritis nodosa is necrotizing panarteritis with inflammation involving intima, media, and adventitia. the inflammatory infiltrate is composed of neutrophils, eosinophils, plasma cells, and lymphocytes and is often associated with a deposition of fibrin. , the vascular lesions are usually seen in various stages of development and healing. disruption of the internal elastic lamina weakens the vascular wall and can lead to aneurysm formation, an important sign used in radiographic diagnosis. of patients with polyarteritis nodosa, % to % have gi tract involvement such as duodenal or gastric ulcer, melena, hematemesis, or small bowel infarcts. gi tract involvement is usually a manifestation of systemic polyarteritis nodosa. however, a growing body of literature has described polyarteritis nodosa apparently limited to one organ, most commonly the skin, gallbladder, or vermiform appendix. [ ] [ ] [ ] [ ] [ ] only a rare patient with polyarteritis nodosa that is initially diagnosed in the gallbladder or appendix develops systemic vasculitis , , , and those who do frequently have elevated serum rheumatoid factor or antinuclear antibodies. phlebitis, churg-strauss angiitis, small vessel vasculitis, buerger's disease, and giant cell arteritis can manifest initially in the gi tract and cause ischemic injury. , phlebitis and small vessel vasculitis are frequently associated with drugs and medications and are often self-limited. , special consideration should be given to an entity referred to as intra-abdominal (lymphocytic) phlebitis. rare cases of ischemic damage to the colon have been linked to a curious form of phlebitis reported under numerous synonyms including lymphocytic phlebitis, necrotizing and giant cell granulomatous phlebitis, idiopathic myointimal hyperplasia of mesenteric veins, mesenteric inflammatory veno-occlusive disease, intramural mesenteric venulitis, and idiopathic colonic phlebitis. , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] since the original descriptions, approximately additional cases have been described. the patients have ranged in age from to years, and no gender predilection is apparent. most cases involve the small intestine and colon but involvement of other organs such as the gallbladder, omentum, and stomach have been described. , four patients received treatment with the drug rutoside. microscopically, specimens show phlebitis and thrombophlebitis mostly involving the submucosal veins and venules ( fig. - ) . the most prominent lesion is infiltrative lymphocytic phlebitis. in approximately half of these patients, the phlebitis has been necrotizing, and in about one third a granulomatous component has been visible. in approximately half of the patients, myointimal proliferation has been described. some investigators have proposed that myointimal hyperplasia represents an end stage of lymphocytic phlebitis. immunohistochemical analysis has been performed on a few cases and demonstrates a mixture of t cells and b cells with a few macrophages. , some of the t cells express the cytotoxic granule-associated protein tia- and granzyme b, findings supporting the contention that the lymphocyte-mediated vascular damage is a central event in the pathogenesis. so far, cases in the literature have had a favorable clinical outcome. the disease usually has been diagnosed and treated by resection in these patients. no patient to date has required repeat resection, nor has any patient developed systemic vasculitis. some patients con- tinue to have abdominal pain, and at least one report noted recurrent phlebitis documented by biopsy. other entities considered part of the spectrum of ischemic bowel disease include necrotizing enterocolitis of the neonate, neutropenic enterocolitis (typhlitis), intestinal behçet's disease, late irradiation enterocolitis, uremic colitis, stercoral ulcer associated with bowel obstruction, potassium chloride-induced ulcer, stress ulcer and ulcers associated with drugs (e.g., nsaids and chemicals), certain infections such as clostridium difficile-associated colitis, and ehec infection. the pathologic features of these disorders resemble ischemic bowel disease and are generally indistinguishable without knowledge of clinical and laboratory information. necrotizing enterocolitis is a serious form of intestinal injury that resembles ischemic bowel disease. although many investigators think it part of the spectrum of ischemia, increasing evidence supports a role for infectious agents in the pathogenesis of the lesion. necrotizing enterocolitis of the neonate generally affects low-birth-weight or premature infants. the disease usually occurs in babies younger than days of age and symptoms usually develop after feeding has begun. the lesions resemble acute ischemic damage. , ulcers that can be patchy or diffuse occur and in general may affect any part of the gi tract. preferred sites of involvement include the terminal ileum, right colon, and stomach. histologically bland transmural necrosis occurs and may be associated with gas cysts within the bowel wall (pneumatosis cystoides intestinalis [pci]) ( fig. - ) . babies surviving the acute episode may later develop fibrous strictures. neutropenic enterocolitis goes by numerous synonyms, including hemorrhagic necrosis of the gi tract, necrotizing enterocolitis, agranulocytic colitis, typhlitis, and the ileocecal syndrome. [ ] [ ] [ ] [ ] the condition is fulminant segmental colitis that is often seen in patients with lymphoma or leukemia receiving immunosuppressive drug therapy, but it has also been seen in association with other chemotherapyinduced or non-neoplastic-induced neutropenias such as cyclic neutropenia. many investigators consider this disorder a form of ischemic bowel disease, but increasing evidence implicates bacteria, especially clostridium septicum, as causative agents. [ ] [ ] [ ] [ ] the pathogenesis is unclear but is likely to be multifactorial with neutropenia, altered mucosal integrity, ischemia, and infection all playing a role. the lesion can occur anywhere in the gi tract, but the preferred sites are cecum, right colon, and terminal ileum. the histologic picture combines infarct necrosis with profound secondary bacterial or fungal overgrowth and is often associated with a paucity of acute inflammatory cells. the prognosis is grave; however, reports of successful surgical and medical management have been published. , , , , nonspecific ulcers of colon investigators have reported benign cecal ulcers and segmental necrosis of the right colon in which the pathologic features are nonspecific or resemble ischemia. [ ] [ ] [ ] the cause of many of these ulcers remains unclear. some may represent inflammatory changes or bleeding vascular lesions in right-sided diverticula. , many are associated with endstage renal disease, hemodialysis, renal or cardiac transplantation, and corticosteroid therapy. some are clearly related to ischemia caused by low-flow states, which are known to occur in patients undergoing hemodialysis. however, a strong correlation exists among cecal and terminal ileal ulcers, renal or cardiac transplantation, immunosuppression, and cmv infection. the possible role of cmv in these ulcers is unknown. the exact cause of many colonic ulcers cannot be determined at present. nsaid use should also be investigated. the term behçet's disease describes the clinical triad of aphthous stomatitis, genital ulcers, and relapsing iritis. behçet's disease is seen worldwide, but most cases have been reported from the mediterranean basin, the middle east, and japan. male patients are twice as commonly affected as female patients and the mean age of onset is in the third decade. , behçet's disease is a systemic disorder; only % to % of patients demonstrate gi tract involvement, [ ] [ ] [ ] usually in the form of intestinal ulcers. the ulcers of intestinal behçet's are described as multiple, deep, and punched-out with a preferential localization in the ileum and cecum. the ulcers often perforate. [ ] [ ] [ ] [ ] the primary clinical differential diagnosis is crohn's disease. intestinal behçet's disease differs from crohn's disease in several ways. behçet's disease is usually not associated with strictures or granulomas. free perforation, common in behçet's disease, occurs rarely in crohn's disease. details of histologic pattern in intestinal behçet's are difficult to find. , the inflammatory infiltrate of behçet's ulcer appears nonspecific. the lesions of so-called acute behçet's ulcer closely resemble acute ischemic bowel disease. the chronic lesion of behçet's disease resembles crohn's disease except the transmural lymphoid aggregates in behçet's disease contain more germinal centers, the bowel wall can be fibrosed, and granulomas are not usually encountered. some authors have reported mononuclear cell infiltrates around capillaries and venules with some areas of fibrinoid necrosis. , indeed, large vessel inflammation with occlusion and aneurysm formation have been described in % to % of patients with behçet's disease. , vasculitis may be the common underlying factor producing the gi lesions of behçet's disease. late complications of irradiation can occur weeks to years after therapy and include colitis, stricture, ulcer, and fistula ( fig. - ) . , many of these abnormalities may be related to ischemia as a result of the effect of radiation on blood vessels. histologically, the mucosa appears atrophic and similar (often identical) to that seen in chronic uc. having said that, the basal plasmacytosis in the lamina propria characteristic of primary ibd is usually but not always absent. ectatic mucosal capillaries with thrombosis and hyalinization are important histologic signs that can point to irradiation effect ( fig. - ). irradiation may be associated with fibrosis of any of the layers of the bowel wall. the fibrosis can appear dense and hyalinized and may contain large, atypical radiation fibroblasts. vascular changes are often prominent. blood vessels may be ectatic in the mucosa and submucosa. more commonly, however, one encounters marked intimal fibroplasia with hyaline thickening of the blood vessel walls that leads to luminal stenosis. sharply demarcated ulcers sometimes with secondary bacterial overgrowth can often be seen in the colon and rectum proximal to obstructing lesions such as invasive carcinoma, or they can be seen in patients with intractable constipation and fecal impaction. , [ ] [ ] [ ] the ulcer, bland in appearance, is thought to result from ischemia caused by a combination of pressure injury related to the hard fecal mass and the physiologic effects of bowel distention. , many drugs or chemicals have been associated with ischemic-type changes in the colon, including hydrogen peroxide and glutaraldehyde (used to clean endo scopes), nsaids, , , cocaine, , , oral contraceptives, and estrogen compounds. colitis can be caused by a host of bacteria including campylobacter species, shigella species, salmonella species, neisseria gonorrhoeae, yersinia species, mycobacterium species, and aeromonas species. although the histologic features of colonic mucosal biopsy specimens can vary greatly in these infections from essentially normal to lesions mimicking idiopathic ibd, large numbers of specimens show active colitis, as outlined earlier in the chapter, that when seen should suggest infectious-type colitis. , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the histologic finding usually consists of the focal active colitis pattern of injury with relative preservation of mucosal architecture except for edema. the active inflammation is associated with little or no mucin depletion. neutrophils can be present within the lamina propria and scattered cryptitis and crypt abscess formation, sometimes with luminal accentuation, can be seen. the definitive diagnosis of infectious colitis requires laboratory documentation by culture, pcr on the paraffin block, or serologic examination. histologic evaluation, although helpful in suggesting infection, can only rarely point to a specific agent. true granulomas can be seen in tuberculosis, syp hilis, chlamydia species infection, and yersinia pseudotuberculosis infection. microgranulomas are described in infection with salmonella species, campylobacter species, and yersinia enterocolitica. isolated mucosal giant cells are nonspecific but have been described in chlamydia trachomatis infection. , some bacterial infections cause little inflammation but can be diagnosed by observation of adherent organisms such as with intestinal spirochetosis and adherent e. coli species. although intestinal spirochetosis (infection with brachyspira aalborgi or brachyspira pilosicoli) can be seen in patients with human immunodeficiency virus (hiv) infection or aids, , it is more frequently encountered in immunocompetent patients in whom its significance remains controversial. , [ ] [ ] [ ] [ ] the microscopic appearance is subtle, consisting of a thickening or accentuation of the colonic brush border that stains deeply with hematoxylin. this effect is caused by adherent spirochetes that align in parallel and embed themselves in the absorptive cells. identification can be enhanced by use of a warthin-starry (or other silver) stain. the organism also cross-reacts with treponema species immunostains. adherent e. coli infections, described in hiv/aids under the term diarrheogenic bacterial enterocolitis, are discussed later. gi involvement with mycobacterium avium-intracellulare complex (maic) is seen in hiv/aids usually as part of disseminated infection. the organism is easily cultured from stool and even blood. maic can affect any part of the gi tract, including the large bowel. , the characteristic histologic feature is infiltration of the lamina propria by foamy macrophages that can mimic muciphages. in addition, maic stains positive with pas, a finding further adding to the possibility of misdiagnosis. maic is easily diagnosed with an acid-fast stain. cryptosporidium parvum infection can occur in immunocompetent persons but is more often seen in hiv/aids , and occasionally can be seen in the colon (fig. - ) . in biopsy specimens, cryptosporidial organisms are identified as basophilic dots measuring approximately µm attached to the surface of the colonocyte. immunocytochemical methods for identification have been described. amebiasis (infection with e. histolytica) is quite common worldwide and is seen in hiv/aids. clinical disease varies widely from asymptomatic conditions to fulminant colitis. early lesions consist of a slight mucosal depression and erosion with adherent exudate containing trophozoites of e. histolytica (fig. - ). focal active colitis can also be seen. more severe disease can cause ulcers that have been described as flask-shaped because they undermine the adjacent mucosa. e. histolytica is easily distinguished from the much larger balantidium coli, which is only rarely seen in humans. in addition, the trophozoite of b. coli is ciliated, the nucleus is much larger and kidney-shaped, and the cytoplasm contains vacuoles. schistosoma species infestation is a major cause of colitis worldwide but is rarely seen in north america. the adult worms elicit no inflammatory response. the colitis and the symptoms are caused by the marked inflammatory response to the eggs that can lead to mucosal ulcers ( fig. - ) . chronic colitis and inflammatory polyps can be seen in areas of endemic infection. strongyloidiasis is usually a self-limiting infection with adult worms residing in the proximal small bowel; the eggs develop into rhabdoid larvae in the lumen that pass in the stool. in immunocompromised patients, the rhabdoid larvae may develop into the infective filiform larvae. these infective larvae may invade intact colonic mucosa or perianal skin and set up a cycle of infection referred to as autoinfection. many such infections are severe and often fatal. in colorectal biopsy specimens the infective larvae can be seen usually associated with marked infiltration of mucosa with the eosinophils (fig. - ) . sometimes an eosinophilic and granulomatous reaction can be seen in biopsy and resection specimens. occasionally intraluminal worms are encountered in the colonic lumen and are removed at colonoscopy. these parasites are usually identified as trichuris (whipworm) or enterobius (pinworm) by examination of the worm and egg morphology. common viral pathogens include adenovirus, rotavirus, coronavirus, echovirus, enterovirus, astrovirus, and norwalk virus. colorectal biopsy is rarely performed in the setting of viral gastroenteritis; therefore, histologic changes are not well documented. norwalk virus and rotavirus are not known to cause morphologic changes in the colon. adenovirus can cause diarrhea in hiv/aids. histologic changes affecting epithelial cells include cellular disorder with loss of orientation and degeneration. eosinophilic viral intranuclear inclusions can be seen in surface goblet cells but are quite subtle. immunohistochemical analysis can be performed as an aid to diagnosis. cmv infection is extraordinarily common in patients with hiv/aids and in other immunosuppressed patients such as those receiving transplants. the characteristic inclusions are seen in endothelial cells, fibroblasts, and smooth muscle cells and are only rarely encountered in epithelial cells. severe infection can lead to vascular thrombosis and ischemic-type damage with ulcers (figs. - and - ) . herpes simplex virus infection is associated with painful ulcers in the distal rectum and perianal skin. , the pathologic changes (ulcer, neutrophils in the lamina propria, cryptitis, and crypt abscess formation) may suggest infec- invasive candidiasis can be seen in severely debilitated patients and in patients with hiv/aids. this disorder is rarely encountered in colorectal biopsy specimens. colorectal histoplasmosis can be seen in immunocompetent patients and in immunocompromised patients such as those with hiv/aids. , in disseminated forms one can see collections of foamy macrophages. some patients demonstrate granulomatous inflammation, which is sometimes suppurative. diagnosis can be made using gomori methenamine silver or other fungal stains. administration of any antibiotic that favors the growth of toxin-producing c. difficile can lead to pseudomembranous colitis. , early studies linked clindamycin and lincomycin to pseudomembranous colitis, but in terms of absolute numbers, most cases are linked to ampicillin, penicillin, the cephalosporins, and the fluoroquinolones (especially the c- -methoxyfluoroquinolones gatifloxacin, and moxifloxacin) because of their far more prevalent use. , currently, the second-and third-generation cephalosporins and the fluoroquinolones are the leading instigators of c. difficileassociated colitis. [ ] [ ] [ ] pseudomembranous colitis has even been associated with antineoplastic chemotherapeutic agents that have antimicrobial activity. in many cases, the source of clostridium difficile infection is the patient's own gut flora. alterations of the gut flora allow the patient's own c. difficile organisms to multiply. however, the spore-forming organism c. difficile is widely distributed in nature. although cases may be acquired by these routes, increasing evidence indicates that c. difficile is often acquired in hospitals. , , indeed, reports of a previously uncommon strain of c. difficile, the nap strain, showed that it is responsible for severe hospital outbreaks and serious disease in otherwise healthy individuals. , [ ] [ ] [ ] [ ] these reports of close-contact transmission, high recurrence rate, young age, bloody diarrhea, and lack of antibiotic exposure suggest changing epidemiologic features. advanced age is a risk factor; patients who are older than years old have an approximately times greater risk of developing c. difficile infection. statistically, c. difficile is associated with only a minority of cases of antibioticassociated diarrhea. in gi diseases other than primary ibd, isolation rates are about the same as in healthy adults. c. difficile is more frequently identified in patients with ibd and may be linked to exacerbations of ibd and fulminant colitis. , symptoms of pseudomembranous colitis usually develop during the administration of antibiotics but in up to one third of patients, the onset of symptoms can be delayed for up to to weeks; occasionally, no antibiotic exposure has occurred. , , , the characteristic endoscopic and histologic lesion is found early in the course of the disease and only in some individuals. the surface of the mucosa is covered by a plaquelike, cream-colored to yellow pseudomembrane ( fig. - ) . the intervening mucosa frequently appears normal but can be hyperemic or edematous. with increasing severity, the membranes can become confluent and linear ulcers can develop. usually the pseudomembranes are evenly distributed throughout the colon but in up to one third of patients, the pseudomembranes can be confined to the right colon. this finding emphasizes the need for total colonoscopy to make an endoscopic diagnosis. histologically, patchy necrosis of the superficial colonic crypts is evident that is not unlike that seen in ischemia. the affected crypts become dilated near the surface and an inflammatory exudate erupts from the surface aspect of the degenerating crypts in an explosive or mushroom-like configuration. the pseudomembrane may cover adjacent virtually normal colonic mucosa (fig. - ). the karyorrhectic debris and neutrophils in the pseudomembranes tend to orient in a curious linear fashion within the fibrin and mucus. early lesions of c. difficile and occasionally the mucosa between diagnostic pseudomembranes can show the focal active colitis pattern of injury frequently described in acute infectious-type colitis (acute self-limited colitis). , , left untreated, some cases of c. difficileassociated colitis progress and can become indistinguishable from ischemic bowel disease. toxic megacolon and perforation can occur. treatment falls into three major categories: ( ) nonspecific therapy, ( ) specific therapy, and ( ) therapies aimed at altering the gut flora. nonspecific treatments include discontinuance of the offending antibiotic, supportive measures, enteric precautions to retard the spread of c. difficile outbreaks, and surgery. surgical excision may occasionally be indicated for patients too sick to take oral antibiotics (the specific therapy), as well as for some patients with relapses and for nonresponders. toxic megacolon and perforation also require surgical treatment. specific therapies include the oral administration of antibiotics such as vancomycin, metronidazole, bacitracin, and the toxin binder cholestyramine. vancomycin, the preferred treatment, is relatively expensive. bacitracin and metronidazole appear to be as effective as vancomycin and are less expensive. theoretical disadvantages to metronidazole therapy include stool levels of the drug that appear far lower than those of vancomycin and the finding that metronidazole has occasionally been implicated as a cause of pseudomembranous colitis. treatment or prevention of pseudomembranous colitis by altering the gut flora with the introduction of such agents as lactobacillus and saccharomyces boulardii , , has been studied. saccharomyces boulardii was shown to decrease the incidence of antibiotic-associated diarrhea but did not change the rate of c. difficile colonization. another provocative approach to prevention is to restrict the use of newer antibiotics such as third-generation cephalosporins and fluoroquinolones in the hospital setting. , patients with c. difficile-associated pseudomembranous colitis who are treated specifically show a % to % response rate usually with defervescence in to days and resolution of diarrhea in days. however, % to % of patients have a relapse and relapse treatment can be challenging. most investigators recommend a second course of the same antibiotic (metronidazole or vancomycin) for days. for a second recurrence, tapered-pulsed vancomycin is advised. for a third or subsequent recurrence, a probiotic or toxin binder is added to tapered-pulsed vancomycin therapy. newer treatments for recurrences include passive immunoglobulin therapy, toxin receptor decoys (e.g., tolevamer), and active immunization against toxin a. stool culture for c. difficile is usually not recommended but some investigators report a significantly higher yield of positive results based on fecal culture followed by toxin assay on positive colonies. cell culture assay for toxin b is not routinely performed. results of latex agglutination tests have been disappointing. enzyme-linked immunoassays (eias) that detect toxin a (meridian diagnostic, vitec, bd, and cambridge) or toxin a and toxin b are widely used. these assays have a % to % positive correlation with cell culture assay. commercially available tests that detect toxin a and toxin b are preferred because % to % of c. difficile strains produce only toxin b. , an alternative and more sensitive but slower approach is to perform an eia for the detection of common antigen (a highly sensitive marker for c. difficile) followed by a cytotoxic assay if the results of the eia are positive. various gene probes and pcr techniques that detect toxin at the dna level are available for research purposes. enterohemorrhagic escherichia coli-associated colitis the clinical syndrome of hemorrhagic colitis is characterized by abdominal cramping, bloody diarrhea, and either no fever or low-grade fever. , patients typically demonstrate right-sided colonic edema, erosion, and hemorrhage and the absence of conventional enteric pathogens. in , investigation of hemorrhagic colitis outbreaks occurring in oregon and michigan implicated a then-rare serotype of e. coli, o :h , as the cause of the syndrome. subsequently, investigations of several additional outbreaks confirmed the association between hemorrhagic colitis and the verocytotoxin-producing e. coli, the most important of which is e. coli o :h . patients with hemorrhagic colitis typically present with the sudden onset of crampy abdominal pain occurring to days after ingestion of contaminated food, usually undercooked hamburger. outbreaks have also been linked to other foods, drinking water, and swimming pools. watery diarrhea, nausea, and vomiting follow within hours. one to days later, grossly bloody diarrhea replaces the watery diarrhea. in almost all patients, the disease resolves spontaneously, usually within days. investigation of the epidemic outbreaks of e. coli o :h infection revealed that not all patients acquire the full syndrome of hemorrhagic colitis. rather, a clinical spectrum exists ranging from asymptomatic carrier or self-limited nonbloody diarrhea to severe cases complicated by hemolytic-uremic syndrome and thrombotic thrombocytopenic purpura. , , e. coli o :h produces several toxins active against vero cells (verocytotoxins) and hela cells that have been termed shiga-like toxins because their mode of action is similar to that of the toxin produced by shigella dysenteriae type i. the toxins interact with the membrane receptor globotriosyl ceramide, are apparently absorbed into epithelial and endothelial cells, and cause damage or cell death by interfering with protein synthesis. , colonic histologic features in ehec infection are the best documented of the diarrheogenic e. coli organisms because ehec infection can lead to hospitalization and it clinically mimics ischemia and primary ibd, thus prompting colonoscopy with biopsy. , , colonoscopy typically demonstrates patchy erythema, edema, and surface ulceration of the colon (fig. - ) . the cecum and right colon are usually described as markedly abnormal, whereas the descending colon typically has mild or no changes. histologically, specimens usually show hemorrhage and edema within the lamina propria. specimens most often show focal necrosis associated with hemorrhage and acute inflammation within the superficial mucosa with preservation of the deep colonic crypts, similar to the pattern of injury described in acute ischemic colitis. specimens from many patients show neutrophils infiltrating the lamina propria and crypts resembling the focal active colitis pattern of injury seen in infectious colitis or acute self-limited colitis ( fig. - ). rarely patients also demonstrate inflammatory pseudomembranes. this combination of ischemic-like and infectious-like injury with capillary thrombi should at least suggest ehec-associated colitis in biopsy specimens. routine stool culture media do not enable one to distinguish e. coli o :h from other strains of e. coli normally found in the stool. physicians suspecting hemorrhagic colitis caused by e. coli o :h should specifically request that stools be screened for this organism. specimens may be examined using sorbitol fermentation as a strain marker; unlike most e. coli organisms, e. coli o :h strains test negative for sorbitol or have delayed positive results. colonies that test negative for sorbitol at hours can be screened with commercial o antisera. additional biochemical tests and h-antigen determinations can be performed later. dna hybridization techniques, pcr, direct immunofluorescence, and latex agglutination techniques have also been described for identification. procedures for detection of free fecal verocytotoxin and more sensitive methods for screening stool cultures for verotoxinproducing e. coli using polymyxin b on colony sweeps have been reported. , other escherichia coli pathogens e. coli organisms are the predominant components of the gut microflora. although most of these organisms are harmless or even beneficial, at least six categories of e. coli intestinal pathogens are recognized (table - ) . , much is known about the microbiology, pathogenic mechanisms, virulence factors, molecular genetics, and epidemiology of intestinal e. coli infections. however, surprisingly little is known about the histopathologic features of the human gut infection. information remains scant because most infectious diarrheas, being self-limited, do not require specific treatment even when an infectious organism is identified. therefore, sophisticated diagnostic tests such as organism identification, virulence factor determination, and endoscopy with biopsy are reserved for outbreaks or for cases with unusual features (e.g., severe or protracted diarrhea, systemic symptoms, need for hospitalization, or a differential diagnosis including serious diseases such as primary ibd or ischemia). analysis of the information available yields limited numbers of reaction patterns in e. coli-associated infection that have been described in the human gut or inferred from in vitro models or from human infection with other organisms having similar virulence factors. these include the following: no histologic change ( etec organisms are thought to adhere to and colonize the surface of the small bowel where they elaborate their toxins. it is possible that a serendipitously obtained small bowel biopsy specimen may contain adherent surface bacteria. histologic appearances of the small bowel and colon are inferred from etec's close relationship with vibrio cholerae, which does not cause histologically recognizable lesions in the small bowel or the colon. therefore, normal colon would be expected in etec-associated diarrhea. it is possible that etec could cause some acute self-limited colitis. recognizing the difficulty in identifying pathogenic e. coli in routine stool culture, it is possible that eiec and ehec could otherwise be responsible for some cases of acute self-limited colitis. as stated earlier, diffuse active colitis was also seen in some examples of documented cases of infectious colitis. these infectious colitis cases were associated with an epidemic outbreak of shigella dysentery. because the pathogenic features of shigella species are virtually identical to those of eiec, it is possible that eiec may on rare occasions cause the diffuse active colitis pattern of injury. that said, eiec infection with this pattern of injury may represent an infectious exacerbation of underlying primary ibd. the ability to adhere to and colonize host enterocytes or colonocytes is requisite for human infection for all six recognized categories of diarrheogenic e. coli. however, epec, eaec, and daec, which are traditionally referred to as the enteroadherent e. coli, are best differentiated on the basis of their adherence patterns to hep- cells in culture. these patterns include localized adherence (epec), aggregative adherence (eaec), and diffuse adherence (daec). the human gut histopathologic features in epec in vivo have been described , and are similar to those seen in experimental animal models. jejunal biopsy specimens have demonstrated variable villous abnormalities without acute inflammation. , adherent bacteria could be identified on routine h&e-stained sections on the luminal surface. adherent surface bacteria have also been seen in colorectal biopsy specimens. the characteristic attaching and effacing lesion can be recognized only by the use of electron microscopy. , , the bacteria intimately adhere to the host cell by an attachment pedestal and cause effacement of the adjacent microvilli. human histopathologic features associated with eaec and daec infection can be inferred only from animal models and in vitro studies. , [ ] [ ] [ ] presumably, bacteria adhere to the surface epithelium of the small intestine and the colon. eaec and daec infection may be associated with variable villous abnormalities. electron microscopic study has shown adherent bacteria in cell culture with a normal microvillous structure. in the colon, eaec produces cytotoxic effects ultrastructurally (microvillous vesic- ulation, enlarged crypt openings, creation of intercrypt crevices, and mucosal epithelial cell extrusion). the most detailed description of human ileal and colorectal infection with epec, eaec, and possibly daec probably is found in a published review of patients with aids. unfortunately, the exquisite light and electron microscopic descriptions of these diarrheogenic bacterial enterocolitides were not complemented by microbiologic studies. therefore, identification of these organisms as e. coli rests on the pathologic similarity to other reported cases and preliminary isolation studies that have shown that at least some of these cases were caused by eaec and daec. the colonic histologic pattern showed surface epithelial degeneration with adherent bacteria (some extremely subtle) without colonic architectural distortion or significant inflammation. electron microscopic examination demonstrated three patterns: ( ) typical adhering and effacing lesions (as with epec), ( ) a loosely adherent pattern with effacement (as with eaec), and ( ) an intercalated pattern with effacement in which vertically oriented bacteria were seen burrowing between intact microvilli. the intercalated pattern could be daec in vivo because it is similar to the pictures and descriptions of daec in other in vitro and animal models. the term intestinal pseudo-obstruction describes a disorder in which patients present with signs and symptoms of intestinal obstruction and in whom no mechanical obstructive lesion can be demonstrated. intestinal pseudoobstruction can be associated with a heterogeneous group of conditions some of which can affect the colon. [ ] [ ] [ ] systemic lupus erythematosus, dermatomyositis, and scleroderma can cause fibrosis of the muscularis externa. , amyloid deposits can also affect colonic motility. visceral myopathies can occasionally be identified in the colon. recognized variants of familial visceral myopathy demonstrate differences in the mode of inheritance (autosomal dominant versus recessive), site of gut involvement, clinical symptoms, and extraintestinal manifestations. visceral myopathies also occur in sporadic form. the intestinal pathologic changes of many familial and sporadic visceral myopathies are identical and consist of muscle cell degeneration, muscle cell loss, and fibrosis of the muscularis externa. the degenerative fibers appear swollen and rarified. collagen may encircle the residual muscle fibers in areas of muscle fiber dropout and impart a vacuolated appearance. , , these changes are often limited to, or are more severe in, the external layer of the muscularis externa. the differential diagnosis of these forms of visceral myopathy includes other entities that cause fibrosis of the muscularis externa and encompasses ischemia, tuberculosis, and scleroderma. ischemia usually is associated with a fibrous stricture and hemosiderin deposits. tuberculosis typically causes strictures and granulomas, often with central necrosis. progressive systemic sclerosis is associated with more patchy bowel involvement than are the visceral myopathies. the fibrosis is often denser and replaces all the muscle layers of the muscularis externa or is accentuated in the inner layer ( fig. - ). vacuolar change is usually not seen with progressive systemic sclerosis. the visceral neuropathies - form a complex group of unusual entities that vary in their pattern of inheritance, the extent of intestinal and extraintestinal involvement, and the nature of the histologic changes in the neural plexuses of the gut. many of the neuronal and axonal changes are subtle; with the exception of inflammatory neuropathies and some neuropathies associated with intranuclear or intracytoplasmic inclusions (e.g., red cytoplasmic inclusions in ganglion cells of mitochondrial neurogastrointestinal encephalomyopathy), they cannot be recognized in routine h&e-stained sections. difficult and unusual cases should probably be referred for consultation to pathology departments with particular expertise in the evaluation of visceral neuropathies. that said, referral is rarely indicated clinically. some sporadic cases demonstrate mononuclear inflammation in the myenteric plexuses, and these cases can be identified by routine light microscopy. , some of these cases are manifestations of paraneoplastic syndromes often linked to small cell carcinoma of the lung. others are postinfectious. the inflammatory neuropathies can sometimes cause acquired aganglionosis and can be associated with circulating antibodies, such as anti-hu, (anna- , antineuronal nuclear antibody- ), anti-ri, and anti-yo (purkinje cell cytoplasmic autoantibody). , an acquired form of hypoganglionosis associated with buserelin-induced formation of anti-gnrh (anti-gonadotropin-releasing hormone) antibodies has been reported. investigators now recognize the role interstitial cells of cajal (iccs) play as gut pacemakers and as mediators of neurotransmission. , iccs stain specifically with the tyrosine kinase receptor c-kit. immunohistochemical analysis for c-kit (cd ) and for cd (which reacts with many c-kit receptors) represents a relatively easy way to study severe constipation and intestinal pseudoobstruction. streutker and colleagues described completely absent or markedly reduced numbers of iccs in some cases of intestinal pseudo-obstruction. reduced volumes of iccs have also been described in some patients with slow-transit constipation. [ ] [ ] [ ] [ ] [ ] although the observations could be an epiphenomenon, they could form the basis of an alternate classification system for these cases. severe intestinal malabsorption for whatever reason (e.g., celiac sprue, cystic fibrosis) can be associated with dark brown or orange-brown discoloration of the bowel wall, , owing to deposits of a granular material that has the characteristics of lipofuscin in the smooth muscle of the muscularis externa and to a lesser degree the muscularis mucosae ( fig. - ). this excessive accumulation of lipofuscin is termed ceroidosis or the brown bowel syndrome. whether this pigment deposition adversely affects muscle function is debated; however, damage to smooth muscle mitochondria has been described, and several reports have linked ceroidosis to intestinal pseudo-obstruction. because of the name brown bowel syndrome, ceroidosis may be confused with melanosis coli (discussed in the next section). melanosis coli is a condition in which macrophages filled with lipofuscin-like pigment are found within the lamina propria or deeper in the wall of the colon (fig. - ) . , these macrophages may be of such numbers as to impart a brown or black color to the colonic mucosa ( fig. - ) . melanosis coli has been associated with increased apoptosis, which is often linked to ingestion of purgatives of the anthracene group (cascara sagrada, aloe, rhubarb, senna, frangula). [ ] [ ] [ ] one group of patients has severe and persistent constipation with no apparent cause (idiopathic constipation or morbid obstipation). these patients, usually women, can have as little as one bowel movement every to weeks, and many of these patients are so uncomfortable that they require colectomy for relief. [ ] [ ] [ ] [ ] because these patients almost invariably take laxatives, melanosis coli is usually found. what is so frustrating to the pathologist is that by h&e staining, the neuronal plexus and smooth muscle in these patients appear normal. distinctive abnormalities of the myenteric plexuses (e.g., loss of "argyrophilic" neurons) have been reported using special silver staining techniques. , , , these tests are best performed by pathology departments with expertise in this area. reduced volumes of iccs have been described in some patients with severe constipation. , cathartic colon is the name given to end-stage idiopathic constipation in which the bowel can no longer contract effectively. the mucosa has the gross appearance of snakeskin and histologically shows melanosis coli. the muscularis propria is thin and atrophic, and neurons are decreased in auerbach's plexus. hirschsprung's disease (aganglionic megacolon) has a predilection for boys and men. approximately % of patients present in infancy with constipation, abdominal distention, vomiting, and delay of meconium stool; diarrhea may occur and some patients may even be affected by lifethreatening enterocolitis. reports suggest that many cases of hirschsprung's disease have a genetic basis. both hirschsprung's disease and men are associated with mutations of the ret proto-oncogene. [ ] [ ] [ ] in cases of men , the mutations are activating and enhance the function of the coded protein, whereas in hirschsprung's disease the mutations are inactivating. several cases of familial hirschsprung's disease have been linked to mutations of the endothelin receptor-b (endrb) gene. at least nine additional genes have been implicated in the pathogenesis of hirschsprung's disease: ntn (seen only with ret mutations), gdnf, edn , ece- , sox , phox b, zfhx b, l cam, and dhcr. despite this progress, mutations of one or more of the known genes are detected in only half the patients with hirschsprung's disease. in the typical clinical picture of hirschsprung's disease, the anus is normal. the anal canal and rectum are usually small and devoid of stool. in classic cases, these physical findings are confirmed by barium enema study. the contrast material flows into an unexpanded distal segment, then passes through a cone-shaped area, and finally passes into the dilated proximal bowel (fig. - ) . the pathologic change is aganglionosis. the narrowed distal segment shows loss of ganglion cells in both the submucosa and myenteric plexuses, usually accompanied by hypertrophy of the muscularis mucosae and muscularis externa and increased numbers of nerve fibers in the submucosa and between the muscle layers of the muscularis externa. hypertrophied nerve fibers (> µm) derived from the extrinsic and sensory fibers are observed in many but not all cases of hirschsprung's disease. in the tapered or cone-shaped region, the number of ganglion cells may be decreased (hypoganglionosis). historically, histologic diagnosis was based on fullthickness rectal biopsy specimens. however, this procedure requires general anesthesia and risks the development of stricture and perforation. because the submucosal and myenteric plexuses stop at about the same level in hirschsprung's disease, , suction biopsy that samples the mucosa and submucosa is now considered the method of choice for the diagnosis. all rectal biopsy specimens for suspected hirschsprung's disease should be serially sectioned throughout the block and each section examined. , if no ganglion cells are found, then some comment should be made concerning the adequacy of the specimen. biopsy specimens devoid of ganglion cells, but in which the amount of submucosa is less than the thickness of the mucosa, should be considered insufficient to diagnose hirschsprung's disease. if the biopsy specimen contains epithelium of the anal canal, this specimen should also be considered inadequate, because the anal canal and distal cm of rectum are normally hypoganglionated or aganglionated. many pathologists prefer to examine frozen section slides stained for acetylcholinesterase in addition to standard h&e-stained sections. in hirschsprung's disease, examination using the acetylcholinesterase stain demonstrates increased acetylcholinesterase-positive nerve fibers in the lamina propria and muscularis mucosae. the utility of this technique as an adjunct to diagnosis is debated. false-positive and false-negative reactions have been reported, and the use of this method is a matter of personal preference. [ ] [ ] [ ] occasionally, ganglion cells may be difficult to identify using light microscopy alone, especially in the neonate. in such cases, a positive immunohistochemical reaction for neuron-specific enolase can be valuable in documenting ganglion cells. we have been successful in performing immunohistochemical analysis for neuron-specific enolase on sections after they have been stained with h&e and examined. other immunostains such as cathepsin d, pgp . (protein gene product . ), ret, bmpr a (bone morphogenetic protein receptor type a), and bcl- decorate ganglion cells. , frozen section is often used as an adjunct to visual inspection to select the site for colostomy. however, use of the frozen section to establish a primary diagnosis of hirschsprung's disease is best avoided because of the high rate of incorrect interpretations. in % of patients with hirschsprung's disease, the aganglionic segment of colon is less than cm in length. the remaining patients have longer aganglionic segments that may extend even into the small intestine. microscopically, the hypertrophied nerve trunks of short-segment hirschsprung's disease are absent, but increased numbers of acetylcholinesterase-positive mucosal nerve fibers are usually but not always seen. ultrashort-segment hirschsprung's disease (segments < cm) reportedly exists but is impossible for a pathologist to document by routine h&e stains of the rectal mucosa and submucosa alone because this segment of the colon is relatively hypoganglionated or aganglionated even in physiologically normal individuals. rectal manometry may be used in the diagnosis of this lesion. recognition of acetylcholinesterase nerve abnormalities similar to those seen in hirschsprung's disease may complement that study. some patients may have internal sphincter achalasia with abnormalities of nitric oxide-induced sphincter relaxation. regardless of pathogenesis, some patients benefit from sphincterotomy. hypoganglionosis is regularly observed in the cone-shaped transition zone between normal and aganglionic bowel in hirschsprung's disease. some authors believe that diffuse hypoganglionosis of the colon may give rise to megacolon similar to that observed in hirschsprung's disease. , no accepted definition of hypoganglionosis exists; however, guidelines were offered by meier-ruge. this study suggested that a decrease by a factor of in the number of ganglion cells per centimeter of bowel as compared with normal ( to myenteric plexus neurons/cm bowel) is diagnostic of hypoganglionosis. other investigators have accepted much higher numbers of ganglion cells as evidence of hypoganglionosis. in general, the condition has not been well characterized and many reports lack quantitation. diverse abnormalities have been described by special silver staining in cases that would have been called hypoganglionosis by h&e staining results, and some cases of "hypoganglionosis" may be similar to those cases reported as severe idiopathic constipation or cathartic colon. intestinal neuronal dysplasia is characterized by hyperplasia of the myenteric plexuses, increased acetylcholinesterase activity in nerves of the lamina propria and submucosa, and increased numbers of ganglion cells with the formation of giant ganglia. , [ ] [ ] [ ] these giant ganglia, typically containing more than to neurons (normal ganglia contain to ), make up only % to % of all ganglia seen in a given case and are usually not seen in the distal cm of the rectum. occasionally, ganglion cells may be found within the lamina propria but this feature should not be considered diagnostic for intestinal neuronal dysplasia because it can be seen in physiologically normal individuals. , the condition may give rise to signs and symptoms similar to those seen in hirschsprung's disease. intestinal neuronal dysplasia may occur in a localized or disseminated form. similar lesions sometimes referred to as ganglioneuromatosis can be observed in patients with von recklinghausen's disease or men b. , , although some clinicians diagnose intestinal neuronal dysplasia based on abnormal acetylcholinesterase staining in specimens containing ganglion cells, others believe that one cannot rely on acetylcholinesterase staining alone for the diagnosis. diagnostic criteria for intestinal neuronal dysplasia and even its very existence are challenged , because % of infants so diagnosed experience normalization of gut motility within year. therefore, many of the observed "abnormalities" could be within normal range and in general the diagnosis should be reserved for florid pathologic cases. in zonal aganglionosis or skip-segment hirschsprung's disease, ganglion cells are found distal to one or more aganglionic segments. [ ] [ ] [ ] evidence suggests that zonal aganglionosis is rare and is likely to be acquired by ischemia (e.g., necrotizing enterocolitis), viral infection, or some other (e.g., immunologic) injury. the problem is that a rectal biopsy specimen may yield ganglion cells in spite of an authentic hirschsprung's disease-like aganglionic lesion. immaturity of ganglion cells , and hypogenesis of myenteric plexuses , have been reported to cause signs and symptoms similar to those seen in hirschsprung's disease. immunostains for bcl- may be helpful in detecting immature ganglion cells. the gi tract is a common site for amyloid deposits; such deposition has been documented in % of primary amyloidosis cases and in half of secondary cases. , , occasionally the deposition is symptomatic and causes ischemic manifestations such as bleeding diarrhea or infarct, sometimes referred to as amyloid colitis. on rare occasions, the deposits can be localized and form a tumor. the aa (secondary) type of amyloid primarily deposits in the lamina propria and walls of blood vessels; the al (primary or myeloma-associated) type tends to accumulate preferentially in blood vessel walls and in the muscularis externa. , amyloid can be subtyped using immunohistochemical analysis. although subcutaneous biopsy or aspiration is an easy way to obtain tissue for diagnosis, rectal biopsy is still widely used to diagnose amyloidosis. , the term pneumatosis cystoides intestinalis (pci) describes the occurrence of gas-filled cysts within the bowel wall. the large intestine is a common site of involvement; benign and fulminant forms have been described. , the fulminant form of pci is most often seen in infants as a complication of ischemia. pci in this setting is caused by mural invasion by gas-forming bacteria and subsequent formation of cysts (see fig. - ) . the fulminant form can be seen in adults sometimes in association with drugs such as antineoplastic chemotherapeutic agents or in pseudomembranous colitis. histologically, one sees ischemic change, bacterial overgrowth, and gas cysts predominantly within the submucosa. it is easy to dismiss the gas cysts as cutting artifact because an endothelial or histiocytic lining is rarely seen in this form of pci. the benign form of pci is most often seen in adults and is usually asymptomatic, although diarrhea, constipation, and rectal bleeding have been documented. , benign pci is often seen with comorbid conditions that either increase intraluminal pressure or provide a breach in mucosal integrity by which the gas can enter the bowel wall. these conditions include chronic obstructive pulmonary disease, emphysema, diverticular disease, appendicitis, cholelithiasis, peptic ulcer disease, abdominal trauma, crohn's disease, and gi tract surgery. , at endoscopy, mucosal broadbased elevations that are sometimes semitranslucent have been described. resection specimens often show crepitance. microscopic analysis demonstrates occasional tears in the submucosal connective tissues, but more often one sees dilated spaces lined totally or partially by endothelium, inflammatory cells, histiocytes, and marked foreign body giant cell reaction (fig. - ). the principal differential diagnostic consideration is the entity referred to as pseudolipomatosis. , pseudolipomatosis resembles fatty infiltration of the lamina propria but ultrastructural study convincingly demonstrates that the spaces are, in fact, gas cysts ( fig. - ). pseudolipoma-tosis has been associated with air inflation used to distend the bowel during colonoscopy. some investigators have implicated cleaning agents used to disinfect colonoscopes. the term fibrosing colopathy has been applied to colonic strictures seen in some patients with cystic fibrosis. , the pathologic change is submucosal fibrosis, which can sometimes extend into the muscularis externa. fibrosing colopathy has been linked to administration of high-dose pancreatic replacement therapy. large bowel agenesis and atresia are extremely rare. , congenital atresia and stenosis are associated with failure to pass meconium, abdominal distention, and vomiting. these conditions are often seen on a background of other congenital anomalies. pathologically, one can see an imperforate septum, a portion of the colon replaced by fibrous cord, or the absence of a segment of colon and associated mesentery. colonic malrotation occurs with malrotation of the small bowel and is associated with abnormal anatomic relationships and fibrous bands; it may predispose patients to volvulus. , mispositioning of the cecum and appendix may lead to delayed diagnosis of acute appendicitis. congenital duplications and diverticula are located within the mesentery and often occur in combination with other congenital malformations. , sometimes, the duplications are tubular, represent doubling of the bowel, and run parallel to the colon and rectum. other duplications can become cystic and are often referred to as enterogenous cysts. patients may have associated spine abnormalities. we classify tubular duplications that communicate with the lumen at one end as congenital diverticula. small duplications and diverticula are usually asymptomatic. larger ones may cause mass lesions, abdominal pain, constipation, or bleeding. hindgut duplications may be associated with complex genital and urinary tract abnormalities. duplications and congenital diverticula usually have organized layers of smooth muscle sometimes with a nerve plexus within their walls. mucosal linings if present resemble colon, respiratory epithelium, or gastric mucosa. as lesions enlarge to become cystic, the lining and the wall can become atrophic. the retrorectal space is a relatively common location for developmental cysts that can become symptomatic in adults. [ ] [ ] [ ] epidermoid or dermoid cysts are unilocular, are lined by squamous epithelium, and may contain adnexal structures (dermoid cyst) and lack smooth muscle in the wall. rectal duplications can become cystic. rectal duplications are also unilocular, are lined by colonic, gastric, or respiratory epithelium, and have an organized muscular wall that recapitulates the muscularis externa. the retrorectal cystic hamartoma is often referred to as a tailgut cyst. this lesion manifests as a multilocular cystic and solid tumor ( fig. - ) . the variably sized cysts can be lined by squamous, transitional, or glandular epithelium. disorganized bundles of smooth muscle are found within the wall. inflammatory changes such as a foreign body giant cell reaction are quite common. developmental cysts in the retrorectal space are susceptible to infection and fistula, and associated malignancy has been reported. therefore, total excision is recommended. the histologic changes associated with acute chemotherapyor radiation-induced colitis are similar. necrosis, ulcer, and inflammation occur usually within weeks of the cessation of therapy. , , epithelium lining the colonic tubules often shows marked enlargement with large atypical nuclei and loss of intracellular mucin (fig. - ). apoptotic bodies similar to those seen with grade gvhd may be prominent. typically, the acute changes subside in to months. , the epithelial atypia seen as a result of acute irradiation and chemotherapy can be alarming and may mimic the appearance of glandular dysplasia and carcinoma. features favoring chemotherapy or radiation effect over dysplasia or carcinoma include ( ) overall preservation of mucosal architecture, ( ) bizarre atypia, ( ) maintenance of a relatively low nuclear-to-cytoplasmic size ratio despite cellular enlargement and nuclear atypia, ( ) a paucity of mitotic figures, ( ) recognition of similar atypia in nearby fibroblast and endothelial cells, and ( ) lack of an infiltration pattern in tumor desmoplasia. the taxanes can cause epithelial changes that can mimic high-grade glandular dysplasia similar to the changes seen with colchicine toxicity. the histologic changes include increased apoptosis, increased mitotic figures, and "ringed" mitotic figures that correlate with metaphase arrest (fig - ). the nuclear stratification and loss of polarity can mimic dysplasia. , late complications of radiation-induced change are better documented than are antineoplastic chemotherapy effects and may occur weeks to years after therapy. these late complications of radiation include chronic colitis, stricture, ulcers, and fistula. the histologic pattern resembles that of ischemic damage with mucosal atrophy and architectural change. mucosal and submucosal blood vessels can become ectatic. fibrosis occurs and often resembles hyalin. fibrosis can affect any bowel layer, including the muscularis externa. atypical radiation-type fibroblasts can persist for years. other vascular changes include intimal fibroplasia, accumulation of foamy macrophages in blood vessel walls, and luminal stenosis. epiploic appendices are pedunculated, serosa-covered accumulations of adipose tissue seen on the lateral aspects of the colon. they can become very large in obese individuals. the pedicles of epiploic appendices are thin and prone to torsion, a feature that can cause infarct and even amputation. infarcted epiploic appendices can appear as fine, graywhite nodules attached to the bowel or even can be found loose within the abdomen at surgery. these appearances can mimic metastatic carcinoma or foreign body. the subsequent biopsy specimen can be confusing to the pathologist who is asked to identify the frozen section. microscopically, these lesions show a central zone of infarct with mummification of the adipose tissue surrounded by a variable amount of fat necrosis and calcification. the outermost portion usually shows fibrosis. pacinian corpuscles occur in many areas of the body. 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different significance acute irradiation in proctitis in man: development of eosinophilic crypt abscesses gastric epithelial atypia associated with hepatic arterial infusion chemotherapy: its distinction from early gastric carcinoma gastrointestinal tract epithelial changes associated with taxanes: marker of drug toxicity versus effect colchicine toxicity: distinct morphologic findings in gastrointestinal biopsies pseudotumoral organization of a twisted epiploic fringe or "hard-boiled egg" in the peritoneal cavity enlarged intra-abdominal pacinian corpuscles simulating tumor implants intra-abdominal pacinian neuroma: a rare lesion in an unusual location intramural extravasation of barium simulating carcinoma of the rectum we wish to acknowledge and thank michele dover for her tenacity and help in transcribing this manuscript. we also thank drs. jeff kneile and amber petrolla for their contributions. key: cord- -xmcwuuk authors: papa, alfredo; gasbarrini, antonio; tursi, antonio title: epidemiology and the impact of therapies on the outcome of covid- in patients with inflammatory bowel disease date: - - journal: am j gastroenterol doi: . /ajg. sha: doc_id: cord_uid: xmcwuuk introduction: it has been hypothesized that people suffering from inflammatory bowel disease (ibd) have an increased risk of coronavirus disease (covid- ). however, it is not known whether immunosuppressive therapies exacerbate the covid- outcome. methods: we reviewed data on the prevalence and clinical outcomes of covid- in patients with ibd. results: covid- prevalence in patients with ibd was comparable with that in the general population. therapies using antitumor necrosis factor-α agents have been associated with better clinical outcomes. discussion: management and treatments provided by gastroenterologists were effective in reducing covid- risk. antitumor necrosis factor-α agents seem to mitigate the course of covid- . the coronavirus disease (covid- ) pandemic, caused by severe acute respiratory syndrome coronavirus (sars-cov- ), has aroused concern in healthcare teams for patients with immunemediated inflammatory diseases, such as inflammatory bowel disease (ibd). there are major questions that are essential for managing patients with ibd. first, does covid- occur more frequently in patients with ibd? second, do therapies for ibd influence clinical course of covid- ? preliminary studies did not report any covid- cases in patients with ibd ( , ) . however, subsequent studies including the surveillance epidemiology of coronavirus under research exclusion-ibd database, which includes global data of patients with ibd and covid- ( ), several cohort studies ( ) ( ) ( ) ( ) ( ) ( ) , and case reports ( ) ( ) ( ) , have reported , patients with ibd affected by covid- . pooled data suggest that covid- does not occur more frequently in patients with ibd than in the general population ( ) ( ) ( ) . indeed, gubatan et al. reported, among their patients with ibd from california, a prevalence of % comparable with . % in the general population ( ). taxonera et al. ( ) found an incidence rate of . covid- cases per patients with ibd in madrid, whereas in the general population, it was . cases per , . however, after adjusting for age, the incidence rate was . per patients with ibd with a lower standardized risk than that in the general population (odds ratio . , % confidence interval . - . ; p , . ) ( ) . finally, a combined italian and french study reported a cumulative incidence of . covid- cases per patients with ibd. this was comparable with that observed in the general population of . per , individuals ( ) . interestingly, clinical symptoms of covid- in patients with ibd seem to be milder, with a relatively lower frequency of serious and complicated cases than in the general population ( - ). allocca et al. ( ) did not report any covid- -associated deaths in their cohort of patients with ibd compared with a mortality rate of % in the general population. furthermore, they reported no significant difference in the standardized mortality ratio between patients with ibd and the general population (odds ratio . , % confidence interval: . - . ; p . ). the clinical and demographic variables associated with unfavorable covid- outcomes, such as old age, presence of comorbidities, and being male, were comparable between patients with ibd and the general population ( , ) . as shown by the study of the italian group for the study of inflammatory bowel disease, which included patients with ibd, aged older than years, had comorbidities, and active ibd were associated with worse covid- outcomes ( ). thus, adding ibd activity at the time of covid- diagnosis is a risk factor for a worse clinical outcome. the impact of ibd-therapeutic armamentarium on covid- incidence and outcome is currently a controversial topic. a retrospective study from the united states including a large cohort of patients with ibd reported that the use of antitumor necrosis factor (tnf)-a agents or thiopurines was not associated with an increased risk of developing covid- ( ) . in fact, the incidence rate of covid- per patients with ibd was of . in patients treated with anti-tnf-a and in those not in treatment with anti-tnf-a (p . ). the literature shows that the therapy's effect on covid- outcome varies across patients (table ). in the surveillance epidemiology of coronavirus under research exclusion-ibd database, we found evidence of greater prevalence of milder covid- cases in patients treated with anti-tnf-a than that in patients undergoing steroid treatments ( ) . as of may , , % of patients treated with anti-tnf-a agents required hospitalization and only a minority ( %) experienced unfavorable outcomes, defined as intensive care unit admission, ventilator use, or death ( ). conversely, % of patients taking oral or parenteral steroids needed hospitalization, with % experiencing unfavorable outcomes ( ) . further support to this theory comes from the results of the italian group for the study of inflammatory bowel disease study, which reported a % reduction in mortality among patients receiving anti-tnf-a antibodies (although not statistically significant); however, corticosteroid use was associated, with a trend toward statistical significance, with covid- -related pneumonia (p . ) and death (p . ) ( ). therefore, the data thus far strongly suggest that the use of anti-tnf-a antibodies as monotherapy is associated with better covid- outcomes than the use of steroids. the rationale for the beneficial effect of anti-tnf-a antibodies on covid- clinical course is closely linked to sars-cov- pathogenesis. the sars-cov- uses the functional receptor angiotensin-converting enzyme (ace ) for host cell entry. this causes increased production of tnf-a and tnf-a-converting enzyme-dependent shedding of the ectodomain of ace that further assists viral cell entry. wang et al. postulated that the use of anti-tnf-a antibodies could be effective in reducing both sars-cov viremia and the consequent organ damage ( ) , considering that ace is overexpressed in inflamed mucosa ( ) . furthermore, anti-tnf-a agents could achieve effective control of inflammatory mediators, which makeup the "cytokine storm" that occurs in severe covid- -related pneumonia, thereby mitigating the course of the disease. further evidence comes from the intentional use of mg/kg doses of infliximab, week apart, in a patient with severe ulcerative colitis and covid- -related pneumonia ( ) . the patient achieved a satisfactory recovery from intestinal and pulmonary disease without complications ( ) . on the other hand, steroid use should be avoided, if possible, or rapid steroid tapering should be considered owing to the risk of respiratory or opportunistic infection that could complicate the course of covid- . although significant uncertainty remains, the data accumulated thus far have demonstrated that the clinical course of covid- and its prevalence in patients with ibd are milder and lesser than those in the general population. the recommendations for reducing covid- risk and the ibd treatments provided by gastroenterologists could potentially explain the reason for this. anti-tnf-a agent use might provide double beneficial effects: first, to maintain ibd clinical remission and second, to mitigate the clinical course of covid- . however, the data we have reported have been obtained from retrospective studies that have not been designed to evaluate the effect of different therapies on covid- outcomes. therefore, we must exercise caution in three patients affected by covid- were under treatment with anti-tnf-a: one pt with adalimumab, one pt with golimumab plus methotrexate, and one pt with adalimumab plus methotrexate: the first patients were admitted to hospital. uneventful course in ibd patients during sars-cov- outbreak in northern italy prevention of covid- in patients with inflammatory bowel disease in wuhan, china secure-ibd database public data update outcomes of covid- in patients with ibd in italy: an ig-ibd study characteristics and prognosis of patients with ixnflammatory bowel disease during the sars-cov- pandemic in the basque country (spain), gastroenterology incidence and patterns of covid- among inflammatory bowel disease patients from the nancy and milan cohorts novel coronavirus disease (covid- ) in patients with inflammatory bowel diseases sars-cov- testing, prevalence, and predictors of covid- in patients with inflammatory bowel disease in northern california impact of anti-tnf-a and thiopurines medications on the development of covid- in patients with inflammatory bowel disease: a nationwide va cohort study a fatal case of covid- pneumonia occurring in a patient with severe acute ulcerative colitis covid- infection in crohn's disease under treatment with adalimumab case report of a sars-cov- infection in a patient with ulcerative colitis on tofacitinib up-regulation of il- and tnf-alpha induced by sars-coronavirus spike protein in murine macrophages via nf-kappab pathway expression of sars-cov- entry molecules ace and tmprss in the gut of patients with ibd key: cord- -qhlatg authors: verma, anukriti; sharda, shivani; rathi, bhawna; somvanshi, pallavi; pandey, bimlesh dhar title: elucidating potential molecular signatures through host-microbe interactions for reactive arthritis and inflammatory bowel disease using combinatorial approach date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: qhlatg reactive arthritis (rea), a rare seronegative inflammatory arthritis, lacks exquisite classification under rheumatic autoimmunity. rea is solely established using differential clinical diagnosis of the patient cohorts, where pathogenic triggers linked to enteric and urogenital microorganisms e.g. salmonella, shigella, yersinia, campylobacter, chlamydia have been reported. inflammatory bowel disease (ibd), an idiopathic enteric disorder co-evolved and attuned to present gut microbiome dysbiosis, can be correlated to the genesis of enteropathic arthropathies like rea. gut microbes symbolically modulate immune system homeostasis and are elementary for varied disease patterns in autoimmune disorders. the gut-microbiota axis structured on the core host-microbe interactions execute an imperative role in discerning the etiopathogenesis of rea and ibd. this study predicts the molecular signatures for rea with co-evolved ibd through the enveloped host-microbe interactions and microbe-microbe ‘interspecies communication’, using synonymous gene expression data for selective microbes. we have utilized a combinatorial approach that have concomitant in-silico work-pipeline and experimental validation to corroborate the findings. in-silico analysis involving text mining, metabolic network reconstruction, simulation, filtering, host-microbe interaction, docking and molecular mimicry studies results in robust drug target/s and biomarker/s for co-evolved ibd and rea. cross validation of the target/s or biomarker/s was done by targeted gene expression analysis following a non-probabilistic convenience sampling. studies were performed to substantiate the host-microbe disease network consisting of protein-marker-symptom/disease-pathway-drug associations resulting in possible identification of vital drug targets, biomarkers, pathways and inhibitors for ibd and rea. our study identified na((+))/h((+)) anti-porter (nhaa) and kynureninase (kynu) to be robust early and essential host-microbe interacting targets for ibd co-evolved rea. other vital host-microbe interacting genes, proteins, pathways and drugs include adenosine deaminase (ada), superoxide dismutase (sod ), catalase (cat), angiotensin i converting enzyme (ace), carbon metabolism (folate biosynthesis) and methotrexate. these can serve as potential prognostic/theranostic biomarkers and signatures that can be extrapolated to stratify rea and related autoimmunity patient cohorts for further pilot studies. www.nature.com/scientificreports/ approach has advantages over the traditional approach for network analysis that can help to simultaneously characterize several protein interaction modules and has the potential to study complex diseases. the vital information obtained in our study from in-silico analysis is cross-validated through targeted gene expression experimental analysis on patient cohorts. this study will help us to obtain clinico-molecular informatics-based outcomes and expand our knowledge regarding the understanding of biological functions for ibd co-existent rea. text mining: data screening and selection. systematic data search and organization was carried out incorporating data identification, data screening and data selection to find target microorganisms involved in inflammatory bowel disease (ibd) and reactive arthritis (rea). data identification was carried out to obtain records through data sources utilising keywords (e.g. "microorganism and inflammatory bowel disease and reactive arthritis") incorporating boolean operators (and/or/not). data screening and selection were carried as part of the manual curation through primary and secondary screening scrutinizing collected data records to obtain organized records relevant for the autoimmune and enteric disorders triggered by microorganisms, especially ibd and rea and the microbial triggers implicated in ibd and rea that were utilised for further metabolic network reconstruction. bottom-up approach consisting of draft reconstruction and manual reconstruction refinement was followed to create metabolic networks of obtained target microorganisms. genome-scale metabolic models simulation, reconstruction and visualization (gemsirv) software that includes reciprocal basic local alignment search tool (blast) of target microorganisms against a template metabolic network of its phylogenetic neighbour and incorporates information from national center for biotechnology information (ncbi), kyoto encyclopedia of genes and genomes (kegg) and transport db was used for creating draft reconstructs. the manual curation of missing links or gaps in the draft reconstruct was done by mapping the incomplete information to other databases such as expert protein analysis system (expasy) and integrated relational enzyme database (intenz) . this fully connected and annotated network was used for further simulation studies . the metabolic networks thus obtained were visualized using celldesigner, a tool for modelling and editing biochemical and gene-regulatory networks. simulation analysis was carried by converting the metabolic networks obtained into a mathematical model and performing the gene deletion analysis to retrieve essential genes. model conversion was through generation of stoichiometric based matrixes consisting of reactions (columns) and metabolites (rows) corresponding to respective genes. upper boundary and lower boundary fluxes i.e. movement of matter across a system were generated for the gene associated reactions and metabolites that was extracted in systems biology markup language (sbml) format. the next step was gene deletion analysis done using the constraint based reconstruction and analysis toolbox (cobra) that runs in matrix laboratory (matlab) for finding the essential genes based upon the gene-reaction matrix and boolean relationship between genes and reactions . the purpose of data filtering is to remove repeats and homologs from essential genes of target microorganisms associated with ibd co-existent rea. the non-homologous protein sequences corresponding to the essential genes of target microorganisms were extracted from pathosystems resource integration (patric) database . refinement of protein sequences was further done using cluster database at high identity with tolerance (cd-hit) suite so as to have % identity non-repeat sequence tolerance stringency. blast-p was further used to remove the homologs from such non-repeats against human database at e-value of - to obtain nonhomologous protein sequences used for further in-silico analysis. essential host-microbe and microbe-microbe interactions. the host-microbe interactions of the non-homologous proteins for the selected target microorganisms were obtained using host-pathogen interaction database (hpidb) , . the host-microbe interactions were visualised using cytoscape. simulation analysis (gene essentiality) was done to obtain the essential host proteins interacting with common microbe proteins of microorganisms triggering ibd and rea utilising the human metabolic model hmr , a cobra compliant metabolic model of human consisting of around , genes, , reactions and , metabolites . this led to profiling of the common host-microbe and microbe-microbe interactions comprehending the complex 'interspecies communication' as complex interaction maps, executed using search tool for the retrieval of interacting genes/proteins (string) , . host-microbe disease network and molecular mimicry studies. the host-microbe disease network is a multilayered archetype that connects the protein-marker-symptom/disease-drug-pathway associations. the contributions of the microorganisms in the co-evolved ibd and rea as part of the disease network was created through the interactive maps of the essential host interaction proteins (verified using literature survey) and the information processed through gene expression data analysis . the information patronised here is mostly scored through the available non-specific protein diagnostic markers of both ibd and rea e.g. c-reactive protein (crp), interleukin (il ) and toll like receptor (tlr ), major histocompatibility complex, class i, b (hla-b) and major histocompatibility complex, class ii, dr beta (hla-drb ) with the essential host proteins determined using string . database genecards was used to assess the role of these interacting partners aka proteins further with symptoms/diseases associated with ibd and rea. the pathways of the above host interacting proteins were found out using kegg database that provides ontologies for proteins related to biological processes www.nature.com/scientificreports/ subsequently, the role of drugs or inhibitors used to suppress the effect of ibd and rea such as indomethacin, prednisone, ciprofloxacin, sulfasalazine, azathioprine, methotrexate and hydroxychloroquine was scored in the disease network through their docking studies against the potential targets (both host as well microbial targets) as per published methodologies , . the host-microbe disease network which is an amalgamation of all the above patterned associations was visualized using cytoscape software . molecular mimicry analysis between the vital targets triggering ibd co-evolved rea, essential human proteins including hla-b , hla-b and hla-drb was done using data repository expasy. this led to retrieval of microbe relayed protein sequences that have been implicated in disease development after sequence alignment performed using emboss . experimental evidences to identify the signature molecules in patient samples. the cross-validation of vital in-silico targets was done in rea patient cohort cases via targeted gene expression analysis. scientific and ethical clearance was taken from amity university ethics committee and institutional ethics committee, fortis noida for handling the patient samples. all experiments were performed in accordance with indian council of medical research (icmr) guidelines constituting the ethics committees. the study was carried out for months on the rare disorder rea patients, with the inclusion criteria as patients having rea according to european spondyloarthropathy study group (essg) and exclusion criteria as patients undergoing treatment from last - months and healthy controls (hc). the participants were inducted in the study design with an informed consent form along with a questionnaire containing information regarding symptomatic and diagnostic history of patient and linked disorders. blood ( ml) was drawn from participants in ethylenediaminetetraacetic acid (edta) vacutainers. these were transported to the laboratory for further analysis. the processing of the samples was done within - h of procurement . peripheral blood mononuclear cells (pbmc's) were isolated from blood using density gradient centrifugation . rna was isolated from pbmc's using trizol method . the quantification of rna was done using nano-drop . the high capacity cdna reverse transcription kit (applied biosystems™) was used for conversion of rna to single-stranded cdna as per the standard protocol . quantitative pcr analysis of target gene was executed using biorad cfx real time-pcr taking human housekeeping gene, gapdh as a reference. previously reported primers for qpcr analysis of target and reference gene were selected for this study , following the standard protocol . relative gene expression analysis from qpcr data was performed using the relative expression software tool (rest® ) that utilises the expression of reference genes to normalize expression of target genes in different samples. the schematic representation of methodology involved in our combinatorial analysis is provided in fig. . text mining: data screening and selection. a systematic literature mining and curation for our thematic connecting autoimmune disorders, inflammatory bowel disease (ibd) and reactive arthritis (rea) was carried out. data identification extracted , records (articles in journals, book chapters, conference papers etc.) corresponding to autoimmune and enteric disorders. data screening extracted records of autoimmune and enteric disorders triggered by microorganisms that belong to class of bacteria, fungi, protozoan, mites, virus, yeast and nematode. data selection yielded ibd, rea and ibd co-evolved rea records. data selection was directed towards the microbial contenders implicated here resulting in target microorganisms namely campylobacter jejuni, escherichia coli o :h , klebsiella oxytoca, salmonella typhimurium, shigella dysenteriae and yersinia enterocolitica, whose genome information was available. the etiopathogenesis in the co-evolved disorders have been documented through gut microbiome associated host-pathogen interactions studies, perpetuating where pathogen microorganisms involve in dysbiosis leading to autoimmunity. the results of text mining are provided in fig. . the list of microorganisms is provided in supplementary table s online. ing of genes along with their corresponding proteins, reactions and metabolites for the selected microorganisms serve as primary set of partial metabolic network information. the missing data persistent in the draft reconstruct obtained through genome-scale metabolic models simulation, reconstruction and visualization (gem-sirv) was manually refined. entirely associated metabolic networks of target microorganisms were obtained (genes, proteins and reactions). the essential genes of microorganisms (vital for survival. sustenance and growth) were obtained after performing simulation on mathematical models consisting of gene associated reactions and metabolites (metabolites, inner cell reactions, exchange reactions and essential genes). due to lack of availability of exchange reactions for campylobacter jejuni, simulation analysis on the partial metabolic network could not be carried out and essential genes could not be retrieved. an alternative approach for finding essential genes of campylobacter jejuni was carried out. the essential genes of campylobacter jejuni were taken from our previous published report and were found out to be . table portrays the results of metabolic network reconstruction and simulation of target microorganisms. the metabolic network and simulation analysis data of target microorganisms is provided in supplementary table s online. the proteins corresponding to essential genes, non-repeats and non-homologs were obtained as stated below according to the parenthesis {proteins corresponding to essential genes, non-repeats, non-homologs}. the essential genes, their corresponding proteins, reactions and metabolites from the curated dataset were refined to create a list of most relevant molecular indicators to assess their coveted role in disease establishment. the non-redundant filtered proteins were utilised further in the computational work-pipeline canvassing the drug targets and signatures in the interspecies communication. essential host-microbe and microbe-microbe interactions. the central mechanism of hostmicrobe/microbe interface conferred through gut microbiome was correlated for the selected microbial species and processed to obtain the common signatures so as to follow the core system of metabolic changes affecting the host harbouring them as either commensal or pathogenic loads. the interactors between human and target microorganisms were obtained. the interactors of escherichia coli o :h were ; klebsiella oxytoca were ; salmonella typhimurium were ; shigella dysenteriae were and yersinia enterocolitica were . there were no interactors for campylobacter jejuni (supplementary table s -s online). table shows the results of filtering and host-microbe interactions of protein sequences corresponding to essential genes of target microorganisms. www.nature.com/scientificreports/ the host-microbe interactors were analysed for all the target microbial species and processed to obtain the common signatures. proteins were found between all target microorganisms having interaction among themselves and with human proteins. the essential host correlative targets to the microbial gene targets were followed by obtaining host essential genes and corresponding proteins from human metabolic model hmr . there were , essential proteins (supplementary table s online) the essential human protein was found out to be kynu having interaction with essential microbial protein nhaa (fig. ) . nhaa was also having interactions with non-essential hcls associated protein x- (hax ), prolyl endopeptidase-like (ppcel), biogenesis of lysosomal organelles complex subunit (hps ) and eukaryotic translation initiation factor alpha kinase (e ak ) proteins of human host. kynu was further mapped with host proteins (direct and indirect) resulting in interactions. out of these the single connected essential protein interactions were and protein interactors were ( fig. and see supplementary table s online). the research design here followed to assess the interaction map of essential proteins in human host to indicate the clinical insights in pathophysiological trends in the autoimmune development. host-microbe disease network and molecular mimicry. the human essential proteome complement with its interacting proteins were analysed further as part of the disease network. human essential protein interactors were found to be associated with ibd and similarly essential protein interactors namely adenosine supplementary table s online) . these proteins can be postulated as probable contenders transcending their role in the simulated network as important regulators in the co-existent disorders. the composite associations of the above proteins with non-specific protein diagnostic markers of ibd and rea were obtained (see supplementary table s online) . this gave rise to a single connected protein network consisting of proteins and , interactions. the association of above with symptoms and diseases linked with ibd and rea were obtained (see supplementary table s online) . apart from non-specific diagnostic markers, the major protein linked with majority of symptoms/diseases is angiotensin i converting enzyme (ace). pathways of the proteins were obtained (see supplementary table s online) in total out of which the pathway associated with majority of proteins was carbon metabolism. another layer of disease network substantiates the role of therapeutic regime followed in the studied autoimmune diseases, so the docking analysis of drugs used to suppress the effect of ibd and rea against nhaa of target microorganisms and kynu of human host was done. the docking analysis resulted in docking scores that represent binding of drugs with host kynu and microbial nhaa of all microorganisms selected in our study. higher the negative docking score more is the binding . escherichia coli o :h nhaa shows highest and lowest docking score with methotrexate (− . ) and azathioprine (− . ); klebsiella oxytoca nhaa with methotrexate (− . ) and azathioprine (− . ); salmonella typhimurium nhaa with ciprofloxacin (− . ) and hydroxychloroquine (− . ); shigella dysenteriae nhaa with methotrexate (− . ) and azathioprine (− . ); yersinia enterocolitica nhaa with hydroxychloroquine (− . ) and azathioprine (− . ) and human kynu with hydroxychloroquine (− . ) and indomethacin ( . ). our results portray methotrexate to have highest docking scores with maximum proteins and therefore can be considered as a vital drug for ibd associated rea. the resultant docking scores are provided in fig. . the extensive interaction pattern of nhaa with kynu along with proteins, markers, symptoms/ diseases, pathways and drugs give rise to a host-microbe disease network of ibd co-existent rea (fig. and see supplementary table s online) . the final league of information processed in this study design was to accommodate the concept of molecular mimicry between the essential host proteins and selected microorganisms. nhaa protein of target microorganisms shows homology with human hla-b , hla-b and hla-drb (fig. ) . peptides homologous to hla-b : peptides homologous to hla-drb : experimental evidences to identify the signature molecules in patients. the in-silico analysis followed for the molecular signature identification till far through gene expression datasets and curated metabolic reconstructs strongly indicate the host protein, kynu being the singular common predictive markers for all pathogenic microbes. kynu has also been indicated in the expression data of inflammatory linked disorder, www.nature.com/scientificreports/ ibd. there is lack of data available regarding kynu differential expression in rea, therefore the experimental evaluation of kynu through targeted expression analysis in rea patients was carried out. a non-probabilistic convenience sampling was followed for our single blind study. this study encompassed individuals: % male with mean age of . and % female with mean age of ( males and females). out of these cases were: with rea and controls were: currently undergoing treatment, with poncet's disease (pd) and healthy control (hc). the clinical characteristics of the patients recruited in the study included inflammatory back pain in %, fatigue in %, fever in %, swollen joint in %, ankylosing spondylitis (as) that affects spine in %, dactylitis that is inflammation in finger or toe in % and poncet's disease (pd) in % of participants. the clinical characteristics of the recruits are provided in table . the expression of kynu in peripheral blood mononuclear cells (pbmc's) of rea cases vs controls was evaluated using relative expression software tool (rest) software that estimated a sample's relative expression ratio in relation to the control housekeeping gene (here gapdh) by calculating an intermediate absolute concentration value: where cp = point at which fluorescence escalates considerably above the background fluorescence. here the cp values for reference and target genes are collectively redistributed to control and sample groups and the expression ratios are calculated based on the mean value. a pair wise fixed reallocation randomisation test is followed for normalisation of the target genes with a reference gene and for calculating the statistical difference of variation between groups . it utilises a bootstrapping technique providing a % confidence interval for expression ratios. it uses a p(h ) test for testing the significance between the samples and controls. according to our analysis, kynu sample group is different to control group where p(h ) = . . kynu was found to be downregulated in sample group (in comparison to control group) by a mean factor of . (standard error range is . - . ) as depicted in the whisker-box plot (fig. ) . kynu expression showed a ~ ninefold decline in rea cases as compared to controls. gut microbiome is pitched to be the central theme housing enormous diversity of microbial species, characterizing the fine balance between healthy and diseased states. the physiological drifts from healthy to diseased and vice-versa is tuned to sophisticated interactive networks of human host and the microbial flora residing the gut. the autoimmune conditions reactive arthritis (rea) and inflammatory bowel disease (ibd) have been linked to prevalent dysbiosis of the gut, where disease development occurs as a perceptive reaction due invading population of microbes. to find out the basal networks of interactions at the host-microbe interface, common microbes affecting the co-evolved diseases with shared characteristics were studied. these involved comprehensive analysis of the bimolecular functional networks including the gene, protein, metabolite molecular signatures engraved at the host-microbe and microbe-microbe interface. this 'interspecies communication' have been linked now with immuno-pathogenesis of most human autoimmune disorders , . www.nature.com/scientificreports/ the etiopathology of these interactions have remained elusive leading to non-specific diagnostic criteria and therapeutic regimes. it is suggested that microbial dysbiosis, pathogenic infection and host-microbe interactions cause incidence of rea. in this study, utilising the combinatorial approach we have compiled a repertoire of microorganisms, biomolecules and pathways that are possibly involved in triggering co-evolved autoimmune disorders ibd and rea. in our study, text mining results convey the presence of microorganisms namely campylobacter jejuni, escherichia coli o :h , klebsiella oxytoca, salmonella typhimurium, shigella dysenteriae and yersinia enterocolitica implicated in both the disorders. the thematic concepts for microbe contribution in host immunity have been explored in our previous analysis of metabolic reconstruction and simulation of campylobacter jejuni and salmonella enterica , . in our current study, we used a designated work-pipeline for metabolic network reconstruction and simulation of target microorganisms. the analysis conducted extracted the information via constraint-based bottom-up approach that was filtered and utilised for further computational analysis. the essential genes, proteins and metabolites of microorganisms represent the promising drug targets as these are speculated to contribute towards infection triggered host physiological drifts leading to development of the co-evolved pattern of autoimmunity in ibd and rea. a thorough curation pattern followed led to provide robust molecular cues in terms of essential proteins and biological networks that are correlated to the 'interspecies communication' using the host-microbe and microbemicrobe interaction profiling. the most closely associated common protein observed in all the selected common microbial species involved in both ibd and rea is na (+) /h (+) antiporter (nhaa), microbial integral membrane protein, catalyzing the exchange of h (+) per na (+) and involved in processes crucial for cell viability. similarly, the common host interacting protein with nhaa is kynureninase (kynu), involved in tryptophan metabolism and whose differential expression (upregulation and downregulation based on the control samples) have been followed in ibd patient cohorts [ ] [ ] [ ] . as per the scientific discourse presented in the studied disorders, the pathological mechanism hypothesizes that after bacterial infection, antigen-presenting cells transport bacterial antigens/peptides into the synovial membrane, where the bacterial components persist causing inflammation. it is suggested that in host-microbe interactions, bacterial proteins entering host cells interact with host proteins and inject their effector components, but has not been proven in rea and ibd. so, this formed a basis of one of the parameters in our study design where we found the physical interactions between nhaa and kynu and predicted that these might be the early host-microbe interactors for establishing pathogenesis in ibd associated rea. this could assist to comprehend the very few reports indicated in the rare autoimmune rea, where gene expression datasets of the co-evolved disorder ibd can serve to incorporate the larger theme of gut-microbiome associations. the theme of gut-microbiome paradigm shifts thus contemplates the vital cues in triggering autoimmunity with indirect linkages to diet and environmental triggers. this is indicative of the identified target molecular signature, kynu, found to be differentially regulated in the patient cohorts with history of infection triggered or ibd co-evolved rea. kynu and nhaa could serve as the robust early and essential host-microbe interacting targets and molecular indicators involved in interspecies communication in ibd associated rea. the investigations further were targeted for parallel analysis of other host-essential protein partners enmeshed to have interaction with host protein kynu indicating the intricate details of host-microbe interaction information. the disease network constructed through our approach consists of single connected essential protein interactors of kynu, where human essential protein interactors are found to be associated with ibd, while of them (adenosine deaminase (ada), catalase (cat) and superoxide dismutase (sod )) are associated with both ibd and rea. ada protein has been reported in juvenile idiopathic arthritis and rea patient cohorts in serum samples . similarly, cat and manganese superoxide dismutase (sod) genes polymorphisms were observed in rea patient cohorts , . these become part of the host-microbe disease network where such molecular elements and co-regulatory pathways represent the intricate biological cross-talk followed during disease development. pathological conditions can also trigger immune cells such as il's and tlr's and various cytokines leading to immune cell infiltration in host and higher levels of inflammation. genetic factors such as hla alleles encode susceptibility, contribute to bacterial persistence and increase risk in rea cases. based on this we also found the interactions of important targets in our study with immunogenic and genetic factors. the host harboured assorted essential proteins were further probed for their association with non-specific protein diagnostic markers as well as with symptoms/diseases linked with ibd and rea, accruing towards a single connected network consisting of interdependent proteins. the reciprocation of these integrated protein indicators to the disease development is conveyed through metabolite monitoring as in the study, angiotensin i converting enzyme (ace) was found to be linked with maximum symptoms/diseases. ace is involved in catalyzing the conversion of angiotensin i into angiotensin ii that is a potent vasopressor and aldosterone-stimulating peptide that controls blood pressure and fluid-electrolyte balance . this could be the indicator of involvement of microbe triggered host physiological drifts. subsequently, the pathways associated with the proteins ramified into pathways of human host speculated to give details of metabolic regulatory checkpoints where carbon metabolism is found to be associated with majority of deduced proteins. carbon metabolism pathway implicated here as the vitally generic pathway for ibd co-related rea confers how diet, balance of gut microbiome, antibiotic exposures can have layered impact on autoimmune disease progression and remissions. kynu is found to be downregulated in rea patients as compared to controls through our targeted gene expression analysis. collectively, the disease network followed here confers interaction of microbial nhaa with host kynu, that is further correlated to proteins, markers, symptoms/diseases, pathways and drugs. docking analysis of drugs used to suppress the effect of ibd and rea predicts methotrexate as an important drug that could be useful for early treatment of ibd co-evolved rea. www.nature.com/scientificreports/ genetic factors found common in both rea and ibd are hla-b , hla-b and hla-drb . the most important mechanism of susceptibility of hla in rea is molecular mimicry that is microbial peptides mimicking hla autopeptides of human host leading to autoimmunity. this mechanism has been observed in rea where reports have predicted microorganism peptides such as chlamydial proteins (clpc, nqra and dnap) and yersinia pseudotuberculosis peptides (yoph) showing homology with human hla-b via bioinformatic analysis . similarly, molecular mimicry has also been observed in ibd cases having extraintestinal manifestations. we performed targeted molecular mimicry analysis in our study using our robust microbial protein (nhaa) with hla-b , hla-b and hla-drb , enhancing the importance of nhaa acting as a trigger for generating ibd associated rea. we generate a putative hypothesis amalgamating key findings with literature. we state that the initial hostmicrobe triggers for ibd associated rea is when pathogenic microbial protein nhaa interacts with host protein kynu that further interacts with human proteins ada, sod , cat and ace and carbon metabolism involving the above host proteins is hampered. methotrexate regulates carbon metabolism and the associated host-microbe proteins reducing effect of ibd associated rea. since carbon metabolism is the most basic aspect of life and therefore an extensive network consisting of sub-pathways, we narrowed down our findings towards a consequentially central and a significant pathway that embrace the carbon metabolism pathway involving the molecular signatures kynu, ada, sod , cat and ace, further is also effectuated by potential drug methotrexate and is associated with ibd/ rea/ ibd and rea cohorts. it is reported that methotrexate is incorporated intracellularly interfering with adenosine concentrations and affecting proinflammatory cytokines in ibd reducing inflammation . in inflammatory arthritis, the mechanisms reported by which methotrexate reduces inflammation include enhanced adenosine release, de novo synthesis of purines and pyrimidines, inhibition of transmethylation reactions, diminished accumulation of polyamines and nitric oxide synthase uncoupling. most of the mechanisms are associated with folate biosynthesis, a type of carbon metabolism . kynu, ada, sod , cat and ace are also found to be involved in folate biosynthesis and metabolism from genecards. apart from the above targets, parallel interactors, pathways and drugs for ibd co-evolved rea obtained in our host-microbe disease network can be utilised further as disease determinants. the experimental validation of these targets in patient cohorts need to be performed on a pilot scale in future to increase the robustness of this network. the intertwined information processed through the knowledge-base created for the linked disorders have given the most elaborate layout of patterns observed in disease diagnosis and analysis. the major information after processing the gene expression profiles, protein markers, molecular networks and metabolic networks involved here have led to chalk out as well as connect the strings for robust gut microbiome paradigm shifts. the current work on host-microbe interactions provides a starting point for researchers and clinicians to investigate inflammatory bowel disease (ibd) associated reactive arthritis (rea). in this study a combinatorial approach is utilised to reveal the interactions of gut microbes with human host extensively sketched through the work-pipeline providing the vital insights for the drug targets, biomarkers, pathways and inhibitors for etiology, prognosis, diagnosis and treatment attributes of pathogenic rheumatic autoimmunity. 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advanced studies, new delhi for providing the facility and technical support during the preparation of the manuscript. we also thank fortis hospital, noida for providing the patient samples. s.s. and b.r. conceived the study concept; s.s., b.r. and p.s. jointly designed and supervised the work; b.d.p. supervised the clinical setting and recruitment of participants; b.d.p. and a.v. recruited the participants and contributed to the sample collection and preparation; a.v. performed the experiments; s.s., b.r., p.s. and a.v. contributed to the analysis and interpretation of data; a.v. generated all figures and tables; a.v. wrote the first draft of the manuscript; s.s., b.r., p.s. and b.d.p. critically reviewed and edited the manuscript; all authors reviewed and approved the final version of the manuscript. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/ . /s - - - .correspondence and requests for materials should be addressed to s.s.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -r usk authors: nan title: research communications of the th ecvim‐ca congress date: - - journal: j vet intern med doi: . /jvim. sha: doc_id: cord_uid: r usk nan saccharomyces boulardii (sb)is a non-pathogenic yeast used in the prevention and treatment of gastrointestinal disorders in human beings and horses. the aim of this study was to evaluate the effect of sb in healthy dogs and dogs with chronic enteropathies (ce). sb was formulated in x cfu capsules. its concentration and viability within the capsules was controlled by yeast culture in subsequent steps until expiration date. four healthy dogs (hd) and dogs with ce ( inflammatory bowel disease -ibd, protein losing enteropathy -ple) were included. in hd sb was administered for days ( x cfu/kg bid); daily clinical evaluation was performed to assess possible adverse effects and quantitative stool cultures for yeasts were performed before, during and after the administration. in dogs with ce a randomized double blind placebo-control study was performed, administering sb ( x cfu/kg bid) or placebo (pl). sb or pl administration was added to standard therapeutic protocols (diet, antibiotics and immunosuppressive drugs), to evaluate its efficacy for the treatment of ibd and ple. complete blood work, abdominal ultrasonography, gastro-duodenal and colon endoscopy and histopathological evaluation of intestinal samples were performed at diagnosis and after days of treatment. validated score system for the clinical signs (ceccai), ultrasonography, endoscopy and histopathology were applied. significance was set for p < . . results in hd showed the absence of sb in the faeces before treatment, its presence after one day, its steady state ( x cfu/g) after days and its complete elimination days after withdrawal of treatment. no adverse effects were reported. in ce dogs the clinical score improved significantly in dogs receiving sb compared to dogs receiving pl (p = . ). in ple dogs the albumin concentration increased significantly (p = . ) in the group receiving sb with respect to pl. the daily frequency of defecation in the sb group was significantly lower with respect to pl after (p = . ) and (p = . ) days of treatment. no statistical differences were found between dogs receiving sb and pl after treatment, based on the endoscopic evaluation of duodenum and colon. no statistical differences were found between the two groups on the duodenal ultrasonographic and histological evaluation after treatment. in conclusion, sb can be safely used in dogs with ce, in addition to standard treatment, to achieve a better control of clinical signs and a significant increase in albumin concentration compared to the standard therapy alone. no conflicts of interest reported. canine inflammatory bowel disease (ibd) is an immune-mediated enteropathy likely triggered by environmental and immunoregulatory factors in genetically susceptible dogs. previous studies suggest a pivotal role for gut bacteria in disease pathogenesis since luminal microbial composition is markedly altered (ie, dysbiosis) at diagnosis. probiotic bacteria appear to be therapeutically effective in some forms of human ibd. controlled studies evaluating the efficacy of probiotic therapy for canine ibd have not been previously reported. the aim of the present study was to characterize the mucosa-associated microbiota and determine the clinical, microbiological, and mucosal homeostatic effects of orally administered vsl# probiotics in dogs with ibd. twenty dogs diagnosed with moderate-to-severe ibd (cibdai score > ) were randomized to receive standard therapy (ie, elimination diet and glucocorticoids) with or without probiotic vsl# . the mucosal microbiota from endoscopic intestinal biopsies of ibd dogs and controls was evaluated by fluorescence in situ hybridization (fish) targeting the s rrna genes of total bacteria, group-specific organisms, and individual bacterial species shown to be relevant in human ibd. epithelial tight junction protein (tjp) expression was studied using immunohistochemistry. clinical signs and changes in mucosal microbiota and tjp expression were assessed before and after probiotic vsl# therapy. ibd dogs showed a reduction in gi signs following weeks of probiotic therapy compared with baseline cibdai scores (p < . ). adherent and sporadic invasive bacteria (eub) were observed in the small intestines and colon of healthy dogs. the diseased canine duodenum was nearly bacteria-free. ibd dogs given probiotic vsl # had altered spatial redistribution of most bacterial groups in the mucus and adherent compartments of the colon. subset analysis showed that lactobacilli were significantly (p < . ) increased in the lumen and mucus post-vsl # , while the number of mucus laden bifidobacteria approached significance (p = . ). expression of tjp showed that occludin was significantly lower in control intestines as compared to duodenal and colonic mucosa obtained from ibd dogs that received probiotic (p = . and p = . , respectively). in contrast, claudin- expression in the colon was significantly higher (p < . ) in control dogs versus vsl # treated ibd dogs. our data demonstrate that probiotic vsl# alters some of the mucosa-associated microbiota in dogs with ibd. these probiotic changes in bacterial composition are associated with up-regulated tjp expression indicative of enhanced epithelial barrier integrity, similar to vsl# -induced disease protection seen in human ibd. the probiotics used in the trial were supplied free of charge by the manufacturer. canine inflammatory bowel disease (ibd) is thought to be partially caused by an aberrant immune response towards the intestinal microbiome. in humans and mice, administration of probiotics can alleviate ibd severity and/or prevent relapse by induction of a more "tolerant"microenvironment. the aim of this study was to investigate the effect of probiotic enterococcus faecium ncimb e (ef) on intestinal microbiome composition. dogs were recruited to receive ef at x e cfu in a double-blinded, placebo-controlled manner in addition to an exclusion diet (hydrolysed protein). seven dogs were included in the probiotics group and dogs in the placebo group. all dogs improved clinically after treatment, however, there was no obvious effect on clinical severity in those that received probiotics. fresh naturally voided faecal samples were collected from all dogs before and after treatment, snap-frozen in liquid nitrogen and stored at - °c until further analysis. genomic dna was extracted from each faecal sample using the mobio power soil dna isolation kit (mobio laboratories), as recommended by the manufacturer. next generation sequencing was performed on the ion-torrent[trademark] (life technologies) platform based upon the v -v region (e. coli position - ) of the s rrna gene with the following primers: forward f: gag-tttgatcntggctcag and reverse r: gtnttacn gcggckgctg. raw sequence data were screened, trimmed, filtered, and chimera depleted with default settings using the qiime pipeline version . and uchime software, in which microbiome composition between treatment groups before and after treatment was compared. microbiota composition was not significantly different between probiotic and placebo treatment groups, and did not change significantly before and after treatment. however, there was large individual variability in the microbiome composition. species richness of faecal samples increased after treatment in both groups, but was only statistically significant in the probiotic treatment group. in conclusion, probiotic treatment in dogs with ibd leads to a significantly increased richness of the faecal bacterial microbiome. a possible additional effect of the change of diet cannot be excluded. further studies should investigate microbiomic changes in healthy dogs fed the same diet to assess if similar changes in the fecal microbiome occur due to dietary changes alone. this study is based on a phd supported by probiotics ltd., somerset, uk (the manufacturer of the probiotic product enterococcus faecium mentioned in this study). the aim of this study was to assess the prevalence and risk factors for faecal carriage of extended-spectrum beta-lactamases (esbl) and plasmidic ampc beta-lactamases (pampc) e. coliproducers in healthy dogs. a -month cross-sectional study was conducted at a private hospital in lisbon, portugal and rectal swabs were obtained from healthy dogs. the dogs included in the study were healthy with no history of antimicrobial consumption in the previous month. esbl and pampc genes were detected by pcr and were sequenced. potential risk factors for esbl-and pampc-producing e. coli faecal carriage were obtained through a questionnaire to the owner regarding reason for veterinary visit, hospitalisation and antimicrobial treatment within the last year, habitat (shelter, dog breeders and private owner), cohabitation with other animals, street access, kennel/hotel access, age and gender. data were analysed by sas software (version . ; sas institute inc., cary, n.c.) and logistic regression models were used. rectal swabs obtained from healthy dogs yielded positive samples for e. coli. about % of the isolates carried esbl genes (bla ctx-m- n = , bla ctx-m- n = , bla ctx-m- n = , bla ctx-m- -like n = ) and % carried pampc genes (bla cmy- n = , bla cmy- -like n = , bla dha- n = ). thirteen dogs carried an e. coli isolate with both an esbl and a pampc gene. dogs previously treated with antimicrobials within the last year were at higher risk of carrying at least one ß-lactamase (p = . ; or = . ; ci %: . - . ) or both ß-lactamases (p = . ; or = . ; ci %: . - . ) than non-treated dogs. dogs in shelters/breeders tended to show a higher incidence of esbl-producing e. coli (p = . ; or = . ; ci %: . - . ) or at least one ß-lactamase producing e. coli (p = . ; or = . ; ci %: . - . ) than dogs from private owners. males tended to be less likely to carry at least one ß-lactamase (esbl or pampc) (p = . ; or = . ; ci %: . - . ) or a pampc enzyme (p = . ; or = . ; ci %: . - . ) than females. this study suggests that dogs may act as reservoirs for resistant bacteria, namely for cephalosporin-resistant e. coli. three potential risk factors associated with the carriage of esbl-and/or pampc-producing e. coli by dogs were identified, which is important for the implementation of effective control measures and judicious antimicrobial therapy. conflicts of interest: dr pomba currently receives research funding from the government and national programmes (fundac ßão para a ciência e a tecnologia). in the past, she has occasionally received research support or honoraria for lectures from pharmaceutical companies including zoetis and atral cipan. she is vice-chair of the antimicrobial working party there are few reports in the literature reporting long-term relapse rate, owner compliance and clinical severity of dogs with chronic enteropathies. the goal of this study was to compare clinical activity index (ccecai), number of relapses and compliance rates - years after diagnosis. food-responsive disease (frd) was defined as dogs that responded to elimination diet alone within weeks after initiating therapy, whereas antibiotic-responsive disease (ard) dogs had an unsuccessful dietary trial before and responded to metronidazole within weeks after initiation of therapy, and steroidresponsive disease (srd)dogs had an unsuccessful dietary and antimicrobial trial before, and required immunosuppressive therapy to control their clinical signs. ccecai was extracted from the medical record database at - years after diagnosis. relapse rate was obtained by requesting the medical records of the referring veterinarians and defined as number of return visits to the referring practice after diagnosis. compliance data was obtained by telephone questionnaire to the owners. the frd group consisted of / dogs ( %), whereas the ard and srd groups consisted of ( %) and dogs ( %), respectively. there was a significant difference in ccecai at follow-up between frd and ard, and frd and srd (median ccecai . (range - ) for frd, (range - ) for ard, and . (range - ) for srd, p = . ). for the frd dogs, % of owners stated that they deviated from the prescription diet on a daily basis, % once a week, and % once a month, with a median ccecai at the time of deviation from the diet of . (range - ). relapse rate was highest for the ard group, when compared to frd and srd ( for ard, . for frd, and . for srd, p = . ). in the frd group, / dogs had been kept on the prescribed diets, and dogs had been changed to supermarket brands. all of the ard dogs had been given immunosuppressive treatment in addition to antibiotics at the time of follow-up, while / srd dogs were still on immunosuppressive treatments, with one dog being in remission with dietary treatment alone. in conclusion, this pilot study indicates that compliance rate for frd dogs is the lowest, with owners willing to tolerate the highest severity of clinical signs related to deviation from the prescription diet. ard dogs had the highest relapse rate in this cohort, indicating poor response to treatment in the long-term. conflicts of interest: dr allenspach has received research funding from bbsrc, american kennel club, comparative gastroenterology society, probiotics ltd uk, laboklin gmbh germany, and bioiberica sp. she has also undertaken paid consultancy work for bioiberica spain and hoffmann-laroche, switzerland. despite the high prevalence of canine pancreatitis in postmortem studies and the introduction of new diagnostic tests, it is believed that the disease, particularly in its chronic form, remains under recognised due to the non-specific nature of presenting signs. histology is considered to be the gold standard for diagnosis of canine pancreatitis, however, most clinicians are reluctant to take pancreatic biopsies due to significant risks to the patient. numerous serum markers have been reported to be elevated in canine pancreatitis, although most lack sensitivity or specificity. consequently, confirmed diagnosis requires results from a range of tests including imaging, serum biochemistry and physical examination. we and others have previously shown in other diseases that performance of individual low specificity markers can be dramatically improved by combining data from multiple markers with clinical information using analytical algorithms. we therefore applied this approach to the detection of pancreatitis in dogs. the activity of two non-specific biomarkers, amylase and lipase, was determined in serum samples from dogs suspected of having pancreatitis by their veterinarian. of these samples were positive by virtue of their pancreatic lipase (cpli) results (cpli > ug/l). the amylase and lipase data was then used to develop a series of algorithms using mathematical data mining and classification techniques. additional algorithms were developed using extra parameters including age, sex, vomiting, diarrhoea and abdominal pain in addition to the two enzyme levels. the performance of the algorithms was assessed using separate blinded serum samples taken from dogs which were scored clinically for acute pancreatitis according to the system described by mccord et al (j vet intern med ; : - ) . these cases presented for evaluation with vomiting, diarrhoea, inappetance and abdominal pain and were included if a clinical history, results of routine haematology, biochemistry, cpli assay and abdominal ultrasound were available. the results of the multifactorial analysis and cpli assay results were compared to the clinical scores. using amylase and lipase data alone, the algorithm gave a sensitivity of . % and specificity of . %, compared to cpli results for the same samples of . % and . % respectively when both methods were referred to clinical scoring. when the presence of additional clinic data was also included into the algorithm, the sensitivity increased to . % with specificity of %. the data suggests that test performance for canine pancreatitis can be dramatically improved when multiple diagnostic parameters are combined using disease specific algorithms. the author receives a salary as editor of the bsava journal companion, and has undertaken unrelated paid consultancies for bayer and merial. the author also receives a salary from avacta animal health, and duties involved working directly on this project. canine chronic enteropathy (cce) can cause significant long-term morbidity. in some cases this is due to intestinal inflammation, resulting from idiopathic inflammatory bowel disease (ibd). currently, the diagnosis of idiopathic ibd and assessment of disease severity relies on results of subjective clinical indices, laboratory data, diagnostic imaging and intestinal histopathology, whilst ruling out known causes of inflammation. in humans with ibd, a number of faecal biomarkers including lactoferrin, aid with diagnosis and determining disease activity. it may therefore be valuable to develop similar non-invasive objective methods to aid diagnosis and clinical assessment of disease severity in dogs with intestinal inflammation due to idiopathic ibd. this pilot study aimed to measure faecal lactoferrin concentration (flc) in dogs with cce and histologically confirmed intestinal inflammation (hcii) and to compare this with control dogs. in addition, the flc in dogs with hcii would be compared with the canine inflammatory bowel disease activity index (cibdai) and wsava standard histopathological criteria for intestinal inflammation to determine whether there was correlation between these methods when assessing disease severity. faecal samples were obtained from dogs with hcii (n = ) having undergone investigation for cce (serum biochemistry, complete blood count, full faecal and urinalysis, serum cobalamin, quantitative cpli, abdominal ultrasound and intestinal biopsies). the control population were dogs presented for reasons unrelated to cce (n = ). analysis was carried out using a faecal lactoferrin elisa previously validated in dogs (techlab, usa). the flc in dogs with hcii (median . lg/g -range . to . ) was significantly higher than control dogs (median . lg/g -range . - . ) (p < . ). a cut-off flc of . lg/g correctly identified / ( %) of dogs with hcii. using this cut-off, there was no overlap between non-cce dogs flc and the hcii group; giving a sensitivity of % and specificity of %. neither the presence of neutrophils nor the extent of inflammation on histopathology showed significant correlation with flc. the cibdai showed moderate correlation with flc in dogs with hcii (r = . , p = . ). the results of this pilot study suggest that flc is able to discriminate between dogs with cce due to hcii and dogs without cce. it is possible that incorporating flc into a panel of faecal biomarkers will enable non-invasive assessment of hcii and could serve as an adjunct to current measures of disease severity in dogs with idiopathic ibd. ryan bettencourt and james boone are employees of techlab, usa. they provided the elisa kits free of charge for this work. there was no other incentive provided and the results have been openly discussed between all parties. there has been no censorship placed on the results by tech-lab and they have been supportive of the work and submission of this abstract. there are no other conflicts to disclose. fecal s a and fecal calprotectin concentrations have been described as biomarkers in dogs with chronic enteropathies [ ]. however, to date there has been no direct comparison of these two markers in dogs with chronic diarrhea. the aim of this study was to evaluate the performance of these two markers in this situation. thirty one dogs presented for a history of chronic diarrhea were prospectively enrolled. the initial diagnostic workup for all patients included a serum biochemistry profile, fecal parasitology, abdominal ultrasound examination, and gastrointestinal endoscopy with collection of endoscopic biopsies. the severity of clinical signs was evaluated using the ccecai scoring index and patients were grouped by having a ccecai of < or ≥ . fecal calprotectin and s a were quantified as previously described [ ] . correlations were evaluated with the spearman rank correlation test. for both markers a receiver operating characteristics (roc) curve was used to select cut-off value that allowed the best discrimination between dogs with a ccecai< and dogs with a ccecai ≥ . sensitivity and specificity were calculated. correlation analyses revealed a significant positive correlation between s a and calprotectin (r = . ; p < . ). the optimal cut-off value for fecal calprotectin concentration was . lg/g, which was associated with a sensitivity of . % and a specificity of . % (auc= . ; p = . ). the optimal cutoff value for fecal s a concentration was . ng/g, which was associated with a sensitivity of . % and a specificity of % (auc= . ; p = . ). the sensitivity for fecal s a was higher than that for fecal calprotectin (p = . ). no significant difference was observed for the specificity of these two markers (p = . ). out of the dogs ( %) had concordant results for s a and calprotectin tests. among these dogs, presented with a ccecai < and of these dogs had both markers below their cut-off values. among the dogs with a ccecai≥ , dogs had both markers above their cutoff values. % of dogs ( / ) presented histologic signs of inflammation. sensitivities for fecal calprotectin and s a concentrations for histopathological intestinal inflammation were % and %, respectively, and specificities were % and %, respectively. at least in this group of patients fecal s a concentration was more sensitive (but less specific) to detect dogs with a cce-cai ≥ or histopathologic intestinal inflammation than fecal calprotectin concentration. weight loss and malabsorption of fat, protein, cobalamin and tocopherol in the face of normal exocrine pancreatic function have been reported in up to - % of cats older than years of age fed a variety of nutritionally balanced dry and wet foods (patil ap and cupp cj. proc. nestle-purina compan anim nutr summit, focus on gastroenterology, [ ] [ ] [ ] [ ] [ ] [ ] [ ] ) . the objectives of this study were to determine if serum cobalamin concentrations increased after oral administration of a cobalamin supplement to affected cats, and the duration of any positive response following cessation of supplementation. the study evaluated cats older than years of age with fat malabsorption demonstrated by either increased fecal fat (> %) or subnormal fat digestibility (< %), but without exocrine pancreatic insufficiency (epi) as assessed by assay of serum trypsin-like immunoreactivity (ftli). a commercially available solution of cobalamin containing mg mixed with ml of a liquid flavor enhancer was added to the food of each cat in a single dose each day for months, after which supplementation ceased. serum cobalamin (assayed by competitive binding assay performed through the gi laboratory at texas a&m university and evaluated using reference ranges derived by that laboratory) was determined immediately prior to initiation of supplementation, then weekly for weeks, then monthly for months. at the start of the study serum cobalamin was subnormal (< ng/l) in of the cats (range < to ng/l) and within the reference range ( to ng/l) in the remaining cats ( to ng/l). serum cobalamin was above the reference range in every cat ( to ng/l) after one week of supplementation and remained above the reference range in every sample collected during the supplementation period, with the exception of two cats with values within the reference range when supplementation was stopped. serum cobalamin , and months after cessation of supplementation ranged from < to , < to , and < to ng/l, respectively. at the end of the study serum cobalamin was subnormal in of the cats. it is concluded oral cobalamin supplementation can effectively increase serum cobalamin concentrations in geriatric cats with idiopathic chronic enteropathy, but that following cessation of supplementation concentrations decrease rapidly and can become subnormal again within as few as weeks. the primary author collaborated with nestle-purinaon the work reported in this abstract, and a co-authoris an employee of nestle-purina. the primary author has previously received funding from iams, mars, hills and nestle-purina. the author also acts as a paid consultant for the gi-lab, texas a&m university. left atrial measurements are crucial in assessing severity of cardiac disease in dogs with myxomatous mitral valve disease (mmvd). however, linear and area dimensions might not provide a comprehensive assessment of patient status, and cannot differentiate between severe subclinical (b ) and clinical disease (chf). estimates of left atrial function could provide additional information to help categorize these patients. we examined dogs with mmvd ( normal, b , b and c) presented for cardiac evaluations by d echocardiography. left atrial linear and area dimensions in right parasternal short and long axis views were obtained at time points -early diastole (la max ), just prior to mitral valve opening, at the onset of atrial systole (la p ) and just prior to mitral valve closure (la min ). we calculated indices of la function: total la emptying fraction (la tef ), active la emptying fraction (la act ), passive la emptying fraction (la pas ) and la reservoir function (la res ) for all sets of measurements. we examined the differences in selected la function indices between different disease stages with a kruskal wallis test with post-hoc multiple comparisons. we also examined the diagnostic accuracy of selected indices of la function in differentiating dogs in stage b and stage c (chf) using roc analysis. three functional indices consistently differed across the various stages of mmvd -la tef , la act and la res . these differences were most apparent in the rpla view for linear measurements and rpsa view for area measurements. dogs with chf had worse function than all other groups, which differed variably depending on the functional index being examined. laarea act showed the best ability to discriminate between b and chf dogs, with a % specificity, % sensitivity and an auc of . , but this was no better than use of la:ao measurements. our data suggest that la function differs between dogs with differing severities of mmvd, but does not provide a clear distinction between dogs with subclinical disease and chf. no conflicts of interest reported. materials and methods: de was used to evaluate consecutive, non-sedated dogs that weighed more than kg. the study population for the morphologic study included normal dogs, and dogs with acvim stages b or b -c mmvd. de image data were digitally recorded and then analyzed offline, using commercially available software. results: de image acquisition was feasible in / ( . %) consecutive dogs. patient anxiety ( ), arrhythmias ( ) and panting ( ) explained failure to obtain a de dataset. fortyone of ( . %) datasets were of analyzable quality. body weight and heart rate were significantly lower in dogs for which it was possible to perform de. dogs with analyzable de datasets were significantly older and weighed less than dogs in which de could not be analyzed. the mitral valve of normal dogs is saddle shaped (annulus height to commissural width ratio (ahcwr): . ae . [mean ae sd]) and has an elliptical annulus (sphericity index (si): . ae . ). the following measurements were significantly related to body surface area (bsa): antero-posterior diameter (apd) (r = . , p < . ), anterolateral-posteromedial diameter (alpmd) (r = . , p < . ), annulus area (aa) (r = . , p < . ), anterior leaflet length (all) (r = . , p < . ), anterior leaflet area (ala) (r = . , p < . ). these variables were indexed (i) to bsa for subsequent statistical analyses. dogs with mmvd had a significantly greater si, non-planar angle, apdi, alpmdi, alai and alli, while having a significantly lower posterior leaflet area (pla), posterior leaflet length (pll), annulus height (ah), tenting height (th), tenting volume (tv), tenting area (ta), and ahcwr compared to normal dogs. ah, tv and ta were significantly greater in normal dogs, compared to dogs with mmvd. si, apdi, al-pmdi, aai, alai and alli were significantly greater in dogs with stages b -c mmvd, compared to normal dogs and those in stage b . pll and pla were significantly lower in b -c dogs, compared to normal dogs. th was significantly different between the three groups; greatest in normal dogs and lowest in dogs in stages b -c, suggesting that flattening of the mv occurs with disease progression. conclusions: de assessment of the canine mv is feasible. morphologic changes associated with mmvd progression are presented. effective regurgitant orifice area (eroa), calculated from a dimensional measurement of the width of vena contracta (vc) as the narrowest portion of the proximal regurgitant jet, might be used to estimate severity of mitral regurgitation (mr). however, this simplified assumption only holds when the eroa is circular, which might not be true in dogs with myxomatous mitral valve disease (mmvd). the aim of the study was to compare measured eroa using color doppler real-time dimensional echocardiography (rt d) with calculated eroa estimated by dimensional echocardiography ( d) in chamber ( ch) and chamber ( ch) views of the left ventricle (lv) in dogs with mmvd. ninety-three privately owned dogs of breeds diagnosed with naturally acquired mmvd were examined using d and rt d. according to the acvim classification of congestive heart failure (chf), dogs were classified with chf ( in class c and in class c ) and dogs without chf ( dogs in class b and dogs in class b ). age ranged from to years (median years), and body weight ranged from . to kg (median kg). fifty-nine males ( %) and females ( %) were included, and heart rate ranged from to beats/minute (median b/min). eroa was calculated from d measurements of vc diameter, in the ch view only (assuming a circular regurgitant orifice), and from measurements of vc diameter in both ch and ch views (assuming an eliptical regurgitant orifice) of lv. bland-altman plots were used to compare eroa measured by rt d with calculated eroa obtained from d ch and ch/ ch lv views. none of the d estimations of eroa showed good agreement with the measured rt d eroa when corrected for bsa, and the difference between methods increased with increasing eroa. the difference between rt d and d methods normalized to the mean eroa value did not increase with increasing eroa, but showed a systematic underestimation of eroa by % ( ch) and % ( ch/ ch), respectively, compared to rt d. the beat-to-beat variation of eroa assessed by rt d (n = ) had a coefficient of variation ranging from . % to % (median %). in conclusion, substituting assessment of eroa with a measurement of vc in or dimensions might underestimate the mr severity in dogs with mmvd. in some dogs, the beat-tobeat variation of the eroa was large, thereby necessitating the need for several consecutive measurements. no conflicts of interest reported. micrornas (mirna) are short ( - nucleotides), singlestranded, non-coding rnas that specifically anneal with complementary sequences in multiple mrna targets, and they silence mrnas and suppress downstream protein translation. a mirna can act as a fine-tuner of gene expression or an on/off switch. these features highlight the potential of mir-nas as therapeutic targets. the role of mirnas in myocardial fibrosis and hypertrophic cardiomyopathy has been widely studied in human patients. however, there is no data available for canine and human myxomatous mitral valve disease (mmvd). the aim of this study was to investigate mirna transcriptomics in canine mmvd by using global transcriptional profiling, mirna target prediction software (diana tool, targetscan . ) and network analysis software (biolayout express d ). four myxomatous mitral valves (ckcs) and controls valves were profiled using the affymetrix canine gene . st array. in total out of mirnas were found to be statistically significantly differentially expressed (down-regulated) based on the false discovery rate, p-value, and fold-change. expression of three mirna (cfa-mir- b, cfa-mir- c, cfa-mir- ) were also validated by quantitative polymerase chain reaction (q-pcr, taqman), and the results were in agreement with the microarray findings. for network analysis and visualization, markov clustering algorithms were conducted in bio-layout express d , and major clusters of mirnas were exported and uploaded to the diana-mirpath (kegg pathway) web-server. the pathways identified in the main cluster were attributed to the biological functions of focal adhesion, cytoskeleton (actin) regulation, tgf-b signalling, glycosaminoglycan biosynthesis, osteoclast differentiation, notch signalling and vegf signalling. the most significantly down-regulated mirna in mmvd was cfa-mir- , which is an endothelial specific mirna shown to regulate endothelial migration and vessel patterning. the top predicted target of cfa-mir- is glucuronic acid epimerase (glce) which is the main enzyme controlling heparan sulphate biosynthesis. other interesting findings were down-regulation of cfa-mir- and members of the cfa-mir- family. cfa-mir- targets multiple extracellular matrix transcripts, such as collagens, elastin, integrin, laminin, mmp (matrix metalloproteinase) and adamts (a disintegrin and metalloproteinase with thrombospondin motifs), whereas cfa-mir- targets hyaluronic acid synthase (has ). since the major pathology of mmvd is aberrant turnover of extracellular matrix proteins, this may be linked to mir-na regulation. dysregulation of valve mirnas might be potential therapeutic targets in the treatment of canine mmvd. no conflicts of interest reported. mitral regurgitation (mr) progresses slowly, but dogs living long enough often develop congestive heart failure (chf). however, tools to predict onset of chf are sparse. echocardiographic examinations in dogs were performed in a longitudinal, multicenter study with a surveillance time of up to . years. client-owned dogswere enrolled at the university hospitals in finland, sweden and denmark (subset to the svep study). left ventricular end diastolic (lvidd) and systolic (lvids) diameters, fractional shortening (fs), left atrial (la) and aortic root (ao) diameters were estimated. values were normalized for body size (nlvidd, nlvids, and nla, respectively) and, for comparison, ratios to aortic root were calculated (lvidd/ao, lvids/ao and la/ao, respectively). a cox's proportional hazard analysis with a counting process approach was used. spline smoothed graphical models were constructed to evaluate linearity of hazards. curves were then used to find cut-off values for interval hazard ratios (hrs). the hr for nlvidd, nlvids and nla (per . unit, % confidence intervals), were . ( . - . , p = . ), . ( . - . , p = . ), and . ( . - . , p = . ), respectively. the hrs for lvidd/ao, lvids/ao and la/ao ( . unit increase) were . ( . - . , p = . ), . ( . - . , p = . ), and . ( . - . , p = . ), respectively. the hr for fs was . ( . - . , p = . ). the relative hazard plot presented a steep increase for fs values above %. hrs for intervals < %, < %, and ≥ % were . ( . - . , p = . ), . ( . - . , p = . ), and . ( . - . , p = . ), respectively. the hr for nlvidd increased linearly. hrs for intervals . < . , . < . , . < . and ≥ . were . ( . - . , p = . ), . ( . - . , p = . ), . ( . - . , p = . ), and . ( . - . , p = . ), respectively. in contrast, the hazard for nlvids remained stable until . , whereafter it increased. the hrs for nlvids ( < . , . < . , . < . and ≥ . ) were . ( . - . , p = . ), . ( . - . , p = . ), ( . - , p = . ), and . ( . - , p = . ), respectively. hrs for values normalized to ao diameter behaved in a parallel way. we conclude that fs, left ventricular and atrial size may be used to predict chf. however, because the value of a hr is dependent on the unit used and, more essentially, does not account for nonlinear change in hazard, interpretation of hazards is challenging. in contrast, interval hazards are only dependent on the reference interval used. therefore they are easier to implement in every day clinical work. no conflicts of interest reported. systemic arterial hypertension is not frequently recognized in dogs with mitral valve degeneration (mvd), although borderline hypertension is difficult to assess, mainly because of different measurement techniques, inter-operator variability and, most importantly, examination-related stress. the object of this study was to evaluate systolic arterial blood pressure (sbp) at initial presentation and at regular intervals in dogs with various clinical stages of mvd. fifty six dogs with mvd that had not received any heart medication prior to admission, were included in the study. based on the isachc staging system, were assigned to class i (group a), to class ii (group b) and to class iii. small-breed dogs and miniature poodles, in particular, were overrepresented. comorbidities that could affect sbp were ruled out prior to enrollment. sbp was measured using a commercially available veterinary oscillometric device, by applying the proper cuff on the cephalic artery. dogs were left to acclimate for - minutes and measurements were always taken by the same investigator, before any other examination was performed, with the dog sitting on the owner's lap. a total of readings were taken, outlier values were discarded and the mean of the remaining measurements was documented. after initial consultation, treatment was customized according to the clinical stage. sbp was then measured every months, up to months after initial admission. at presentation, all class i dogs had sbp > mm hg, with only / having spb ≥ mm hg, whereas all class ii dogs had sbp < mm hg. of class iii dogs, had sbp > mm hg, and had sbp ≥ mm hg. a linear mixed effects model was used to assess the temporal variability of the measured parameters between groups. groups were matched for gender, age and body weight. blood pressure measurements, for the duration of the study, were higher in group a dogs, compared to groups b and c (p < , ). at the same time, group c had significantly higher sbp values than group b dogs (p < , ). asymptomatic mvd dogs seem to have higher sbp measurements, compared to those with clinical evidence of heart failure. whether this difference is stress-related, a maladaptive mechanism of sympathetic and raas activation to mvd or idiopathic remains to be elucidated. no conflicts of interest reported. sarcoplasmic reticulum (sr) ca + -atpase and its regulatory proteins are pivotal determinants of myocardial active relaxation via calcium uptake against the sr-cytoplasmic gradient. the lowered density of the sr ca + -atpase has been well demonstrated in many species during chronic hemodynamic overload. the genes linked to sr calcium uptake were reported not only being expressed in peripheral blood but serving as potential cardiac biomarkers in dogs with chronic mitral regurgitation, such as sr ca + adenosine triphosphatase isoform a (ser-ca a), phospholamban (pln), and hs- associated protein x- (hax- ). the aim of this study is to determine whether the target genes expressed in the blood will be translatable to the myocardial setting as cardiac biomarkers. the mrna expression levels of the target genes (serca a, pln, hax- ) from biopsied left ventricle (lv) and peripheral white blood cells (pwbc) in surgical mitral valve repair cases were estimated with quantitative real-time pcr using comparative ct method with gapdh. the gene expression levels in lv and pwbc were compared and their clinical relationships were evaluated. the diagnostic power of the genetic expressions in pwbc was analyzed by comparing to those of normal dogs. the levels of all target genes expressed in lv and pwbc were highly correlated each other in linear regression analysis (p < . ; serca a, r = . , r = . ; pln, r = . , r = . ; hax- , r = . , r = . ), although lv and pwbc showed different expression levels in a paired comparison (p < . ). according to the severity of the heart failure (isachc), the expression levels of all genes were gradually and significantly reduced in both lv and pwbc (p < . ). especially, the serca a and pln expressed in pwbc could clearly discriminate all isachc groups from the control (p < . ). multivariate regression adjusted by age and body weight revealed that serca a and pln in lv were negatively associated with lv internal systolic dimension (p = . , adjusted r = . and p = . , adjusted r = . , respectively). pln was also negatively related with lv internal diastolic dimension (p = . , adjusted r = . ). additionally, receiver-operating characteristic analysis using pwbc showed high area under the curve (auc) values for all target genes on overall isachc groups (p < . ; serca a, auc= . ; pln, auc= . ; hax- , auc= . ). in conclusion, the transcriptional changes of the calcium uptake related genes in pwbc may be able to reflect myocardial hemodynamic stress as well as to be utilized as promising cardiac biomarkers. no conflicts of interest reported. the aim of this study was to estimate heart-rate normalized pulmonary transit times (nptt) in cardiomyopathic cats with or without congestive heart failure (chf), to assess potential associations of echocardiographic variables and nptt, and to evaluate nptt as a test for presence of chf. privately owned cats were included. nptt was measured using echocardiography and the ultrasound contrast media sonovue â in groups of cats: healthy cats (group ), cats with cardiomyopathy (cm) but without chf (group ), and cats with cm and chf (group ). receiver operating characteristic curves (roc) were created for nptt, left atrial diameter (lad) and the left atrial to aortic root ratio (la:ao) to assess and compare their usefulness as tests for presence of chf. interrelations between pulmonary blood volume (pbv), nptt, stroke volume (sv) and echocardiographic variables were investigated by means of uni-and multivariate analysis. nptt values in group , group and group were . (interquartile range (iqr) . - . ), . (iqr . - . ), and . (iqr . - . ), respectively. values were significantly different between all groups. pulmonary blood volumes in group , group , and group were . ml (iqr . ml- . ml), . ml (iqr . ml- . ml) and . ml (iqr . ml- . ml). sv, pbv and shortening fraction < % were significant predictors of nptt. nptt and la:ao ratio, not sv were the main predictors of pbv. analyzing roc for nptt as a clinical test for chf yielded an auc of . which was similar for la:ao ratio. nptt may be useful test for the presence of chf in cats with cm and as a measure of cardiac performance. nptt and la: ao ratios predict chf with equal accuracy. increased pbv is significantly associated with higher nptt and la:ao ratios. both decreased sv and increased pbv explain the increased nptt in cardiomyopathic cats. the author received a travel scholarship from zoetis to attend this congress. acute arterial thromboembolism (aate) occurs commonly in cats, and less frequently in dogs, mostly resulting in limb paresis or paralysis. diagnosis is based typically on physical examination and advanced imaging. diminished affected-limb peripheral blood flow induces changes in several analytes concentrations in affected limb venous samples, compared to their peripheral venous concentration. we hypothesized that in aate, local, affected-limb venous glucose concentration decreases below reference interval, while systemic glucose concentration remains unaffected. the study included groups for each species: paralytic aate cases, non-ambulatory controls with limb paralysis of orthopedic or neurologic disorders, and ambulatory controls diagnosed with various diseases. systemic and peripheral, affected-limb blood glucose concentrations were measured. group absolute (dglu) and relative (% dglu) differences were compared. no procedure-associated complications or pain were noted. peripheral blood glucose concentrations were decreased (p ≤ . ) only in cats and dogs with aate. dglu and %dglu were higher in the aate groups in both cats and dogs compared to their respective control groups (p < . , p < . , respectively), with no differences between the control groups. receiver operator characteristics analysis of dglu and %dglu as predictors of aate in cats had areas under the curve of . and . , respectively, and . and . , in dogs, respectively. dglu cutoffs of mg/dl and mg/dl, in cats and dogs, respectively, corresponded to sensitivity and specificity of % and % in cats, respectively, and % in dogs. dglu and %dglu are extremely accurate, readily-available, simple diagnostic markers of aate in cats and dogs. no conflicts of interest reported. glycaemia determination is usually included in routine biochemisty panels. no works are devoted to the evaluation of pheripheral glycaemia in animals suffering from arterial thrombosis. the aim of this study was to document the pheripheral glycaemia variations in hypoperfused limbs of patients affected by mriconfirmed arterial thrombosis. eleven dogs referred for monoparesis or paraparesis were recruited. inclusion criteria were a clinical examination supportive of limb hypoperfusion and availability of blood cell count, biochemical profile and urine analyses. before mri examination, peripheral glycaemia was tested. two blood samples were obtained, one from the affected limbs and one from a healty limb.plasmatic glycaemia was measured using an automated glucose analyser. all the patients underwent a total body mri (mri intera . t, philips medical systems) that provided the final diagnosis. the arterial thrombosis location was documented and the entity was scored. all the eleven patients were diagnosed with a peripheral thrombosis involving an arterial vessel and in some cases the relative branches. the thrombus was located: in the abdominal aorta ( / ), in the subclavian artery ( / ), in the axillary artery ( / ), in the iliac arteries ( / ). of the total amount of abdominal aortic thrombosis, / involved also the internal iliac arteries, / the external ones and / both internal and external. the extent of the thrombosis was classified as grade (g ), when the greatest portion of the thrombus did not reach half of the vessel lumen ( / patients); grade (g ), when the greatest portion of the thrombus was between / and / of the vessel lumen ( / ); grade (g ), when the thrombus exceded / of the lumen ( / ). a substantial decrease in pheripheral glycaemia values was found in sampling arising from the thrombosisaffected limbs. comparing thrombosis-affected limbs values with healthy limbs measurements from the same patient, the reduction was found from . % to . %. accounting only the g scored patients, the percentage of reduction was found up to the . % suggesting a proportional decrease related to the grade of occlusion. results from this study suggest that peripheral glycaemia values are affected by limb hypoperfusion disorders. if an arterial thrombosis is suspected, samples from the affected limbs and the healthy ones could be used to compare glycaemia values and to support the early stage therapy in anticipation of diagnostic imaging. further studies are needed to confirm the proportional relation of the decrease with thrombus entity. no conflicts of interest reported. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease mainly affecting west highland white terriers (whwt). pulmonary hypertension (ph) may develop secondary to hypoxic vasoconstriction and/or pulmonary parenchymal infiltration. in the absence of measurable tricuspid regurgitation (tr), this co-morbid condition may be difficult to diagnose non-invasively. the degree of cardio-pulmonary impairment in cipf dogs can be evaluated through blood gas analysis (bga) and minute walking test ( mwt). a new echocardiographic index, the right pulmonary vein to pulmonary artery ratio (pv/pa) has been described for the detection of pulmonary venous hypertension. the aim of this study was to investigate pv/pa in cipf in order to determine its utility in the detection of ph and in the assessment of cardio-pulmonary disease severity. this prospective clinical cohort study included whwt with cipf (group a), healthy whwt (group b) and healthy dogs from other breeds (group c). diameters of right pv and pa were measured, in bi-dimensional (bd) and m-modes (mm), in a parasternal right long axis view, at the end of the t wave. other echocardiographic parameters for evaluation of ph were also measured: speed of tr, acceleration time to ejection time ratio of the pulmonary flow (at:et) and pulmonary artery to aorta ratio (pa/ao). bga was performed in dogs ( , and in groups a, b and c) and mwt in dogs ( , and ). values are given as meanaesd. in bd and mm mode, the pv/pa ratio was lower in group a (mm: . ae . , bd: . ae . ) compared to group b (mm: . ae . , bd: . ae . , p ≤ . ) and group c (mm: . ae . , bd: . ae . , p ≤ . ). the changes in pv/pa were both due to an increase of pa (p ≤ . ) and a decrease of pv (p ≤ . ). tr was found in % of dogs with cipf; mean pressure gradient was . ae . mmhg. at:et was lower in group a ( . ae . ) compared to group c ( . ae . , p = . ) and tended to be lower compared to group b ( . ae . , p = . ). pa/ao was not statistically different between groups. pv/pa was correlated with arterial po values (b mode: r = . , p = . ) and results of the mwt (b mode: r = . , p = . ). pv/pa was also correlated with at:et and the speed of tr, but not with pa/ao. in conclusion, in whwt affected by cipf, pv/pa is a useful indicator of ph and could serve in the assessment of disease severity. no conflicts of interest reported. ventricular septal defect (vsd) is the fourth most common congenital cardiac defect in dogs and the most common in cats. the aim of this study was to evaluate the long-term outcome in vsd patients. case records of animals were reviewed, of these re-evaluated echocardiographically and followed up by phone interview only. out of dogs pug was the most common breed ( %) followed by border terrier ( %). out of cats domestic short hair was most common ( %) followed by main coon ( %). isolated vsds were present in dogs and cats. complex defects (cds) were present in cases, most frequent anomalies being sub-aortic stenosis ( dogs, cat), pulmonic stenosis ( dogs, cats), tetralogy of fallot ( dogs, cats), cushion defects ( cats) and double-chambered right ventricle (dcrv) ( dogs, cats; in / dogs not present initially supporting the cause-and-effect theory). eisenmenger was observed in dog and cats. aortic insufficiency, not considered a cd, was noted in dogs. in dogs and cats ( % of isolated vsds and cat with a dcrv) the defect closed spontaneously. nine dogs and cats ( %) died of non-cardiac causes with an age at death of to (mean . ) months; dogs and cats died due to cardiac causes with an age at death of . to (mean . ) months. cardiac deaths were sudden ( dogs with cds) or euthanasia for left sided congestive heart (chf) failure associated with cds ( dogs, cat); right sided chf associated with cds ( cats); biventricular failure ( cat with cd); weakness (eisenmenger, dog and cat; fallot dog; cd cat). two cats developed chf due to unrelated hcm. only one dog with an isolated vsd was euthanized for chf. these results indicate that spontaneous vsd closure occurs more often than previously thought, most patients with isolated restrictive vsds live a normal life span without surgical intervention, but non-restrictive vsds or complex defects can be associated with significant morbidity and mortality. echocardiography early in life is crucial to identify the anomaly and cds, as well as useful to prognosticate long-term outcome and to identify patients where a surgical intervention should be considered if available. follow-up echocardiography is indicated to corroborate the prognosis, to detect complications due to the vsd and to detect unassociated acquired cardiac diseases. no conflicts of interest reported. centronuclear myopathy (cnm) is the most prevalent congenital inherited disorder affecting skeletal muscles in labrador retrievers. this disabling condition segregates worldwide and a recessive loss-of-function founder mutation was identified in the protein tyrosine phosphatase-like, member a gene (ptpla/ hacd ). the objectives of this study were ) to describe ptpla expression pattern in hearts from homozygous wild type (wt), heterozygous (het) and homozygous mutated (cnm) dogs, ) to assess and compare the left myocardial function in aging wt, het and cnm dogs. for this purpose, seven wt, four het and eleven cnm dogs were included in the study. ptpla mrna levels were assessed by rt-pcr and rt-qpcr. all dogs were examined using conventional echocardiography, d color tissue doppler imaging (tdi) and tdi-derived strain imaging. we found that the expression of the two wild type ptpla splice isoforms increased post-natally in wt dogs. their levels were halved in het dogs and drastically reduced in cnm dogs. in both het and cnm dogs, a slight left ventricular hypertrophy was detected using conventional echocardiography. tdi and strain imaging revealed that the left ventricular myocardial function was significantly altered in both het and cnm dogs compared to wt dogs. moreover, these functional defects were associated with significantly higher values of systemic arterial blood pressure, although maintained within normal ranges. in conclusion, subclinical myocardial alterations were detected in both het and cnm aging dogs from our french pedigree, suggesting a role for ptpla in long-term cardiovascular homeostasis. these findings prompt globalized confirmation in additional ptpla"'deficient dogs, which may thus be considered as a new large-size model for human left ventricular sub-clinical myocardial dysfunction. no conflicts of interest reported. mitral regurgitation (mr) secondary to degenerative mitral valve disease (dmvd) is the most common heart disease in dogs. in dogs with mr, mitral valve prolapse caused by degeneration of the mitral valve leaflet, chordae tendinae extension and/or rupture and mitral annulus dilation are observed. however, limited data are available on morphological changes in dogs with mr. currently, there are no studies confirming the anomaly of the mitral complex via direct observation in living dogs with mitral regurgitation. at our institution over the last ten years, approximately dogs have undergone mitral valve repair. during surgery, the anomaly of mitral complex can be observed macroscopically (directly visualized). to our knowledge, this is the first study evaluating the anomaly of the mitral valve leaflet and chordae tendineae in dogs undergoing mitral valve repair. animals: dogs that underwent mitral valve repair with cpb at nihon university between february and june were included in this study. methods: confirmation of chordae tendineae rupture was visually confirmed during surgery. the sites of chordae tendineae rupture were also recorded at that time. septal chordae and mural chordae were divided three division depend on the site (s , s , s and m , m , m respectively). results: ninety eight dogs were included in this study. the mean age and body weight were . ae . years and . ae . kg, respectively. of the dogs, ruptured chordae was observed in dogs ( . %). septal leaflet chordae was ruptured in dogs ( . %) and mural leaflet chordae was ruptured in dogs ( . %). chordae of both leaflets were ruptured in dogs ( . %). no chordal rupture was observed in dogs ( . %). in the dogs with ruptured septal chordae, the chordae between s and s was most often ruptured (n = , %). in this study, rupture of the septal chordae tendineae was most commonly observed. this is the first pilot study to visually evaluate the anomaly of the mitral valve leaflet and chordae tendineae in dogs undergoing mitral valve repair. future studies comparing pathological changes and molecular biological analysis to gross findings of mitral chordae tendineae in dogs undergoing mitral valve repair may be useful in advancing the understanding of the disease. no conflicts of interest reported. echocardiographic aortic valve (ao) measurements are routinely obtained during cardiac evaluation of patients. cardiologists commonly use diastolic ao measurements to obtain ratiometric weight-independent estimates of dimensions of other cardiac structures, most commonly the left atrium (la). however, no consensus exists about the point in diastole at which ao measurements should be obtained -immediately after closure of the aortic valve, when la size is largest (ao max , but often with least distinct margins), during the p-wave of the ecg (ao p ) and at the onset of ventricular electrical systole, when la size is smallest (ao min ). we examined the linear and area dimensions of the ao (aod and aoa) to determine if clinically significant differences exist at distinct diastolic time-points, or if these measurements could be interchangeable. we examined patients ( dogs and cats) presented for cardiac evaluations by d echocardiography. three replicates of each time-point linear and area measurement (ao max , ao p , ao min ) were obtained in each patient and averaged for analysis. only patients with aortic valve disease and those with atrial fibrillation were excluded from analysis. beat-to-beat variability of the ao measurements was determined. standard and normalized limits of agreement (loa) plots were generated for each pairwise comparison. the frequency of each ao measurement being the largest or smallest within-patient measurement was determined, and compared via repeated measures anova. all pairwise agreement plots of both aod and aoa demonstrated heteroscedasticity; normalized aod plots showed % loa to be % of the mean aod measurement, with a bias of approximately . % for aod max -aod min , % for aod max -aod p , and % for aod p -aod min . normalized aoa plots showed %loa to be % of the mean aoa measurement, with a bias of approximately % for aoa max -aoa min , % for aoa max -aoa p , and % for aoa p -aoa min . aod max was the largest measurement in / ( %) patients and aod min was the smallest measurement in / ( %) patients; aoa max was the largest measurement in / ( %) patients and aoa min was the smallest measurement in / ( %) patients. rmanova confirmed that ao max >ao p >ao min (p < . ). median within-patient within-measurement variability was % for aod and % for aoa measurements. our data suggest that ao measurements differ throughout diastole, with ao max >ao p >ao min . the disparity is greater for area than linear estimates. the degree of disagreement between ao max and ao p is small and similar to the within-measurement variability. thus, using either ao max or ao p measurements should result in similar ratiometric estimates of cardiac dimensions. no conflicts of interest reported. feline hypertrophic cardiomyopathy (fhcm) is the most common heart disease in cats. hcm is considered an inherited disease of the sarcomere and fhcm has been linked to mutations in one sarcomere protein i.e. mybpc . however, the pathophysiologic mechanisms behind disease development and progression are largely unknown. in this study we investigate whether mitochondrial morphological changes in the myocardium accompany mitochondrial dysfunction and enhanced oxidative stress formation that we recently found in fhcm. myocardial tissue from the left ventricle (lv) was obtained immediately after euthanasia from cats diagnosed with primary hcm on echocardiography ( maine coon, british shorthair, exotic shorthair, norwegian forest cat) and age-matched control cats ( maine coon, norwegian forest cats). ultrastructural examination was performed by the use of transmission electron microscopy. in hcm cats, marked ultrastructural changes of the cardiomyocytes were observed. the population of subsarcolemmal mitochondria (ssm) was absent in large cellular areas in cats with moderate and severe lv hypertrophy. flattening of the sarcolemma was a common finding, causing disorganization of the t-tubular system. interfibrillar mitochondria (ifm) were disorganized but not depleted. additional changes in cardiomyocytes from cats diagnosed with fhcm included remodeling of sarcomeres, disorganization of myofibrils, convolution of gap junctions, accumulation of intracellular z-disc material, perinuclear lipofuscin granula and extensive extracellular deposits of collagen. in healthy mammalian cardiomyocytes, the t-tubular system upholds cellular structure, prevents mitochondrial reticulum formation and provides calcium, oxygen and substrates, necessary for normal functioning muscle. disorganization of the sarcolemma and t-tubular system may cause the depletion of ssm. possible mechanisms are atrophy or disruption of the mitochondria or altered fusion-fission dynamics. calcium cycling and substrate supply are likely to be compromised by the observed structural changes. we propose this to be related to mitochondrial dysfunction and oxidative stress formation that occurs in fhcm, however a causative relationship remains unknown. in conclusion, morphological changes of mitochondria and extra-sarcomeric structures are common in fhcm, regardless of breed, genotype and phenotypic disease expression. moreover, mitochondrial subpopulation-specific changes occur in fhcm with depletion of ssm. ultrastructural and functional changes of cardiac muscle mitochondria are considered important molecular mechanisms, responsible for the development and progression of fhcm and may be relevant future treatment targets. no conflicts of interest reported. patent ductus arteriosus (pda) is one of the most common congenital cardiac defect in the dog. ductal patency is associated with pulmonary overcirculation, left ventricular volume overload and can rapidly determine congestive heart failure if untreated. several devices to close the pda have been used, with amplatzer canine duct occluder (acdo©) being considered the safer device with lowest complication rates. echocardiography represents the cornerstone of pda diagnosis, but its role has been recently expanded to wider field of application: device sizing and intraoperative monitoring, as well as a tool to quantify cardiac morphology and function. speckletracking echocardiography (ste) has been used to evaluate cardiac function in a wide variety of diseases in human and veterinary patients, however no study has evaluated its usefulness in dogs affected by pda both before and after percutaneous closure of pda. the aim of our study was therefore to assess standard m/bmode derived parameter of cardiac function and ste derived longitudinal, radial and circumferential strain and strain rate before and after pda closure. twenty-five dogs of different breeds, age and weight were prospectively recruited and a complete echocardiographic evaluation was performed before and hours after pda closure. end diastolic and systolic diameters indexed for body surface area (edvi/esvi) both derived by m-mode and b-mode views, allometric scaling derived allod and allos, sphericity index (si) and pulmonary to systemic flow ratio (qp/qs) were assessed both pre and postoperatively. ste derived parameters assessed were longitudinal, radial and circumferential strain and strain rate. a statistically significant difference was found in all standard parameters of cardiac function before and after pda closure (p < . ), with a general decrease in values hours postoperatively. ste derived parameters of cardiac function showed a trend toward a decrease back to normal values, which was statistically significant (p < . ) for circumferential and radial strain and strain rate, while longitudinal strain and strain rate did not reach statistically significance. based on our results, no cardiac dysfunction was identified by the use of ste derived parameters both before and after pda closure, with an increased contractility as identified by higher than normal ste values before pda closure and a decrease back to normal strain and strain rate values for both circumferential and radial immediately after percutaneous closure. longitudinal strain persists on higher than normal values, refusing the hypothesis of systolic dysfunction after pda closure and suggesting a longer reverse remodeling process after pda closure. dr bussadori receives royalties from esaote (florence, italy) related to an european patent (nr ) he developed for xstrain software. the study was not funded by a research grant. cardiac cachexia which is characterized by progressive weight loss and depletion of lean body mass, is an independent predictor of survival in human patients with congestive heart failure. chronic degenerative mitral valve disease (cdmd) is one of the most common cardiac diseases in dogs. the aims of this study were to evaluate the prevalence and the effects of cardiac cachexia in survival of dogs with cdmd. medical records of client-owned dogs with cdmd were reviewed. the mean age at entry was . ae . years; were females, and were males. data obtained from the records including breed, sex, body weight, age at diagnosis, complete blood counts, biochemical profiles, urinalysis, systemic blood pressure, thoracic radiographs, electrocardiograms, ultrasonography and echocardiographic examinations at initial visit and survival time. diagnosis of cdmd was based on echocardiographic characteristics and categorized by modified new york heart association (nyha) functional classification. cardiac cachexia was defined as presence unintentional weight loss (> % within months after diagnosis) together with anorexia and muscle weakness, anemia (red cell count < . /ll, hemoglobin < g/dl, or both), hypoalbuminemia (plasma albumin < . g/dl), and azotemia (blood urea nitrogen > g/dl, creatinine > . g/dl, or both). dogs with other cardiac disorders and other systemic disorders those would cause anemia and hypoalbuminemia were excluded from this study. prevalence of cardiac cachexia, anemia and azotemia was . %, . % and . %, respectively. these conditions were the most prevalent in nyha class , followed by nyha classes and . the prevalence of hypoalbuminemia was not significantly different among classes. the one-year body weight change was found in the nyha classes (increased . ae . %), (decreased . ae . %) and (decreased . ae . %). the difference between classes and was significant. results of the cox proportional hazard model indicated that survival time was significantly positively associated with nyha functional severity at diagnosis (p < . ), presence of cardiac cachexia, weight loss, anemia, hypoalbuminemia and azotemia (p < . , p = . , p = . , p = . and p = . , respectively). the prevalence of cardiac cachexia was common in advanced cdmd dogs, and the parameters of cardiac cachexia, namely weight loss, anemia, hypoalbuminemia and azotemia were strong prognostic factors associated with survival. no conflicts of interest reported. mitral valve disease (mvd) is the most common cardiovascular disease in dogs. it's characterized by myxomatous degeneration, which causes mitral valve prolapse (mvp), mitral regurgitation (mr) and a left apical systolic murmur (lasm). mvd affects small breed dogs with a very high prevalence in cavalier king charles spaniels (ckcs). the main goal of this study was to determine the prevalence of lasm, mvp and mr in the maltese, the most presented breed among dogs with mvd in taiwan. the correlation between these measurements and the influence of age, gender, reproductive state, and body weight were also investigated. study results were compared to other mvd prevalence studies in europe and north-america. client-owned maltese dogs ( males and females; body weight . - . kg; age - yrs) with no signs of heart failure were recruited. the intensity (grade - ) of lasm was recorded. grade of mvp (mild/severe) and mr severity (mild/moderate/ severe) were evaluated by echocardiography. logistic regression was used to determine the correlation between age and presence of lasm, mvp and mr. a chi-square test was used to evaluate whether sex and reproductive-status were related to prevalences of lasm, mvp and mr. spearman's correlation coefficient was used to assess the relationships between age, body weight, lasm intensity, grade of mvp and severity of mr. the prevalence of lasm, mvp and mr were . %, % and . %, respectively. all have positive correlation with age (p = . ). the age at which % of the dogs had lasm, mvp and mr was . , . , and . years, respectively. the lasm intensity, mvp grade and mr severity were all positively correlated to age (all p = . ) and had no correlation with bw and reproductive status. females had a significantly higher prevalence of lasm than males ( % vs. . %, p = . ). maltese dogs in taiwan have a very high prevalence and an early development of mvd as compared to other small breed dogs, similar to mvd in ckcs in other countries. since we only recruited asymptomatic dogs, this study may underestimate the prevalence of mvd in the whole maltese population. to our knowledge, this is the first report to document the high prevalence of mvd in taiwanese maltese.the maltese may be a new canine model for genetic, pathology, and natural history studies in mvd. boehringer-ingelheim sponsored the author's accommodation costs for this congress. esvc-o- cardiorenal syndrome in dogs with chronic valvular heart disease: a retrospective study. e. martinelli , p. scarpa , c. quintavalla , c. locatelli , p. brambilla . university of parma, parma, italy, university of milan, milan, italy in human medicine, primary disorders of the heart often result in secondary dysfunction or injury to the kidneys. the coexistence of the two problems in the same patient is referred as cardiorenal syndrome (crs). just little information about crs is available in veterinary medicine. the aim of this study was to define the prevalence of chronic kidney disease (ckd) complicating chronic valvular heart disease (cvhd) in dogs and to investigate the relationship between class of cardiac insufficiency (acvim) and class of renal insufficiency (iris). medical records of dogs presented at the cardiology service of the department of veterinary science and public health, university of milan, between january and december were retrospectively evaluated. dogs with a complete physical examination, thoracic radiographs, a cvhd diagnosis based on echocardiographic examination, and a serum biochemical panel, including assessment of serum creatinine (scr) and serum urea (bun), were included in the study. dogs with other heart disease, neoplasm or systemic diseases were not included in the study. one hundred eighteen dogs of both genders ( males and females), to years of age ( . ae . years), to kg of bodyweight ( . ae . kg) fulfilled the inclusion criteria. the % of males and the % of females were neutered. the most represented breeds were mongrel ( %), miniature poodle ( . %), york shire terrier ( . %), shih -tzu ( . %), pinscher ( . %) and dachshund ( . %). dogs were classified as follow: % acvim a, % acvim b , % acvim b , % acvim c and % acvim d. while the % of the dogs were normoazotemic (scr < , mg/dl), % were staged in iris , % in iris and %in iris . statistical analysis was performed using jmp . (sas institute inc.). a p value < , was considered significant. the prevalence of ckd associated with azotemia in dogs affected by cvhd was %. there was a statistically significant direct correlation between acvim and iris class (pearson test p = . ). unexpectedly, the % of dogs receiving drugs for medical management of heart failure (acvim class c and d) were normoazotemic. despite a definite conclusion about the role of cvhd on the induction and/or progression of ckd cannot be drawn from this cross-sectional study, these results suggest that there is a direct correlation between the severity of ckd and cvhd. no conflicts of interest reported. there is growing evidence of breed differences in concentrations of several blood variables in dogs. the aim of the study was to investigate breed differences in plasma concentrations of components of the renin-angiotensin-aldosterone system (raas), endothelin- (et- ) and serum cortisol concentration in healthy dogs. healthy, privately-owned dogs of nine breeds were examined at five centers as part of the european lupa-project. absence of cardiovascular or other clinically relevant organrelated or systemic disease was ensured by thorough clinical investigations. plasma concentrations of et- and aldosterone, renin activity, and serum concentration of cortisol were measured by ria or elisa assays. overall significant breed differences were found (p < . for all variables). bonferroni-corrected pair-wise significant differences between breeds were found in % of comparisons for et- , % for cortisol, % for renin and % for aldosterone. for et- , the highest median concentration was found in newfoundlands with values > times higher than most other breeds, while renin was highest in dachshunds, > times higher than in newfoundlands and boxers, which had the lowest concentrations. aldosterone was especially low in belgian shepherds with median concentration < times than the other breeds. cortisol was highest in finnish lapphunds, almost times higher than boxers with the lowest concentration. in conclusion, considerable inter-breed variation in concentrations of et- , components of raas and cortisol was found in healthy dogs. these differences are likely influenced by genetic factors and should be taken into account when designing clinical trials and tests. breed-specific reference ranges might be necessary. no conflicts of interest reported. most studies that assess weight management in obese dogs only examine the early stages of weight loss, and this may not properly reflect a complete weight management regime. the aim of the current study was to examine the kinetics of a complete weight management cycle in obese client-owned dogs. dogs referred to the royal canin weight management clinic, university of liverpool, for the management of obesity, were eligible for inclusion. all dogs were followed until they had either completed (i.e. reached target weight) or the programme was discontinued. rate of weight loss, percentage weight lost, and energy were assessed at different time points. a total of dogs were included, with a range of breeds, ages and sexes represented. rate of weight loss steadily decreased throughout the weight loss period (d : . ae . %/wk; d : . ae . %/wk; d : . ae . %/wk; d : . ae . %/wk; d . ae . %/wk; d : . ae . %/wk; p < . ). the energy intake required to maintain weight loss also progressively decreased (p < . ). by day , mean aesd weight loss was ae . %, and compliance was good, but most had not com-pleted ( % completed, % ongoing, % discontinued). thereafter, more dogs completed, but the number of discontinuing also increased (d : ae . % weight loss, % completed, % ongoing, % discontinued; d : ae . % weight loss; % completed, % ongoing, % stopped). initial weight loss is good in obese dogs but, thereafter, steadily worsens. thus, studies examining only the first few months of weight loss are not fully representative of the entire weight loss process. conflicts of interest: the following conflicts of interest apply: the diet used in this study is manufactured by royal canin.whilst vb is employed by royal canin. vb and ss are employed by royal canin. ajg's readership is funded by royal canin. obesity and obesity-related metabolic dysfunctions are increasing in humans as well as in dogs. obese dogs become affected by chronic diseases at young age, have a decreased quality of life and a shorter life-span. the aim of the study was to describe the metabolic and hormonal response to a feed-challenge test in lean and overweight dogs. twenty-eight healthy intact male labrador retrievers aged . ae . years with varying body condition score (bcs, scale - ) were included. twelve dogs were classified as lean (bcs - ), ten as slightly overweight (bcs ) and six as overweight (bcs . - ). an overnight fasting period and blood sample collection was followed by a high fat meal. after food intake, blood samples were collected hourly for four hours. a glucagon elisa was validated for use in dogs. the assigned bcs was supported by positive association with serum leptin concentrations. postprandial triglyceride concentration was significantly higher in the overweight group. a tendency to higher cholesterol concentration was seen in the overweight group but cholesterol was not affected by food intake. glucagon concentration rose after food intake and resembled the response seen in humans after a mixed meal. glucose and insulin concentrations followed the same pattern while free fatty acids had declined one hour after the meal. in this study, the metabolic and hormonal response to a high fat meal was similar between lean and slightly overweight dogs, whereas the response of overweight dogs differed. studies on the health significance of postprandial hypertriglyceridemia in dogs are warranted. conflicts of interest: the study was financially supported by the swedish veterinarian federation, the companion animal research foundation, and the foundation of thure f. & karin forsberg. feline weight-loss programs are often hindered by compliance issues and sedentary lifestyle. the purpose of this study was to assess the effectiveness of a new dietetic weight management food (ndwmf)* in achieving weight loss in overweight/obese, client-owned cats. the objectives were ) to evaluate weight loss parameters in cats fed the ndwmf* and ) to describe the owner's perception of the cat's quality of life. overweight/obese, otherwise healthy, client-owned cats (> / body condition score -bcs) were enrolled in the study (n = ). initial veterinary evaluation comprised a physical examination, nutritional assess-ment, determination of ideal body weight (ibw), and development of weight loss feeding plan. daily energy requirement (der) for weight loss was calculated as der = . x ( x ibw kg . ). initial and follow-up evaluations (monthly for months) consisted in determination of body weight (bw), bcs, body fat index (bfi), muscle condition score (mcs), and current feeding practices. quality of life assessment by owners included cat's level of energy, happiness, appetite, begging behavior, flatulence, stool volume, and fecal score. statistical analysis encompassed scatterplots, regression analysis, summary statistics as appropriate for the type of analyses (continuous or categorical variables, distribution), a mixed model anova was used to assess changes over time (statistical significance at p < . ). eighty three percent of the cats (n = ) lost weight with an average weight loss of % (sem, . %) over months and an average weekly weight loss rate of . % (sem, . %). a significant decrease in bcs from week - and in bfi from week - compared to baseline was observed. mcs did not change. average duration of weight loss was days (sem, . days) with days (sem, . days) between visits. fourteen percent of cats achieved ibw ( . , ci: . - . ). seventy nine percent of cats ate more than the recommended der (median fed above der= %), and the majority of these cats still lost weight. owners perceived a significant increase in energy and happiness (>week ) compared to baseline in the cats that lost weight without changes in appetite or begging behavior. no significant changes were seen in scores for flatulence, stool volume, and fecal score. in conclusion, this clinical study showed that feeding the ndwmf* to client-owned, overweight/obese cats resulted in weight loss. owners reported significant improvements in cat's quality of life without negative side effects. * porphyrias are a group of inborn errors of metabolism resulting from accumulation of porphyrins due to deficient activities of specific enzymes in heme biosynthesis. in humans, they are clinically classified as either erythroid with cutaneous involvement or hepatic with acute neurovisceral attacks. here we describe the clinical, biochemical, and molecular genetic studies in porphyric cats from new brunswick, canada. from to , three separately identified adult domestic shorthair cats from the city of saint john in new brunswick were found to have erythrodontia (brown discolored teeth which fluoresced pink) and pigmenturia. a mild compensated hemolytic disorder with numerous small dark blue irregularly shaped erythrocyte inclusions was noted. there was no evidence of acute lifethreatening neurovisceral attacks or cutaneous lesions. necropsy of one cat revealed massive deposition of porphyrins in all bones and teeth. urine and edta blood samples from one cat were metabolically studied, while molecular genetic studies were performed in all cats either from edta blood or a formalinized splenic tissue block. urinary d-aminolevulinic acid, porphobilinogen, uroporphyrin i, and coproporphyrin i concentrations were increased in the cat studied, suggesting an acute intermittent porphyria (aip). the erythrocytic hydroxymethylbilane synthase (hmbs) activity in erythrocytes was approximately half normal suggesting a dominant enzymopathy, while the erythrocyte uroporphyrinogen iiisynthase activity was normal. sequencing the feline hmbs gene revealed a heterozygous intronic base deletion (c. - _- del) which results in an insertion in the mrna and would predict a truncated protein. in conclusion, these three domestic shorthair cats had the same hmbs mutation causing an autosomal dominantly inherited aip. cats with discolored teeth and normal or mild hemolysis may have either acute intermittent porphyria or congenital erythroid porphyria. interestingly, seven disease-causing mutations have now been found by us in the hmbs gene -more than in any other gene in cats. the biochemical and molecular characterization facilitates clinical screening of affected cats to reach a specific diagnosis. supported in part by nih od . urs giger and raj karthik are also part of the laboratory that offers dna testing for this mutation. fibrinogen decreases when coagulation is activated to form fibrin, while fdps and d-dimers represent the products of fibrinolysis. in humans, activation of coagulation and fibrinolysis develops in all type of ascites and it is also associated with signs of systemic fibrinolysis.these results have lead to the suggestion that ascitic fluid is inherently fibrinolytic. preliminary studies showed similar results also in dogs (javma nov. , ecvim proceedings . in addition, in an old experimental study conducted in dogs, inoculation of blood or of a solution containing fibrinogen and thrombin into the pleural cavity resulted in the activation of the coagulation system followed by fibrinolysis. therefore, the objective of the present study was to determine whether the activation of coagulation and fibrinolysis (i.e. low fibrinogen and elevated fdps and ddimer) occurs not only in the ascitic fluid, as alredy been demonstrated, but also in all type of pleural effusions in dogs. thirty-three dogs referred to the san marco veterinary clinic with pleural effusion, but without ascites, were studied. fibrinogen, fdps, and d-dimer concentrations were measured and then compared in both pleural fluid and venous blood via wilcoxon signed ranks test. the dog's pleural effusions were categorized based on pathophysiology of fluid formation into dogs with transudate ( due to increased hydrostatic pressure and due to decreased osmotic pressure), with an exudate (of which due to septic causes), with a haemorrhagic pleural effusion, and with a chylous effusions. the fibrinogen concentration in the pleural effusion (median: mg/dl; range: - ) was significantly lower (p < . ) than the plasma fibrinogen concentration (median: mg/dl; range: - ). in all dogs, the fibrinogen pleural fluid concentration was lower than the plasma concentration. the fdp concentration in the pleural effusion (median: mg/dl; range: . - ) was significantly (p < . ) higher than plasma fdps concentrations (median: . mg/dl; range: . - . ). in case, the fdps pleural fluid concentration was lower than the plasma concentration and in cases the pleural fluid concentration was higher. the d-dimer concentrations were significantly(p < . ) higher in the pleural effusion (median: . lg/ml; range: . - . ) than in the plasma (median: . lg/ml; range: . - . ). in one case, the d-dimer pleural fluid concentration was lower than the plasma concentration and in cases was higher. these findings support the hypothesis that activation of coagulation followed by fibrinolysis occurs in all type of pleural effusions. no conflicts of interest reported. during primary hyperfibrinogenolysis (phf), fdps production is increased but production of d-dimer is not. therefore, elevated fdps and normal d-dimer are considered an indicator of phf. in humans and dogs, activation of coagulation and fibrinolysis develops in all type of ascites and it is associated with systemic phf, suggesting that ascitis is inherently fibrinolytic. preliminary data have shown that activation of coagulation followed by fibrinolysis occurs also in all type of pleural effusions (pe). the objective of this study was to determine if systemic phf occurs also in dogs with pe. thirty-three dogs referred to the san marco veterinary clinic with pe, but without ascites, were studied (group ). from the electronic data-base of the clinic dogs for inclusion in control groups (healthy dogs) and (sick dogs without pe or ascites) were randomly selected and individually matched to group dogs for age, sex, and breed. fibrinogen, fdps, d-dimers, c-reactive protein (crp), fibrinogen/crp ratio, and prevalence of phf (i.e., dogs with elevated plasma fdps and normal d-dimer) were determined. differences between the groups were analyzed using anova (fibrinogen), chi-square (fdps and prevalence of phf) and kruskal-wallis test (crp, fibrinogen/crp ratio, and d-dimer). post-test analysis were performed by tamhane and mann-whitney test. fibrinogen concentration in group was significantly increased compared to group (p < . ), but not compared to group (p = . ). fdps concentration in group was significantly increased compared to groups (p < . ), but not compared to group (p = . ). d-dimers concentration in group was significantly increased compared to group (p < . ), but not compared to group (p = . ). crp was significantly increased in group compared to group and (p < . for both comparison). fibrinogen/crp ratio was significantly decreased in group compared to group and (p < . for both comparison). prevalence of phf was significantly higher in group compared to groups (p = . ), but not compared to group (p = . ). these results support the hypothesis that phf occurs significantly more often in dogs with pe compared to healthy dogs. despite there was a trend of increased phf also in dogs with pe compared to sick dogs, this difference did not reach significance. nevertheless, the decreased in fibrinogen/crp ratio in group compared to group , in the face of a similar d-dimer concentration, would suggest that phf is also more prevalent in dogs with pe compared to sick control dogs. no conflicts of interest reported. the systemic inflammatory response syndrome (sirs) refers to clinical signs of systemic inflammation in response to (non-) infectious insults. current diagnosis of sirs is based on clinical and basic laboratory data and is a sensitive screening to identify patients at risk. c-reactive protein (crp) is a major canine acute phase protein with concentrations related to disease severity and underlying cause. crp rises in response to proinflammatory cytokines, mainly interleukin (il)- and tumor necrosis factor (tnf)-a, which are considered the main triggers of sirs. we therefore evaluated crp, il- and tnf-a kinetics in canine emergency sirs patients hypothesizing that crp is ( ) increased in dogs with a clinical sirs-diagnosis, ( ) correlated with il- and tnf-a concentrations, ( ) influenced by the underlying etiology, and ( ) a prognostic marker. canine emergencies with clinically diagnosed sirs were prospectively included. serum and plasma were immediately stored at - °c after sampling at presentation, after (t ), (t ), (t ) and (t ) hours, and at a control visit (t m) over one month after discharge. serum crp was measured with a caninespecific immunoturbidimetric crp assay. plasma il- and tnfa were measured using a bioassay measuring biologically active cytokine concentrations. disease categories were infection (i), neoplasia (n), trauma (t), gastric-dilation and volvulus (gdv), other gastrointestinal (gi), renal (r) and miscellaneous (m) diseases. statistical analysis was performed with sas. concentrations of inflammatory cytokines were expressed logarithmically, with univariate analysis confirming normal distribution. a correlation procedure, mixed procedure on a linear model and a logistic procedure were performed (p-value < . ). sixty seven dogs (i = , n = , t = , gdv= , gi= , r = , m = ) were included. forty-three patients survived (seven died, seventeen were euthanized). twenty patients had a control visit. crp was elevated in . % of dogs at presentation, and only remained within reference range ( - . mg/l) throughout hospitalization in four dogs ( . %). crp concentrations were significantly higher from t ( . ae . mg/l) to t ( . ae . mg/l) decreasing at t ( . ae . mg/l), and returning within reference range at t m ( . ae . mg/l) in all but one dog ( . mg/l). crp was significantly correlated with logarithmical concentrations of il- and tnf-a, however, these did not change significantly over time. none of the evaluated parameters was associated with disease category, nor outcome. crp appears useful to diagnose sirs in emergency patients, and tends to decrease during hospitalization. however, crp, neither il- nor tnf-a concentrations appear useful to predict the underlying disease and outcome in sirs patients. no conflicts of interest reported. calprotectin (s a /a complex) belongs to the s /calgranulin family, and is primarily released from activated neutrophils and macrophages. serum calprotectin concentrations (cp) were shown to be increased in dogs with inflammatory diseases such as inflammatory bowel disease, pancreatitis, systemic inflammatory response syndrome, and sepsis. canine cp thus appears to be a biomarker of inflammation. considerable day-today variation of fecal canine cp was found in both healthy dogs and dogs with chronic gastrointestinal disease. however, the biological variation of canine cp in serum has not been reported. the aim of this study was to determine the biological variation of serum canine cp and its minimum critical difference (mcd). eleven healthy dogs were used for this study. biological variation of serum canine cp was evaluated over a . -months period. tests for outliers were carried out at levels (within-run analytical variance, intra-, and inter-individual variation). a nested analysis of variance (anova) model was used to calculate analytical (cv a ), intra-individual (cv i ), inter-individual (cv g ), and total variation (cv t ), and to determine the index of individuality (ii), index of heterogeneity (ih), and mcd. a total of serial specimens were collected from dogs, serial samples from dogs, and serial samples from dogs. four within-subject outliers were detected and excluded from further analysis, yielding a total of serum samples and slightly right-skewed data. no outlying observations (cochrane test) or outliers among mean concentrations of subjects (reed's criterion) were detected. cv a was calculated as . %, cv i as . %, and cv g as . %, resulting in a cv t of . %. index of individuality (ii) was determined to be . and ih was . , yielding a one-sided mcd of . mg/l. the analytical goal of cv a ≤ ½ cv i was satisfied. although serum canine cp remained within a relatively narrow concentration range in healthy dogs, moderate individuality was detected. moderate changes in serum canine cp ( . mg/l) between sequential measurements are needed to be considered clinically relevant, and using a population-based reference interval may or may not be appropriate for serum canine cp. using the mcd with the previously determined median canine cp concentration ( . mg/l) for the reference sample group yielded a serum canine cp concentration close to the upper limit of the previously established reference interval ( . mg/l), showing that the reference interval for serum ccp ( . - . mg/l) is within reasonable limits. the assay used in the study was developed at the gi laboratory, texas a&m university. most authors also work at the gi laboratory, texa a&m university. canine leishmaniasis (canl) is a multisystemic disease that is endemic in the mediterranean region. in the past, concentrations of acute phase proteins (apps), and specifically c-reactive protein (crp), haptoglobin (hp), ceruloplasmin (cp), serum amyloid a (saa) and albumin (alb), have been reported to change in dogs with leishmaniasis, and revert to normal after successful treatment, highlighting the intrinsic inflammatory reaction of the host to the parasite. since the spectrum of clinical and laboratory derangements is broad, it is possible that apps are increased specifically because of certain clinicopathological syndromes associated with canl. a total of dogs with canl, diagnosed on the basis of cytological amastigote identification and ifat serology, were retrospectively included in the study. in all of them, crp, saa, hp and alb were measured at interlab-umu, murcia, spain, in aliquots of serum, which were stored in - °c for - years (median: years). results for each of the apps were correlated to laboratory and clinical parameters (n: ), clinical and parasitological scoring (n: ), ehrlichia and leishmania serology (n: ), and clinical staging according to leishvet (n: ), using an array of linear and ordinal regression models, as well as one-way anova, t-test and fisher's lsd test. crp and alb were by far the apps most frequently correlated with clinical and laboratory abnormalities such as nutritional status, lethargy and skin ulcers (p < , ), as well as urinary protein to creatinine ratio (upc), total serum protein, and urine specific gravity (p < , ). there were limited associations between hp, cp, saa and clinicopathological parameters. a minor linear relationship was observed between crp and clinical scoring. crp and alb were also correlated with parasitological scoring in bone marrow, but not lymph node cytology (p < . ). dogs with ehrlichia titers had higher crp, cp and lower alb concentrations. finally, crp concentrations were higher in later compared to earlier stages of the infection, as defined by the leishvet criteria. the inflammatory component to leishmania infection doesn't seem to be exemplified by the reaction of a particular tissue, with the possible exception of glomerulonephritis. the magnitude of increase in crp and decrease in albumin is correlated with clinical staging and bone marrow parasitological scoring. no conflicts of interest reported. the consequences of abnormal platelet function in dogs and cats can be devastating and the use of anti-thrombotic therapy to prevent thrombotic events is increasingly common. the ability to measure platelet function and the efficacy of anti-thrombotic therapy is difficult due to limited availability of equipment and inability to delay platelet function analysis. the aim of this study was to adapt and validate test procedures and protocols previously developed for humans for use in dogs and cats. residual samples of citrate anticoagulated blood were used from dogs and cats presented to a specialist referral centre for various reasons unrelated to clotting abnormalities. initially the blood was stimulated using specific combinations of either arachidonic acid/epinephrine (aa/epi) or adp/u , designed to assess the effects of the anti-thrombotic agents aspirin and clopidogrel respectively. after minutes stimulation, the blood samples were fixed using a patented platelet fixative solution developed for human platelets, which allows the delayed analysis of p-selectin an established marker of platelet activation. all analysis was performed by flow cytometry. in order to do this, specific antibodies were selected for the recognition of both canine and feline platelets. cd was used as a platelet identifier antibody while appropriate cd p (p-selectin) antibodies for each species were chosen. fixed samples were repeatedly analysed at time points between to days following fixation to establish the stability of the fixed samples. thirteen dogs and three cats were analysed. high p-selectin expression was detected following stimulation with aa/epi and adp/u in both dogs and cats following fixation. this was significantly different to unstimulated blood (p < . ). there was no significant difference in detectable pselectin expression following storage of the fixed samples at any time-point up to days. this confirmed the fixative was suitable as a preservative of canine and feline platelets. a limited number of dogs were evaluated whilst receiving antithrombotic medication. there was a significant difference in the activation of platelets in the dogs treated with either aspirin (p < . ) or clopidogrel (p < . ) compared with untreated dogs following stimulation with aa/epi (aspirin group) or adp/ u (clopidogrel group). our results show that fixation and delayed analysis of platelet function in dogs and cats is possible for up to days. this demonstrates an exciting opportunity to analyse platelet function remotely and to determine the efficacy of thromboprophylaxis in animals presenting to clinics that do not have on-site platelet analysers. no conflicts of interest reported. several authors consider thyroid hormone supplementation as a valid initial treatment option for dogs with aggression related problems. indeed, mood and behaviour modulating properties of thyroid hormones may, in part, be mediated through the interaction of thyroid hormones with neurohormones such as serotonin and prolactin. at present, prospective trials evaluating neurohormonal status or behaviour in hypothyroid dogs before and after thyroid supplementation are lacking. therefore, the aims of this study were to assess behaviour and measure serum serotonin and prolactin concentrations in dogs with spontaneous hypothyroidism before and after treatment.twenty three client-owned dogs diagnosed with spontaneous primary hypothyroidism were prospectively included in our study. after diagnosis all dogs were treated with levothyroxine ( micrograms/kg bid). behaviour of dogs was screened at initial presentation, at weeks and months after initiation of therapy. owners had to fill in a hard copy of the standardized canine behavioural assessment and research questionnaire (c-barq) consisting of scored questions evaluating seven behavioural categories. the average score on all questions was calculated for each dog at each of the three time periods and a paired t-test was used for comparison. serum serotonin and prolactin concentrations were evaluated at each time period using a commercially validated elisa kit and heterologous ria, respectively.results of the c-barq after six weeks of thyroid hormone supplementation when compared with the time zero demonstrated a significant increase (p < . ) in excitability, activity and aggression, which most likely became unmasked owing to improved overall activity of dogs. conversely, at six months period when compared with the time zero no significant changes in any of the behavioural symptoms were observed. serum serotonin was measured in / dogs colorimetrically at nm. at time zero, weeks and months serum serotonine was . (range, . - . ), . (range, , - . ) and . (range, . - . ). no significant difference was noted between week and month period comparing to time zero (p = . and p = . ). serum prolactin concentration measured in / dogs at time zero, weeks and months was . ng/ml (range, . - . ), . ng/ml (range, . - . ) and . ng/ml (range, . - . ) and did not differ significantly in either time period when compared with time zero (p = . and p = . ).altogether, results of this study failed to demonstrate a significant role of thyroid supplementation on the majority of evaluated behavioural symptoms as well as neurohormonal status of hypothyroid dogs during months of therapy. no conflicts of interest reported. iatrogenic hypothyroidism is a recognized complication of radioiodine treatment of hyperthyroidism in cats, but no prospective studies of the prevalence, clinical features, routine laboratory findings, or results of thyroid function tests have been reported in a series of hypothyroid cats. in this study, we describe the features of hypothyroidism in cats treated with radioiodine over a -month period (october -march ). during this same period, we treated % hyperthyroid cats with radioiodine, providing a prevalence rate of %. hypothyroidism was diagnosed - days (median, days) after i treatment, with doses ranging from - mbq (median, mbq; median pretreatment t , nmol/l). the hypothyroid cats ranged in age from - years (median, years). all were dsh/dlh; ( %) were female and were males (p = . ). clinical signs in these cats included overweight/obesity in ( %), lethargy/dullness in ( %), poor appetite in ( . %), and polyuria/polydipsia in ( %). abnormalities on physical examination included dermatologic signs (dry coat, seborrhea, matting) in ( %) and bradycardia (< bpm) in . twenty-two cats ( %) had no noticeable clinical features of hypothyroidism. routine laboratory abnormalities included hypercholesterolemia (> mmol/l) in ( %) and new or worsening azotemia (> lmol/l) in ( %) and ( %) cats, respectively. median serum concentrations of total t ( . nmol/l; reference interval [ri], - nmol/l), t ( . nmol/l; ri, . - . nmol/l), and ft ( pmol/l; ri, - pmol/l) were all in the low end of the ri. normal ri values for t and ft were maintained in ( %) and ( %) of the cats, respectively. serum ctsh values were high in all cats (median, . ng/dl; range, . - . ng/dl; ri, . - . ng/ml). thyroid scintigraphy showed less-than-normal amounts of residual tissue, as well as low values for thyroid-to-salivary ratio and %-uptake of pertechnetate, in ( %). of those cats with normal scintiscans, serum ctsh decreased into the ri without treatment when retested - months later. in conclusion, this study confirms that i-induced hypothyroidism is not uncommon, with an apparent female sex predilection. serum t and ft remain normal in most cats, but high serum ctsh values and thyroid scintigraphy aid in diagnosis. unless cats have overt, long-standing hypothyroidism, most cats with subclinical disease are relatively asymptomatic, other than worsening azotemia. subclinical hypothyroidism will be transient in some cats, with normalization of ctsh values within a few months. no conflicts of interest reported. iatrogenic hypothyroidism is a recognized complication of radioiodine treatment for hyperthyroidism in cats. at our clinic where we use a variable -i dosing protocol (based on tumor volume and severity of hyperthyroidism), the prevalence of overt or subclinical hypothyroidism is at least %. during the -month period from october to march , we treated cats with iatrogenic hypothyroidism, which had developed - days (median, days) after treatment with radioiodine (median dose, mbq). these cats ranged in age from - years (median, years); all were dsh/dlh; ( %) were female and were males. new or worsening azotemia (> lmol/l) was documented in ( %) and ( . %) cats, respectively. diagnosis of hypothyroidism was based on the following: ) low to low-normal serum concentrations of t , ft , and t ; ) high serum tsh concentration (> . ng/dl); and ) less-than-normal amounts of residual tissue on thyroid scintigraphy. all cats were given thyroid hormone replacement as a liquid l-t preparation (leventa; merck animal health). cats were monitored at - month intervals by repeating serum t and tsh concentrations - hours after the morning l-t dose. ten of the cats were started on a once-daily l-t regimen ( lg); of these, only ( %) had suppression of high serum tsh values into the reference interval (ri). of the cats that had persistently high tsh values, were switched to twice-daily administration ( - lg, bid), which successfully lowered high tsh concentrations in cats. the remaining cats were started on twice daily l-t ( lg, bid); of these, normalization of tsh occurred in cats. overall, l-t treatment was successful in normalizing tsh concentrations in ( %) cats, with once-daily and with twice-daily dosing. peak serum t concentrations of ≥ nmol/l were needed in most cats to normalize tsh values. higher serum t and lower tsh concentrations were achieved when l-t was administered on an empty stomach rather than given with food. a significant decrease (p < . ) in serum creatinine occurred after treatment with l-t . in conclusion, our results indicate that twice-daily administration of l-t is needed in most cats with iatrogenic hypothyroidism to normalize high serum tsh concentrations. many cats appear to absorb l-t rather poorly, which can be enhanced by giving the drug on an empty stomach. the azotemia that commonly develops in cats with hypothyroidism improved or stabilized with adequate l-t supplementation. no conflicts of interest reported. congenital hypothyroidism (ch) has been reported in many species; the hereditary forms can be divided into thyroid dysmorphogenesis and dyshormonogenesis. while thyroid hypoplasia has been described in dogs and cats, the molecular basis remains unknown. in contrast few breeds of dogs with goiterous ch were found to have deficient thyroid peroxidase (tpo) activity. the purpose of our study was to characterize a family of domestic shorthair cats with goiterous ch and disease-causing tpo gene mutations. clinical features included dwarfism and dullness, known as cretinism and seen with ch in all species, but also constipation and megacolon which are unique to cats with ch. pedigree analysis documented an autosomal recessive mode of inheritance. affected kittens developed a goiter and had low serum thyroxine (t ) and triiodothyronine (t ) when compared to controls, but high thyroid stimulating (tsh) hormone levels indicating thyroid dyshormonogenesis. oral thyroid supplementation corrected the progression of clinical signs and prevented further constipation and reversed the megacolon. the tpo enzyme activity was extremely low in hypothyroid cats when compared to that of normal cats. genomic dna and cdna from affected, carrier, and normal cats were extracted and sequenced based upon primers developed from the feline genome database. a homozygous missense point mutation (c. g>a) in tpo, which results in an amino acid change (p.ala thr), was discovered in affected cats and the mutant allele segregated within the family with goiterous ch. this is the first report of a tpo deficiency in cats. other unrelated domestic shorthair cats with goiterous ch did not have this same tpo mutation. the prevalence of this tpo mutation in the domestic cat population seems low, but ch is likely underreported in cats. supported in part by nih od . some of the authors are members of diagnostic laboratories (penngen). supported in part by the nih od # . glucagon-like peptide- (glp- )is a gastrointestinal hormone released in response to food intake that increases insulin secretion, inhibits glucagon secretion, slows gastric emptying and induces satiation. it is also assumed to stimulate beta-cell proliferation. glp- agonists are successfully used in humans with type diabetes mellitus usually either in combination with insulin or other anti-diabetic drugs. in healthy cats twice daily (exenatide) as well as once weekly (exenatide extended-release (er)) application of glp- agonists induced pronounced insulin secretion. benefits of exenatide er are the regimen of once weekly injection and less side effects. the objective of the study was to assess whether administration of exenatide er in addition to standard treatment leads to improved glycemic control and higher remission rates in cats with newly diagnosed diabetes. the study was designed as a prospective, placebo-controlled clinical trial. cats were randomly assigned to two groups receiving exenatide er (group : bydureon â , mg/kg, q d, sc) or . % saline solution (group : q d, sc). both groups additionally received insulin glargine (lantus â , initial dose: ≤ kg: . iu, q h; > kg . - . iu, q h) and diet (purina dm â ). exenatide er was applied over weeks or, in case of remission, for additional weeks after cessation of insulin application. cats were rechecked , , , and weeks after starting therapy. remission of diabetes was defined as absence of clinical signs of diabetes and normal blood glucose and fructosamine concentrations for at least weeks after discontinuing insulin injections. so far cats have completed the study. mild and transient side effects in group (n = ) were reduced appetite (n = ), nausea (n = ), vomitus (n = ), tiredness (n = ) and hiding in dark spots of the house (n = ). in group remission was achieved in / ( %) cats and good metabolic control in / ( %) nonremission cats. in group remission was achieved in / ( %) cats and good metabolic control in / ( %) non-remission cats. median insulin dose given during the study period was . iu/ kg/day in group and . iu/kg/day in group . the preliminary results suggest that exenatide er can be used safely in diabetic cats. a tendency for higher remission rate, better metabolic control and lower insulin requirement was seen when exenatide er was added to the standard treatment regimen. further cases need to be evaluated to verify the potential beneficial role of exenatide er. no conflicts of interest reported. feline diabetes mellitus shares many similarities with human type diabetes mellitus (t dm), including clinical, physiological and pathological features of the disease. domestic cats spontaneously develop diabetes associated with insulin resistance in their middle age or later, with residual but declining insulin secretion. humans and cats share the same environment and risk factors for diabetes, such as obesity and physical inactivity. moreover, amyloid formation and loss of beta cells are found in the diabetic cat pancreas, as in humans. subsequently, studying the molecular mechanisms in the failing beta cells may contribute to a better understanding of the pathophysiology of t dm in both cats and humans. the aim of the present study was to develop a method to study mrna expression of islet-specific genes in healthy and diabetic cats. previous attempts in isolating feline islets with different collagenase-based protocols have led to damaged islets or islets coated with exocrine acinar cells, which either way compromise the results obtained from gene expression studies. by using the laser microdissection technique, we were able to sample islets that were not contaminated with exocrine tissue, from both healthy and diabetic cats. high rna quality was confirmed with gel electrophoresis. by quantitative real-time pcr (qrt-pcr), mrna levels of the islet-specific genes insulin, pdx- , iapp, chga and ia- were detected in both healthy and diabetic cats. we used actin b, gapdh and rps as internal reference genes for normalizations of our qrt-pcr data. the laser microdissection technique allows studies of islets without contamination of acinar cells, as shown in this study, and is of great advantage since it is difficult to get pure feline islets from collagenase-based isolation. differences in gene expression in healthy and diabetic cats may reveal underlying mechanisms for beta cell dysfunction and decreased beta cell mass in human and feline type diabetes. conflicts of interest: the study was financially supported by the swedish juvenile diabetes foundation, the fredrik and ingrid thuring foundation, the magnus bergvall foundation, the lars hierta memorial foundation, and the foundation for research, agria insurance company. feline acromegaly is an increasingly recognised endocrinopathy among diabetic cats, caused by chronic excessive growth hormone secretion by a functional somatotrophinoma in the pars distalis of the anterior pituitary gland. the majority of human somatotrophinomas are sporadic, however up to % of familial isolated pituitary adenomas are caused by germline mutations of the aryl-hydrocarbon-receptor interacting protein (aip). feline acromegaly has phenotypic and biochemical similarities to human familial acromegaly with aip mutations, such as male predominance, somatotroph macroadenoma and resistance to octreotide therapy. the objective of this study was to identify the feline aip gene, identify single nucleotide polymorphisms (snps) within this gene and compare any snps with reported human aip snps. stored pituitary tissue from an acromegalic cat was used to create feline aip cdna using feline specific aip primers. stored edta blood from acromegalic cats (diagnosis of insulin resistant diabetes mellitus, serum igf- > ng/ml and pituitary mass > mm identified using pituitary computed tomography or necropsy) and control cats (no history of diabetes mellitus and greater than years of age) were selected, dna extracted and genotyped using pcr, agarose gel electrophoresis and sanger sequencing. the feline aip gene was identified, encoding a amino acid protein with % homology to the human aip protein. a blast search revealed this gene contained exons and exon specific primers were created to enable sequencing. a single nonconservative snp was identified in exon (aip:c. g>t), encoding for an amino acid change from aspartic acid to glutamic acid in / acromegalic patients and / control cats. two additional conservative snps were also identified (aip:c. t>c and aip:c. t>c). exon encodes for a region of the aip protein considered essential for aip-aip receptor interaction. although different human aip mutations have been identified to date, a human aip:c g>t mutation has not yet been identified. the aip n-terminal is required for the stability of the aip protein-aip-receptor complex, and essential for the regulation of translocation into the nucleus, where it binds to aryl hydrocarbon receptor nuclear translocator leading to activation of genes thought to act as tumor suppressors. loss of normal aip activity is thought to promote somatotrophinoma development. it is therefore possible that the detected aip:c. g>t mutation predisposed to somatotrophinoma tumorigenesis in the two affected patients, and a study containing a larger number of cases is indicated. no conflicts of interest reported. hypersomatotrophism (hs) is an important cause of feline diabetes mellitus (dm). in humans surgical removal of the somatotrophinoma is generally recommended, though hypophysectomy programs have suffered from significant initial morbidity and mortality given a documented steep learning curve in newly established programs. hypophysectomy as treatment for feline hs has thus far only been described in a handful of cases, all having been treated by one single experienced hypophysectomy team. this study's aim was to evaluate the learning curve of a de novo established hypophysectomy program, through analysis of peri-and post-operative morbidity and mortality, and endocrine outcomes in the first cohort of cats with hs treated. from owners of diabetic cats with confirmed hs (igf- > ng/ml, pituitary mass) presented at the royal veterinary college were offered hypophysectomy. all cats undergoing surgery were operated by one neurosurgeon with previously only cadaveric experience of the procedure, through an adapted transsphenoidal approach referencing bony landmarks to computed tomographic scans reconstructed on neuronavigation software. the somatotrophinoma was extirpated using fine surgical tools. all cats received intense electrolyte and blood pressure monitoring, peri-and post-operative ddavp and intravenous insulin and hydrocortisone infusion, transitioning to subcutaneous glargine, conjunctival ddavp, oral hydrocortisone and levothyroxine. between april -february , cats underwent hypophysectomy (median + range age: . years, . - . ; igf- : ng/ml, -> ; pituitary height . mm, . - . ). all displayed uncontrolled dm due to hs (median fructosamine: umol/l); none displayed overt central neurological deficits. two cats ( %, cats and ; pituitary height (mm): . and . ) required mechanical ventilation post-operatively and both were euthanized. post-mortem magnetic resonance imaging revealed brain herniation and cerebral ischaemia was suspected. one cat suffered cardiac arrest post-operatively at time of jugular catheter placement, though made an uneventful recovery. four other cats developed congestive heart failure within days, which was successfully treated not necessitating ongoing therapy. temporarily diminished tear production was seen in cats. seven of the ten surviving cats went into diabetic remission within a median of . days ( - ); others saw reduction of insulin needs by %. serum igf- normalised rapidly and significantly in all but one cat (median serum igf- ng/ml within days). persistent neurological deficits or palatal wound breakdown were not encountered. starting a hypophysectomy program to treat feline hs was associated with some risk of mortality, though surviving cases benefited from the procedure with a high incidence of diabetic remission. no conflicts of interest reported. pituitary dependent hypercortisolism (pdh) in dogs is frequently associated with high serum phosphate and parathormone concentrations. the pathogenesis of such abnormalities remains unknown and the evaluation of the urinary fractional excretion of phosphate and calcium in pdh dogs might be helpful in enhancing the knowledge regarding this issue. the aim of the present study was to evaluate the serum and urinary concentrations and the urinary fractional excretion of phosphate and calcium in dogs with pdh. medical records from one referral center were retrospectively evaluated between and . the diagnosis of pdh was confirmed using the cortisol to creatinine ratio, the ldds test and/or acth stimulation test, the plasma acth concentration, ultrasonography of the adrenal glands and computer tomography (ct) of the pituitary and the adrenal glands in dogs with consistent clinical signs. only newly diagnosed dogs, before treatment for pdh, were evaluated. two control groups were included: one healthy and one sick control dog (without pdh) for each dog with pdh were included. healthy control dogs (hcd) and sick control dogs (scd) were matched for age (ae months), breed, sex and sexual status. data were analysed using non-parametric tests and expressed as median and ranges. significance was set at p < . . one-hundredsixty-seven dogs with pdh were eligible for inclusion in the study. the median age at diagnosis was years (range: - ) and the median body weight was . kg (range: . - . ). there were female ( spayed) and male ( castrated). serum phosphate concentration ( . mg/dl, . - . ) was significantly (p < . ) higher compared to hcd ( . mg/dl, . - . ) and scd ( . mg/dl, . - . ). serum calcium concentration ( . mg/dl, . - . ) was significantly higher compared to scd ( . mg/dl, . - . ) but not different compared to hcd ( . mg/dl, . - . ). urinary fractional excretion of phosphate ( . %, . - . ) was significantly lower compared to hcd ( . %, . - . ) and scd ( . %, - . ). urinary fractional excretion of calcium ( . %, - . ) was significantly higher compared to hcd ( . %, . - . ) and scd ( . %, - . ). urinary calcium to creatinine ratio ( . , - . ) was significantly higher compared to hcd ( . . - . ) and scd ( . , - . ), while urinary phosphate to creatinine ratio were not significantly different in pdh dogs, hcd and scd. in conclusion pdh dogs have lower phosphaturia and higher calciuria compared to control dogs. this findings suggest that, at least in part, the high serum phosphate concentrations are related to the renal retention of phosphate. no conflicts of interest reported. four cortisol-based methods of monitoring trilostane treatment of canine hyperadrenocorticism were compared to the results of a clinical scoring scheme based on an owner questionnaire. cases of canine hyperadrenocorticism that had received a consistent dose of trilostane for more than one month were recruited from first opinion and referral practice. each dog was used only once. owners were asked to complete a questionnaire that assessed clinical control. the dogs were then categorised as being over-controlled, well-controlled, moderately-controlled and poorly-controlled. cortisol was measured in serum samples taken pre-trilostane (peak), hours post-trilostane (trough) and hour post-acth injection. dogs that had an increase in cortisol after trilostane administration were excluded. a scoring system was developed for each of these measurements. a fourth scoring system was developed using a novel algorithm that combined the peak and trough cortisol (peak-trough). the results of each of the scoring systems categorised the dogs into those that would be expected to be over-controlled, well-controlled, moderately-controlled and poorly-controlled. weighted kappa was calculated to assess the agreement between the categorisation according to each of the methods compared to the categorisation using the owners score. the pearson correlation coefficient was calculated to assess relationships between the various parameters. in total tests were analysed. when compared to the results of the owner's questionnaire , , and dogs were correctly categorised using the peak-trough, peak alone, post-acth and trough alone respectively. amongst the miscategorised results , , and dogs were incorrect by category and , , and dogs by categories using the peak-trough, peak alone, post-acth and trough alone respectively. all methods correctly recognised the over-controlled dog that had been identified by the owner's score. the weighted kappas for post-acth and trough cortisol categories compared to the owner score categories were . and . respectively (defined as slight agreement). in contrast the weighted kappas for the peak and peak-trough categories were . and . respectively (defined as fair agreement). there were no significant correlations between the absolute clinical scores and cortisol concentrations. there were significant correlations between the cortisol measurements. the novel methods of peak-trough and peak cortisol better reflected the level of clinical control of hyperadrenocorticism identified by the owners' questionnaire than either post-acth stimulation or trough cortisol. peak-trough and peak cortisol concentrations should be further investigated as monitoring methods for trilostane. financial support from dechra pharmaceuticals. the prognosis of canine adrenocortical insufficiency is generally regarded to be excellent. however, there is paucity of sur-vival analyses in the literature. the aim of the present study was to evaluate the survival of dogs with the diagnosis adrenocortical insufficiency based on data from a cohort of , swedish client-owned dogs insured in one insurance company (agria pet insurance, stockholm, sweden) during the time period - . dogs were identified by search for insurance claims with the register code for adrenocortical insufficiency. dogs were excluded from analysis if they had a previous history of hypercortisolism, and if they were born before begin of the study period. kaplan-meier survival analysis was performed. dogs were regarded as censored when the registered cause of death was other than adrenocortical insufficiency or hypercortisolism that was registered after the first claim for adrenocortical insufficiency. data from dogs was included. one hundred twenty-four dogs were registered to be dead. in dogs the cause of death was related to the adrenocortical insufficiency. the -year estimated survival-rate was % ( % ci, - %). the -year estimated survival-rate was % ( % ci, - %). the -year estimated survival-rate was % ( % ci, - %). twelve dogs ( . %) were still alive after years. in conclusion, the long-term survival of dogs with adrenocortical insufficiency was reasonably good. however, the diseases-related mortality was higher than expected, and occurred mainly during the first years after diagnosis. conflicts of interest: this study was supported by grants from the swedish research council and the foundation for research, agria insurance company. the concomitant occurrence of two or more endocrine tumors and/or hyperplasias, known as multiple endocrine neoplasia (men) is a well-known entity in humans. multiple gene mutations have been identified. the two major forms are men and men . in men , the main affected organs are parathyroid, pancreas and pituitary gland. men occurs in clinical variants: men a, characterized by medullary thyroid carcinoma (mtc), pheochromocytoma and primary hyperparathyroidism; men b, characterized by mtc, pheochromocytoma and additionally abnormalities; familial medullary thyroid carcinoma. in dogs and cats only a few cases have been reported and it is unknown whether hereditary men-like syndromes exist in these species. the aim of this study was to evaluate the prevalence of multiple endocrine tumors in dogs and cats at our institution, to identify possible breed and sex predispositions and to investigate similarities with the human men syndromes. autopsy reports of dogs and cats from until were reviewed. animals with at least two endocrine tumors/hyperplasias (eth) were included. autopsy reports of dogs and cats were examined. dogs had eth affecting a single organ, had multiple eth; cats had single eth, had multiple eth. in dogs with multiple eth, the most common breeds were west highland white terrier (whwt, / ), poodle, golden retriever, mixed-breed dogs (each / ). / were male ( intact); / were female ( neutered). median age was years (range - ). the most common combination was multiple testicle tumors of various types ( / ). the most common affected organs were the adrenals ( / ). adrenal cortical adenomas/carcinomas/hyperplasias were mainly associated with pheochromocytomas ( / ), testicle tumors ( / ) and insulinomas ( / ). all whwts had adrenal adenomas. both poodles had pheochromocytoma associated with pituitary adenoma or adrenal hyperplasia. dogs showed tumor combinations similar to the human men syndrome: pituitary adenoma and insulinoma; pituitary adenoma and parathyroid hyperplasia. / cats were domestic short/long hair, / were persians. / were male ( castrated); / were female ( neutered). the median age was . years (range - ). the most common affected organs were thyroid glands ( / ), combined mostly with lesions of parathyroid ( / ) and adrenal glands ( / ). none of the cats had combinations similar to the human men syndromes. the prevalence of multiple eth in dogs and cats was . % and . %. men-like syndromes were extremely rare in dogs and non-existing in cats. no sex predisposition was observed. possible breed predispositions need further investigations. no conflicts of interest reported. canine angiostrongylosis is an increasingly reported disease worldwilde, including many european countries, possibly due to climatic factors, presence of foxes (acting as reservoir) or more simply, to the availability of more accurate diagnostic methods. although detection of the first-stage larvae (l ) using the baermann technique on faecal samples (preferably collected over three consecutive days) remains the gold standard, recently developed serological and molecular tests (quantitative polymerase chain reaction, qpcr) are now available. until now, the prevalence of canine angiostrongylosis among healthy and coughing dogs in belgium was unknown. the aims of the present study were ( ) to describe a clinical series of recent autochtonous cases and ( ) to retrospectively assess angiostrongylus vasorum qpcr in bronchoalveolar lavage fluid (balf) samples, collected over the last years from a larger series of dogs, healthy or with other respiratory conditions, in order to investigate the past prevalence of the disease in belgium. seven dogs, living in southern or eastern belgium, were recently diagnosed as having angiostrongylosis (mean age= . y, mean body weight= . kg). they all presented with respiratory signs of variable severity. in dogs, balf was obtained and qpcr was positive in all of them, at moderate or high level (ct from , to , ) while larvae were detected in the faeces of only animals. in the remaining two dogs, no balf was obtained, but coproscopy was positive. all dogs responded to medical treatment, consisting in a -week course of fenbendazole and/or two spot-on application of moxidectin at -month interval. balf samples were collected between and from asymptomatic client-owned dogs and dogs with various respiratory conditions, including dogs with confirmed bordetellosis, dogs with eosinophilic bronchopneumopathy (ebp), dogs with chronic bronchitis and dogs with bacterial bronchopneumonia, and were retrospectively assessed with a a. vasorum qpcr assay. amongst those dogs, only one balf, from a dog with ebp, yielded a positive qpcr result. in this dog, faecal analysis was negative. the present data show that, based on balf qpcr and coproscopy, presence of angiostrongylosis in healthy and coughing dogs was negligible in belgium until the last months. it is now considered as an emerging condition and must be included in the differential diagnosis in coughing dogs. the present results also support that qpcr detection of a. vasorum in balf, when available, is an adequate and reliable detection technique. no conflicts of interest reported. ligneous membranitis is a rare chronic inflammatory disease associated with congenital plasminogen deficiency. it has only been described in six unrelated dogs. the objective of this study is to report the presentation, clinicopathological and post mortem findings in three related scottish terrier puppies with ligneous membranitis. ligneous membranitis is well described in humans, where it is inherited in an autosomal recessive manner. patients commonly present as infants. ocular, oral and genital lesions are most common, but other organs are occasionally involved and congenital obstructive hydrocephalus is reported in some individuals. numerous mutations and polymorphisms in the plasminogen gene have been identified in affected individuals. the affected scottish terriers (two male and one female) presented at months of age with severe proliferative and ulcerative conjunctivitis and gingivitis/stomatitis; biopsy confirmed ligneous membranitis. other clinical signs included increased upper respiratory tract noise, nasal discharge and lymphadenopathy. one male was cryptorchid. clinical pathological findings included neutrophilia, proteinuria and hypoalbuinaemia. serum plasminogen activity was measured in two dogs, and was low in one. the dam and sire of the affected dogs had normal serum plasminogen activity and no history or clinical signs consistent with ligneous membranitis. no significant clinical improvement was evident following treatment with antibiotics, glucocorticoids, topical ciclosporin or heparin. one dog died of cardiopulmonary arrest in the hospital and the two other dogs were euthanized due to progressive clinical signs. post-mortem evaluation of the affected dogs revealed multiple abnormalities including severe proliferative fibrinous lesions affecting the trachea, larynx and epicardium, and multiple fibrous adhesions throughout the thoracic and abdominal cavities. the male dog had internal hydrocephalus and lacked a cerebellar vermis. this is the first report of ligneous membranitis in related dogs and the first report in scottish terriers. sequencing the plasminogen gene in the affected dogs, their parents and unrelated control dogs to identify polymorphisms or mutations that may be associated with ligneous membranitis in dogs is ongoing. the author received a travel scholarship from zoetis to attend this congress. health screening of elderly dogs is often recommended, but scientific information on clinical and laboratory abnormalities in senior and geriatric dogs is scarce. this study was undertaken to describe blood pressure measurement, physical examination (pe) abnormalities and routine laboratory test results in senior and geriatric dogs that were apparently healthy for the owner. because life expectancy in dogs is related to body size, the inclusion of dogs was based on a human/pet analogy chart to determine whether a dog was senior (n = ) or geriatric (n = ). to verify health status, owners were asked to complete an extensive questionnaire. systolic blood pressure (sbp) was measured using the doppler technique according to the acvim guidelines. subsequently a thorough pe was performed, including body and muscle condition scoring, orthopedic examination, neurologic evaluation, indirect fundoscopy and bilateral schirmer tear test. complete blood count, serum biochemistry and urinalysis (including urinary sediment, urinary protein:creatinine ratio (upc) and bacterial culture) were evaluated. in of dogs sbp exceeded mmhg, none of the dogs had fundoscopic lesions secondary to hypertension. body condition score was abnormal in animals, were overweight or obese. physical examination revealed a heart murmur in , submandibular lymphadenopathy in , moderate to severe dental plaque in and one or more (sub)cutaneous masses in dogs. twenty-three dogs were leukopenic, had a decreased phosphorus, an increased serum creatinine and one dog a decreased total thyroxine (with concurrent increased thyroid stimulating hormone). crystalluria was commonly detected ( / ) and mostly due to low numbers (< /high power field) of amorphous crystals ( %). struvite crystals were present in % of the crystalluric dogs. overt and borderline proteinuria were detected in and of dogs, respectively. four dogs had a positive urinary culture. sbp was not significantly different between the senior and geriatric group. there was no significant effect of obesity or gender on sbp. the platelet count (p = . ), total thyroxine concentration (p = . ) and the frequency of orthopedic problems (p = . ) and cutaneous masses (p = . ) were significantly higher in the geriatric compared to the senior dogs. hematocrit (p = . ) and body temperature (p = . ) were significantly lower in the geriatric group. these findings indicate that physical and laboratory abnormalities are common in apparently healthy senior and geriatric dogs. this underlines the necessity for regular health screening in elderly dogs and the urgent need for reliable and maybe age specific reference intervals in veterinary medicine. the cost of examinations reported in this study were covered by hill's pet nutrition belgium. systemic lupus erythematosus, sle, is a chronic autoimmune disorder with varying clinical manifestations and diagnosis is based on both clinical signs and laboratory findings. other systemic rheumatic diseases, referred to as sle-related diseases or immune-mediated rheumatic disease (imrd), are also described. the most common clinical signs in dogs are stiffness and pain from varying joints. one hallmark of sle and sle-related diseases in both dogs and humans is high titres of circulating antinuclear antibodies (ana), which can be demonstrated by the indirect immunofluorescence (iif) ana test. earlier studies have shown that canine iif ana positive samples may be divided into two main subgroups: homogenous (ana h ) and speckled (ana s ) iif ana fluorescence pattern. in humans, further determination of the specificity of ana positive sera is frequently employed to characterize the ana reactivity. some of these ana specificities have been demonstrated in man to strongly associate with different systemic autoimmune diseases and also with different iif ana staining patterns. presence and character of antinuclear antibodies in canine sle-related diseases are not well described. the aim of this work was to further characterize the ana specificity in dogs with sle-related disease/imrd. sera from anapositive dogs, including different breeds, were analyzed with elisa and line blot techniques (elisa and euroline ana profile, euroimmun, germany). the five most prevalent breeds were german shepherd dog, nova scotia duck tolling retriever, cocker spaniel, crossbreed and golden retriever. sera displayed a homogenous and a speckled iif ana fluorescence pattern. several specific ana-reactivities earlier characterized in human patients were identified. the majority of ana h , n = , %, showed reactivity against nucleosomal antigens and ( %) against dsdna when conducted on line blot. these sera also reacted against nucleosomes and dsdna on the elisa. there were some additional positive with the elisa, so in total the elisa identified % with nucleosomal and % with dsdna reactivity. in few cases, other reactivities identified were against histones, pcna, jo- and rnp. in the ana s subgroup, the sm+rnp antigen evoked the most frequent reactivity, n = , % with both line blot and elisa. in few cases, reactivity against dsdna, pcna, jo- , pmscl kd, scl- , ssa and ssb were identified. in several dogs no specific antigen was identified. further studies are in progress in order to in more detail characterize and identify subtypes of already known and unknown antigens with clinical importance in canine autoimmunity. one of the authors, erik lattwein, is employed by euroimmun where the analyses were performed. chronic kidney disease (ckd) has a high prevalence in cats. routine renal markers, serum creatinine (scr) and urea are not sensitive or specific enough to detect early ckd. serum cystatin c (scysc) has advantages over scr for the detection of early kidney dysfunction, both in humans and dogs. a significant higher scysc concentration in ckd cats has been demonstrated. the objective of this study was to determine the effect of age, gender and breed on feline scysc and to establish a reference interval for feline scysc. in total, healthy cats between one and years were included. serum cysc was determined with a validated particleenhanced nephelometric immunoassay (penia). serum cr, urea, urine specific gravity (usg), urinary protein: creatinine ratio (upc) and systolic blood pressure (sbp) were also measured. to test for difference between the groups, the f-test was used. the lower and upper value of the % reference interval were obtained as the . % and . % quantiles of the scysc observations. no significant differences in scysc concentration were observed between young, middle-aged and old cats; between female, female neutered, male and male neutered cats; and between purebred and domestic short-or longhaired cats. the % reference interval for feline scysc was determined as [ . - . mg/l]. there was a significant difference in scr concentration between domestic short-or longhaired cats and purebred cats. the sbp was significantly influenced by gender as well as age, while urea was influenced by both age, gender and breed. this study showed that the biological factors age, gender and breed have little or no impact on feline scysc, in contrast to scr and serum urea, making it an interesting marker. therefore, further studies are warranted to evaluate the diagnostic value of scysc as a renal marker in cats. this study recieved support from the institute for the promotion of innovation by science and technology in flanders (iwt) through a bursary to l. ghys. assessment of renal function is often needed, however existing methods including urine and plasma clearances are invasive, cumbersome and time consuming. in this pilot study the feasibility of a transcutaneous glomerular filtration rate measurement was investigated. the transcutaneous disappearance rate (expressed as half-life) of fluorescein-isothiocyanatelabelled sinistrin (fitc-s) was measured in three healthy research dogs and three healthy research cats. plasma clearance of sinistrin ( data points) was performed in both species as previously described (res vet sci ; : - and j fel med surg ; : - ) and half-life was calculated using a -compartment model with a freely available pharmacokinetic calculator (comput meth prog bio ; : - ) . renal elimination of fitc-s was measured transcutaneously for hours ( - data points) using a miniaturized device as described previously for the same purpose in rats (kidney int : - ). the procedures were performed in awake, freely moving animals using escalating doses of fitc-s ( mg/kg, mg/kg, mg/kg) with a wash-out period of at least h in each animal. to find the best position for the device, multiple devices were placed on each animal. the resulting fitc-s disappearance curves were visually assessed to determine the most suitable location and the appropriate dose to reach an adequate transcutaneous peak signal for kinetic analysis. in both species mg/kg were adequate for kinetic calculation. the most suitable place for the device was the lateral thoracic wall in dogs and the ventral abdominal wall in cats, respectively. transcutaneous fitc-s clearance was then repeated using the optimal dose and location and in parallel with the plasma sinistrin clearance. plasma sinistrin clearances [ml/kg/min] were . , . and . in the three dogs, respectively. corresponding plasma elimination half-lives [min] were , and , and corresponding transcutaneous elimination half-lives [min] were , and , respectively. plasma sinistrin clearances [ml/kg/min] were . , . and . in the three cats, respectively. corresponding plasma elimination half-lives [min] were , and , and corresponding transcutaneous elimination half-lives [min] were , and , respectively. in conclusion, transcutaneous fitc-s clearance is a feasible method for assessment of gfr in awake dogs and cats. it is noninvasive, well tolerated and easy to perform even in a clinical setting with results being readily available. a dose of mg/kg of fitc-s seems adequate for kinetic assessment. further studies are now needed to establish reference values and evaluate transcutaneous renal clearance in various conditions. conflicts of interest: zhp and sg are supported by the ec fp marie-curie programme: nephrotools. the device development was supported by the fp activity: place-it.ng is owner of a patent covering fitc-sinistrin and the technology for its measurement. excretion of urinary biomarkers of renal damage should occur at an early stage of chronic kidney disease (ckd), thus facilitating earlier diagnosis of renal disease. albumin and cystatin c in the renal ultrafiltrate are mostly reabsorbed by the proximal tubular cells, therefore increased urinary excretion of albumin and cystatin c (uac and ucysc) would be expected to correlate with the presence of renal tubular damage and ckd. the aim of this study was to establish biological validity of two particle enhanced turbidimetric assays (petias) for the measurement of albumin and cystatin c (previously validated for use in feline urine) by comparing the uac and ucysc between non-azotaemic cats and cats with azotaemic ckd. blood and urine samples were obtained from cats at three uk first opinion practices as part of a geriatric screening programme. haematology, serum biochemistry (including total thyroxine concentration (tt )) and urinalysis (including urine protein:creati-nine ratio (upc)) were performed. dental disease score (calculus and gingivitis) and body condition score (bcs) were recorded. cats with tt > nmol/l, evidence of pyuria or bacteruria, or significant systemic disease were excluded. uac and ucysc were determined in non-azotaemic cats (n = ) and cats with azotaemic ckd (n = , defined as a serum creatinine concentration > lmol/l and concurrent urine specific gravity < . ). comparisons between the non-azotaemic and azotaemic ckd groups were made using the mann whitney u test. correlations were assessed by spearman's correlation coefficient. data are presented as median [ th , th percentile] and statistical significance was defined as p < . . uac was significantly higher in the azotaemic group than the non-azotaemic group ( . [ . , . ]x - vs. . [ . , . ]x - ; p = . ), whereas upc was not significantly different between the groups (p = . ). unexpectedly, ucysc tended to be lower in azotaemic cats than non-azotaemic cats ( . [ . , . ]x - vs. . [ . , . ]x - ; p = . ). uac was weakly positively correlated with serum urea concentration (r s = . , p = . ), but was not correlated with serum creatinine concentration. ucysc was not significantly correlated with serum concentrations of urea, creatinine or tt . uac was also weakly negatively correlated with dental calculus score (r s = - . ; p = . ) and bcs (r s = - . ; p = . ). uac appears to be a more sensitive test for azotaemic ckd than upc, however the apparent low specificity may limit the utility of uac as a urinary screening test for ckd. increased ucysc (determined by petia) would not appear to be a marker of azotaemic ckd in cats. no conflicts of interest reported. cystinuria is an inherited metabolic disorder that causes defective tubular reabsorption of the aminoacids cystine, ornithine, lysine and arginine (cola). the low solubility of cystine in acidic urine promotes formation of cystine crystals and uroliths in the urinary tract resulting in the clinical signs of stranguria, urinary obstruction and renal failure in affected individuals. cystinuria occurs in > breeds of dog and has been classified into types ia (newfoundland, landseer, labrador), iia (australian cattle dog), ib (miniature pinscher) and iii (androgen-dependent; e.g. mastiff, irish terrier). the kromfohrl€ ander is a medium-sized companion dog, bred initially as a cross between a wire fox terrier and a grand griffon vend een, first recognised internationally in . cystinuria has been suspected in this breed but no cases have been reported in the literature to date. we determined urinary cola concentrations in adult kro-mfohrl€ ander dogs aged - years comprising intact and castrated males, and intact and spayed females. a total of ( %) intact males aged . to . years had cola values > lmol/g creatinine and several developed cystic calculi. furthermore, intact male dogs had increased cola but normal cystine levels. all castrated males had normal cola concentrations. no females had increased cola and cystine concentrations or formed any cystine calculi. we conclude that cystinuria with cystine calculi occurs frequently in adult intact male kromfohrl€ ander dogs but neither is seen in females. this appears to be an androgen dependent type iii cystinuria, as seen in mastiff-type dogs and irish terriers. thus, castration may resolve the increased urinary cola excretion and risk for cystine calculi formation and obstruction. the precise mode of inheritance is still unclear. all adult intact male kromfohrl€ ander dogs should be screened by urinary cola testing. work carried out at the author's previous place of employment (university children's hospital, frankfurt, germany). acs, rk, em, md and ug provide a diagnostic service for cystinuria and other inborn errors of metabolism in companion animals. ureteral urolithiasis is an emerging medical concern in cats. there are few reports on epidemiology, diagnosis or medical management of ureteral calculi in cats, particularly in europe. cats diagnosed with ureteral urolithiasis in the teaching hospital of the veterinary school of alfort from to were included in this study. diagnosis was confirmed with radiographs, ultrasound scan and/or laparotomy. signalment, clinical signs, clinicopathologic and diagnostic imaging findings, medical treatment and outcome were recorded. epidemiological data were compared to a reference population of cats. eighty three cats were included in the study. the occurrence of ureteral urolithiasis was significantly higher in birman to the author knowledge, it is the first time that a higher prevalence of ureteral calculi in birman cats is reported in europe. spontaneous elimination of calculus is associated with a small size (< . mm). if the size of calculi tends to be bigger in cats with no improvement of renal function after medical treatment, prospective studies are still needed to determine the best medical treatment. no conflicts of interest reported. feline immunodeficiency virus (fiv) infection has been associated with kidney disease, mainly characterised by an increased prevalence of proteinuria in fiv-infected cats. however, studies evaluating renal variables in fiv-positive cats are scarce. recently, a higher systolic blood pressure (sbp) was reported in a small number of fiv-infected cats. hypertension is an important cause of proteinuria and a frequent cause of renal disease in human immunodeficiency virus (hiv) positive patients. therefore, our main objective was to describe sbp in clinically ill fivpositive cats. secondly we aimed to evaluate routine renal variables in this population. naturally infected clinically ill fiv-positive cats were prospectively included. the doppler ultrasonic technique was used to measure sbp according to acvim guidelines. serum creatinine (screat) and urea (surea) concentrations, urine specific gravity (usg) and urinary protein:creatinine ratio (upc) were determined. the study included cats, with a mean age of . ae . years and a mean body condition score of . ae . on a nine-point scale. the sbp ranged from to mmhg, with a mean of ae mmhg. only two cats were hypertensive (sbp > mmhg). both had isosthenuric urine, were borderline proteinuric (upc . - . ) and one of them was mildly azotemic. mean screat was . ae . lmol/l (reference interval (ri) . - . lmol/l) and mean surea concentration . ae . mmol/l (ri . - . mmol/l). thirteen cats showed increased screat levels, with decreased usg (< . ) in eight, proteinuria (upc > . ) in seven and increased surea concentrations in ten of them. five out of ten azotemic cats were proteinuric with a decreased usg. mean upc was . ae . , with a wide range from . to . . borderline proteinuria was present in / ( . %) and proteinuria in / ( . %). half of the proteinuric cats had a decreased usg. mean usg was . ae . . one third of all cats had a decreased usg, with isosthenuria in seven of them. these results demonstrate that proteinuria and poorly concentrated urine are common in naturally infected clinically ill fivpositive cats, confirming previous reports in cats and humans. however, longitudinal studies of (borderline) proteinuric patients are needed to elucidate the clinical relevance. the low number of hypertensive patients and low mean sbp in our study indicate that hypertension is uncommon and unlikely to be the cause of renal damage in clinically ill fiv-infected cats. aratana therapeutics nv financially supports a clinical trial on the use of antivirals in fiv cats at our university. screeninng examinations repoorted in this trial were part of the required pre-trial investigations for that study. the presenting author is also funded by a scholarship from aratana therapeutics av. the objective of this study was to identify the prevalence of bacterial species and the susceptibility of isolates to doxycycline, trimethoprim-sulfamethoxazole (tms), ampicillin, amoxicillinclavulanic acid (amc), cephalothin, and enrofloxacin in cats with urinary tract infections (uti) with and without predisposing comorbidities. a retrospective analysis of case records between and was performed and resulted in inclusion cats into the study: cats with a systemic comorbidity, cats with indwelling urinary catheters, cats with other local comorbidities, and cats with no comorbidity. the most commonly isolated bacteria were escherichia coli (e. coli), streptococcus species (spp.), staphylococcus spp., and enterococcus spp.. the proportion of gram-negative isolates was significantly higher in the cats with systemic comorbidities than in cats with indwelling urinary catheters (p < . ) and cats with other local abnormalities (p < . ), whereas gram-positive isolates were significantly more commonly isolated from cats with indwelling urinary catheters and other local comorbidities than in cats with systemic comorbidities (p < . ). the proportions of isolates susceptible to amc, enrofloxacin, and tms and the antimicrobial impact factors (if) were higher than the proportions of isolates susceptible to doxycycline, ampicillin, and cephalothin and the respective if. based on these findings, amc and tms would be the firstchoice antimicrobial agents for empiric treatment of bacterial the increasing rates of resistance exhibited by uropathogens represent a serious problem for the selection of an appropriate antibiotic. the aim of this study was to determine secular trends of companion animal urinary tract infection (uti) that involve extended-spectrum b-lactamase (esbl)-and carbapemenase-producing gram negative bacteria (namely, escherichia coli, klebsiella pneumoniae, proteus mirabilis, acinetobacter baumannii), methicillin-resistant-staphylococci (mrs) and ampicillin and high-level-gentamicin-resistance (hlgr) enterococci. nine hundred and twenty two uropathogenic bacteria were isolated from dogs and cats, between january and march , at the veterinary teaching hospital of the faculty of veterinary medicine and at veterinary private practices in the lisbon area. isolates were identified using standard commercial systems. susceptibility testing was performed using the disk diffusion and broth microdilution methods. clsi breakpoints were applied. extended-spectrum b-lactamases (esbl) production was screened by double-disk synergy test. the esbl, plasmid-mediated ampc, carbapemenases, meca and aac( ')-ieaph( '')-ia genes were detected by pcr and gene enzymes were sequenced. among enterobacteriaceae . % were dhaproducers, . % were esbl-producers and . % were cmyproducers. all isolates were also multidrug-resistant. cefalosporinases-producer enterobacteriaceae were detected in , the first being a cmy- -producer e. coli. all the esbl-producers were e. coli or k. pneumoniae producing ctx-m-group enzymes. ampicillin-resistance in enterococci was present throughout the years ( , %, n = ). hlgr appeared in enterococci in and was confirmed by the detection of the bifunctional enzyme that confers high level resistance to aminoglycosides ( out of isolates). in this study we showed that in the last decade the emergence of resistance to critically important antimicrobials among uropathogens from companion animals is a concerning fact. the multidrug-resistant enterobacteriaceae may compromise effective therapeutic options, namely third and fourth generation cephalosporins, fluoroquinolones, trimethoprim/sulpha combinations. the emergence of mrsa/mrsp and hlgr among uropathogens is also a therapeutic challenge. the detection of uropathogens with antimicrobial resistance is not only an animal health issue but also a matter of public health, since companion animals may act as reservoirs of antimicrobial resistant bacteria or resistance genes for humans. the author currently receives a phd grant funded by the portuguese foundation for science and technology. in the past, the author received once research support and honoraries from portuguese merial for a project on canine vector borne diseases. the aim of the present study was to use insurance data to estimate morbidity and mortality related to kidney disease in the swedish dog population. insurance company data from veterinary care-insured and lifeinsured dogs during the years - were studied retrospectively. incidence-and mortality rates were calculated for the whole group of dogs as well as divided by sex and breed. for the breeds with the highest incidence-and mortality rates, respectively, the reasons for kidney disease were investigated by dividing the diagnoses into ethiology groups. the total number of veterinary care-insured dogs was , . the total incidence rate of kidney disease in this group of dogs was . ( . - . ) cases/ , dog-years at risk. the number of dogs in the life insurance was , and in this group the total kidney-related mortality rate was . ( . - . ) deaths/ , dogyears at risk. the most commonly reported ethiologies of kidney disease were "ethiology not determined"and "infectious/ inflammatory". the breeds with the highest incidence rate of kidney disease were the bernese mountain dog, miniature schnauzer and boxer. the breeds with the highest mortality caused by kidney disease were the bernese mountain dog, shetland sheepdog and flatcoated retriever. in conclusion, the epidemiological information provided in this study concerning kidney disease in dogs can assist clinicians in establishing diagnoses, and can assist breeders in defining priorities for preventative measures. it can also provide valuable information for future research. the senior author has received money from the insurance company we have used data from to write our study, for another project. jens h€ aggstr€ om and ingrid ljungvall have received financial support for research from sante animale, agria insurance ltd, sveland insurance ltd, forsgren research foundation. both of these authors have also undertaken paid consulatcny work for boehringer-ingelheim, ceva sante animale. mast cell tumours represent the most common cutaneous tumour in the dog. diagnosis of a mast cell tumour can be achieved through cytological examination of fine needle aspirate. however the grade of the tumour is an important prognostic marker and requires so far histologic assessment. a -tier histologic grading system based on number of mitoses, multinucleated cells, bizarre nuclei and karyomegaly was recently proposed by kiupel et al. the aim of this study was to assess if the cytomorphological criteria proposed in the -tier histologic grading system are applicable on cytology specimens. ninety-three mast cell tumour specimens of grade i or grade iii according to patnaik with both histological specimens and fine needle aspirates were retrospectively taken from a data set and histologically and cytologically re-evaluated. according to the kiupel grading system thirty-six were diagnosed histologically as high grades and fifty-seven were considered low-grade mast cell tumours. the cytologic examination of the corresponding specimens revealed thirty-one high grade and fifty-five low-grade tumours. an agreement between histologic and cytologic diagnosis based on the kiupel grading system was achieved in eighty-six cases (accuracy . %, specificity . %, sensitivity . %). five high-grade tumours ( . %) were considered as low grade on cytology. cytologic grading of mast cell tumours in the dog has satisfactory accuracy, sensitivity, and specificity. histologic grading of canine mast cell tumours still remains the gold standard, but cytology already gives reliable information. no conflicts of interest reported. in canines mastocytomas are among the most frequently diagnosed neoplasms of the skin. high grade mastocytomas (grade iii, patnaik classification) are characterized by an uncontrolled growth of neoplastic mast cells (mc) and a poor prognosis. recently, the kit-targeting tyrosine kinase inhibitors masitinib and toceranib have been approved for the treatment of canine mc tumors. these drugs are able to induce responses in mastocytoma patients. however, in many patients, relapses are seen. therefore, research is focusing on new drug targets. recently, the transcription factor stat has been reported to play an important role in the proliferation and survival of human neoplastic mc. the aim of the present study was to evaluate the jak -stat pathway in canine mastocytomas. to address this aim, the canine mastocytoma cell lines c and ni- as well as inhibitors directed against jak or stat were employed. as assessed by immunocytochemistry, c cells and ni- cells were found to express pstat in their cytoplasm and nuclei. intracellular expression of pstat was confirmed by flow cytometry. interestingly, c cells were found to express higher levels of pstat compared to ni- cells. next, we treated c cells and ni- cells with various concentrations of the stat inhibitors piceatannol and pimozide and the jak inhibitors azd and tg . as assessed by h-thymidine uptake, all compounds were found to inhibit the proliferation of canine mc in a dose-dependent manner. drug effects were found to vary in different cell lines, with the following rank-order of potency (ic values): tg : . - . lm; pimozide: . - . lm; azd : - lm; piceatannol: - lm. to further explore the mechanism of drug-induced inhibition of proliferation, we examined cell cycle progression and apoptosis in drug-exposed cells. whereas all drugs tested induced only moderate cell cycle arrests in the g phase, these drugs were found to induce substantial apoptosis in c cells and ni- cells as evidenced by microscopy and annexin-v/pi staining. together, our data show that jak -and stat -targeting drugs exert anti-proliferative and apoptosis-inducing effects in canine mastocytoma cells suggesting that this signaling pathway may be a promising new therapeutic target in canine mastocytomas. the clinical relevance of this observation remains to be determined. no conflicts of interest reported. subcutaneous mast cell tumours (sqmct) in dogs are relatively uncommon compared to their cutaneous counterparts. the veterinary literature describes these tumours as a specific pathological entity with, in general, a low probability of aggressive progression. surgery is considered the main treatment modality, while medical treatment has not been described. the purpose of this study was to determine progression free survival (pfs) for a chemo na€ ıve cohort of dogs presented with non-resectable and/ or metastasized sqmct, which all underwent masitinib-based therapy. data were collected for patients with sqmct presented to participating centres in the netherlands and the uk from / / to / / , which received masitinib-based therapy. treatment protocols employed, included masitinib alone (m), masitinib and prednisolone (mp), masitinib plus vinblastine and/ or lomustine and prednisolone (mpc). response to therapy was measured conforming to recist . . adverse events were graded using vcog-ctcae . . patients were grouped according to presence or absence of metastasis, treatment protocol used, previous surgery, and remission status achieved; simple comparisons were made to evaluate possible significance. twenty-five cases were identified. / were female. median age of occurrence was years ( - ). diagnosis was made by histology in / ; proliferation indices were defined in only dogs. fourteen cases exhibited metastasis at initiation of therapy. pfs for all cases ranged from - days. median/mean pfs (days) according to treatment was m: / d (n = ), mp: / d (n = ), mpc: / d (n = ). median/mean pfs according to metastasis status was m : / d (n = ) and m : / d (n = ). dogs who underwent previous surgery (n = ) had a median/mean pfs of / d compared to those who had no surgery / d. looking at remission status, median pfs of patients who achieved a complete remission was not reached, with a mean pfs of d (n = ). median/mean pfs of patients with partial remission was / d (n = ), stable disease / d (n = ), and progressive disease / d (n = ). / cases experienced suspected adverse events. three dogs, two of which ultimately died, had seven grade - adverse events (anaemia (n = ), hepatotoxicity (n = ), gastrointestinal toxicity (n = )). masitinib-based treatment is effective in the management of sqmct perceived to be aggressive. patients do not appear to benefit from prior surgery. metastatic status did not influence outcome. adding chemotherapy negatively influenced pfs. complete remission is a very favourable prognostic development. the authors have received financial support from ab science to help with the costs of statistical analyses in an unrelated project advances in distinction between morphological subtypes of canine non-hodgkin's lymphomas (nhl) have provided a better understanding of this cancer in dogs. diffuse large b-cell lymphomas (dlbcl) are the most frequent form of nhl in dogs including some distinguished morphological subtypes (mainly centroblastic polymorphic and immunoblastic) according to the who classification. few clinical studies reported dlbcl clinical outcomes under treatment while survival times of the centroblastic polymorphic subgroup were reported. the aim of this retrospective study was to evaluate the response of dlbcl to a standardized multi-agent chemotherapy protocol. medical records from dogs with a diagnosis of dlbcl between and were retrospectively reviewed. inclusion criteria were the availability of complete initial and follow-up information and the application of a standardized multi-agent l-cop chemotherapy protocol as previously described. dogs which received corticosteroids before the initiation of treatment and dogs which died for other reasons than their related disease before the end of the induction period ( d) were excluded. response to chemotherapy was evaluated every week during the induction of treatment, then every to weeks. statistical analysis was performed using kaplan-meier analysis. thirty cases of dlbcl meeting all inclusion criteria were included from the initial population. seven dogs were in clinical stage iii according to the who classification, in stage iv and in stage v. nineteen dogs were in substage a, and in substage b. on dogs which have an adequate response evaluation, dogs ( . %) achieved a complete remission. the median and mean duration of first remission were d and d, respectively (range - d). the median and mean durations of survival time were respectively d and d (range - d). one year and -years survival rates were % and % respectively. according to the statistical analysis, neither clinical stage (p = . ) nor substage at presentation (p = . ) or morphological subtype (immunoblastic vs centroblastic polymorphic, p = . ) were considered as a significant prognostic factor regarding the duration of the first remission and the overall survival time. a complete response was significantly associated with longer survival times (p = . ). to conclude, the dlbcl displayed a good clinical response to l-cop protocol, with a median survival time of days. the only significant prognostic criterion identified was a complete clinical response to the treatment. a prospective controlled study on a larger population is warranted to confirm these results. no conflicts of interest reported. canine histiocytic sarcoma (hs) is an aggressive round cell neoplasm with a poor prognosis. both lomustine and doxorubicin have been evaluated as first line chemotherapy agents with response rates of up to % and median survival times around - months. the aim of this study was to evaluate the response to epirubicin in a population of dogs with hs pre-treated with lomustine. medical records of dogs with a diagnosis of hs that were treated with lomustine and subsequently epirubicin were retrospectively evaluated. fifteen dogs received lomustine followed by epirubicin. there was a measureable response to lomustine in seven of dogs with evident disease, % ( cr & pr). an additional dogs achieved stable disease for an overall biological effective response of %. median ttp following lomustine could be assessed in dogs and was days (range - ). all fifteen dogs received epirubicin as a rescue agent: nine following progressive disease and three with stable disease on lomustine. one dog received epirubicin after completing six doses of lomustine in complete remission and two dogs in partial remission changed to epirubicin due to hepatotoxicity associated with lomustine. response rate to epirubicin was % and biologic effective response was % ( cr, pr, sd, pd) in dogs. one dog was euthanized due to epirubicin associated gastro-intestinal toxicity and dog stopped treatment with no assessment of response. median duration of response to epirubicin was days (range: - ) and dog is still alive and in remission ( days). overall median survival time for dogs receiving epirubicin following lomustine was days (range: - ). single agent epirubicin is a valid rescue therapy after lomustine for canine histiocytic sarcoma and results in modestly improved overall survival times in responding patients. the author received a travel scholarship from zoetis to attend this congress. the incidence of melanocytic lesions is increasing among canine population. canine malignant melanoma could have an aggressive behavior, metastasize early in the course of the disease and be resistant to most current therapeutic regimens leading to the need of finding markers with potential as therapeutic targets. the overexpression of cox- seems to play a key role in melanocytic tumours, having being described an association between high cox- immunoexpression and the malignant behavior. in order to contribute to the understanding of the role of cox- in melanocytic tumours, three main pathways were investigated: angiogenesis, tumour cell proliferation and inflammatory microenvironment (t-lymphocytes and macrophages). fifty one ( ) melanocytic tumours [ cutaneous ( malignant melanomas and melanocytomas) and oral malignant melanomas] were included. all the samples were submitted to immunohistochemical staining carried out by the streptavidinbiotin-peroxidase method, with a commercial detection system with or without melanin blanching, for detection of the following markers (cox- , ki- , factor viii, vegf, cd and mac ). in melanocytic tumours (n = ), both cox- labelling extension and intensity revealed a statistically significant association with angiogenesis by factor viii (p < , ), vegf (p < , ); ki- (p < , ), cd + t-lymphocytes (p < , ) and mac (p < , ). considering only malignant melanomas (n = cases), cox- labelling extension revealed a statistically significant association with angiogenesis (p = , ) and cd + t-lymphocytes (p = , ). cox- intensity was also positively associated with angiogenesis (p = , ) and with mac (p = , ). present study demonstrated a link between high cox- immunoexpression and increased angiogenesis and tumoural t-lymphocyte and macrophage infiltration in malignant melanomas. these findings reinforce the usefulness of using selective cox- inhibitors as a valuable therapeutic tool in malignant melanocytic tumours. this study received financial support from a company (merial). neuter status and risk of malignant neoplasia is not well evaluated in the canine population, when excluding neoplasia not normally believed to be sex-hormone dependent. denmark and the scandinavian countries have a high proportion of intact dogs compared to populations from other parts of the world. in the present study it was hypothesized that there would be no difference in gender and neuter status between the population of canine patients with a non-sex-hormone dependent malignant neoplasia reported to the danish veterinary cancer registry and a general population. from august to march , canine neoplasms were reported to the danish veterinary cancer registry. the total number of malignant ( ) and benign ( ) were comparable ( % and %). malignant neoplasms totalled , when tumors from areas of distribution with known sex-hormone dependency (reproductive organs, mammary gland, perineal), and cases with unknown area of distribution were excluded. the overall distribution of malignant neoplasia was ( %) intact male dogs, ( . %) neutered male dogs, ( %) intact female dogs and ( . %) neutered female dogs. the distribution was even between male and female dogs ( . % and . %). compared to a known standard population of dogs, there was an overall statistically significant association of malignant neoplasia with neuter status in both sexes. for both genders this was significant for lymphoma, mast cell tumors and osteosarcomas,. for neutered females, but not males, there was increased risk of hemangiosarcoma, squamous cell carcinoma and malignant melanoma. these findings indicate that there might be an association between neuter status and development of malignant neoplasia but larger prospective studies are needed to evaluate the risk of non-sex hormone dependent cancers in neutered dogs. no conflicts of interest reported. serum acute phase proteins (apps) are considered biomarkers of the acute phase reaction, and are being increasingly used in human and veterinary medicine in diagnosis and monitoring of neoplastic diseases. in the cat, serum amyloid a (saa) is considered a positive major app, haptoglobin (hp) a moderate app, and albumin and insulin-like growth factor- (igf- ) negative apps. the aim of the present study was to characterize the apps response in cats with mammary tumours. for that purpose, saa, hp, igf- and albumin serum concentrations were determined in female cats with malignant mammary tumours. cats with history of previous tumours or with concomitant tumours or other diseases were excluded. information on cats age, gender, breed, tumour type, histological grade, tumour size and location, skin ulceration, vascular neoplastic infiltration, necrosis, metastasis to regional lymph nodes, thoracic or abdominal organs, and survival time from diagnosis was assessed. blood samples were collected before surgery in all cats, and whenever possible, serial samples collected on control visits. owners gave informed consent. studied population included domestic short-haired cats with ages ranging from eight to years ( , + /- , ). all had carcinomas, including solid carcinomas (n = ), tubulopapillary carcinomas (n = ), one cribiform carcinoma and one carcinosarcoma. at the time of diagnosis, % of cats had an increase in serum concentration of hp and % of saa, and % had a decrease in concentration of albumin. mean and standard deviation values were of , + /- , g/dl for albumin (reference range , - , g/ dl), , + /- , lg/dl for igf- , , + /- , lg/ml for saa (reference value ˂ lg/ml), and , + /- , g/l for hp (reference value ˂ g/l). a positive correlation (r = , ) was detected between increases in serum concentrations of saa and hp. the increase in the size of tumour was significantly associated with the concentration of saa (p˂ , ). serum hp concentrations were significantly increased in tubulopapillary carcinomas (p˂ , ), and igf- decreased in solid carcinomas (p˂ , ). in the cats where serial determinations were performed, development of thoracic metastasis was significantly associated with a decrease of serum concentration of albumin (p˂ , ), and with an increase of saa (p˂ , ). this study suggests that feline mammary tumours are associated with an acute phase response. according with the results obtained, saa, hp, albumin and igf- might be important serum biomarkers in diagnosis and monitoring of the evolution of feline malignant mammary neoplasias. no conflicts of interest reported. histiocytic sarcoma (hs) is a neoplastic proliferation of interstitial dendritic cells or tissue macrophages. dogs with hs can present with local disease or with multifocal (disseminated) involvement. disseminated hs is poorly responsive to therapy and almost always fatal. little is established regarding the aetio-pathology of histiocytic sarcoma in dogs. the purpose of this study was to establish and characterise a hs cell line from fresh tumour samples obtained from a dog with disseminated hs in order to further clarify disease pathogenesis and behaviour. with owner consent, treatment-na€ ıve tumour sections were collected from a dog with disseminated hs that was euthanased. tumour tissue was assessed with immunohistochemistry (ihc) using antibodies against canine cd , cd , and pax- to support the diagnosis of histiocytic sarcoma. primary cell cultures (hscs), established from the tumour were cultured and maintained in modified eagle's medium with % fetal bovine serum, l-glutamine, penicillin and streptomycin, in standard conditions. hscs were characterised by alpha naphthyl acetate esterase (anae) and lysozyme staining while pcr was used to detect cell markers cd a, cd c, mhc ii, cd , ccr , e-cadherin, and cd . cell surface markers were compared to an established canine hs cell line (dh ). phagocytic activity of hsc cells was assessed using cellular uptake of carboxylated fluorescent beads and documented using flow cytometry and fluorescent microscopy. tumour tissue was strongly cd positive and negative for cd and pax- . cultured cells exhibited morphological characteristics consist with dendritic cells, such as projections and pleomorphism. hsc cells stained positively for non-specific esterase (anae) and lysozyme, and pcr indicated cells were positive for cd a, cd c, mhc ii and cd and negative for cd and e-cadherin. hsc cells were positive for mhc ii and ccr while dh cells were negative. phagocytic activity was evident. a novel hs cell line (hsc) was established and characterized from primary tumour tissue collected from a dog with disseminated disease. hsc cells were most consistent with interstitial dendritic cell origin based on cd a, cd c, and mhc ii staining as well as demonstrable phagocytic activity. hsc cells also displayed expression of ccr , unlike the established dh line, supporting a notion that hs consists of a variety of subtypes. ccr has been linked to hs growth and metastasis, suggesting it may represent a possible therapeutic target. further studies establishing and characterising canine hs cells may contribute to the elucidation of mechanisms of tumourigenesis. no conflicts of interest reported. virulent-systemic (vs)-fcv that induce cutaneous edema, ulcerations of the head and feet, and occasionally jaundice have been described in the usa and europe. here we characterize for the first time vs-fcv outbreaks in cats in switzerland and liechtenstein. the four outbreaks occurred in three geographically separated locations: schaan (liechtenstein, shelter ), zurich (switzerland) and lausanne (switzerland, shelter ) between november and january . pcr (fcv and feline herpesvirus- , fhv- ), virus isolation and felv/fiv testing were performed on saliva and blood samples collected from clinically affected cats. furthermore, saliva for pcr was collected from additional cats in shelter . phylogenetic analyses were performed based on the capsid (vp ) gene sequence of fcv. vs-fcv isolates were tested for virus neutralization with sera raised against common fcv vaccine strains. outbreak occurred in a cattery in liechtenstein and involved five non-vaccinated, -months old siblings with fever, edema, skin and tongue ulcerations. outbreak occurred in a small animal clinic in zurich. a -year old cat presented with severe paw edema, fever, tongue and skin ulcerations, progressive hypoproteinemia and hyperbilirubinemia. outbreaks and happened in a cattery in lausanne five months apart and involved two litters of non-vaccinated, -to -months old kittens. the cats presented with fever, nasal discharge, edema and skin and oral ulcerations. all affected cats tested fcv-positive but negative for fhv- , felv and fiv, except for one kitten from outbreak (fiv-positive). all cats in outbreaks and recovered, whereas all cats in outbreaks and died or were euthanized because of clinical deterioration. each outbreak was caused by a phylogenetically distinct vs-fcv strain. in shelter , the queen and three in contact cats remained asymptomatic although infected with the same vs-fcv strain. furthermore, / other cats of shelter were infected with closely related, but distinct fcv strains. the vs-fcv isolates from the two outbreaks in shelter were distinct but phylogenetically related. all vs-fcv isolates from cats from the same outbreak showed a similar virus neutralization pattern, but neutralization differed between different outbreaks. in conclusion, all vs-fcv outbreaks involved multi-cat environments. the same vs-fcv strains with similar virus neutralization patterns were isolated from cats from the same outbreak. not all cats infected with a vs-fcv strain developed disease and mortality varied between the outbreaks. the sera for virus neutralization were provided by merial, france. defined herein as presence of sneezing, nasal-and/or ocular discharge, conjunctivitis and/or keratitis), but also oral cavity lesions, chronic stomatitis, limping syndrome and, rarely, virulent systemic disease. the aims of the present study were to compare cats suspected of fcv (fcv-sc) based on clinical symptoms and healthy controls (controls) and to investigate potential risk and protective factors, such as co-infection with feline herpesvirus- (fhv- ), mycoplasma felis, chlamydophila felis, bordetella bronchiseptica and feline retroviruses, vaccination, gender, age, breed, housing and corticosteroid and antibiotic treatment. oropharyngeal, nasal and conjunctival swabs from fcv-sc and controls were collected into transport medium, processed within hours after collection and analyzed for fcv by virus isolation and for all tested pathogens using molecular assays. the samples were collected by randomly selected veterinary practices in different areas of switzerland ( fcv-sc and controls/ area). to record clinical data, retroviral status and vaccination history of the cats, a questionnaire was filled out by the private veterinarian. the seven tested pathogens were found in the investigated population. the prevalence (fcv-sc vs. controls) was: fcv % vs. %; fhv- % vs. %, c. felis % vs. %, b. bronchiseptica % vs. %, m. felis % vs. %, feline leukemia virus % vs. % and feline immunodeficiency virus % vs. %. fcv-sc were positive for fcv significantly more often compared with controls (or . ) and shed more fcv. co-infections with up to four pathogens were detected; fcv-sc were significantly more frequently co-infected ( %) compared with controls ( %). gingivostomatitis and oral ulceration but not urtd were highly associated with fcv infection. in contrast, c. felis was associated with urtd; fhv- was associated with nasal and ocular discharge and m. felis with conjunctivitis and ocular discharge. risk factors for fcv infection were housing in groups (especially ≥ cats), an intact gender, maine coon breed and corticosteroid therapy. fcv-positive cats with gingivostomatitis were older and more commonly vaccinated than fcv-positive cats without gingivostomatitis. moreover they shed more fcv than cats with urtd. vaccination and primary immunization defined as two vaccinations - weeks apart with the same vaccine brand were protective factors against fcv but not fhv- infection. vaccination was associated with a decreased incidence of urtd in fcv-infected cats (or . ). further analyses will investigate cross-neutralization patterns of the prevailing fcv isolates. conflicts of interest: the study was partially funded by merial, france, and biokema, switzerland. antibody preparations are commonly used for the treatment of feline upper respiratory tract disease (furtd), although their efficacy has not been proven. the aim of this study was to evaluate efficacy of a commercial serum containing antibodies against feline herpesvirus- (fhv- ) and feline calicivirus (fcv) in cats with acute viral furtd. this prospective, randomized, placebo-controlled, double-blind study included cats with acute (< days) clinical signs of fhv- and/or fcv infection (confirmed by quantitative pcr). all cats received symptomatic treatment and either hyperimmune serum (n = ) (≤ weeks ml, > weeks ml, subcutaneously q h, topically into eyes, nostrils, and mouth q h) or saline (n = ) for three days. clinical signs, including a 'furtd score' and general health status, were recorded daily (day to and on day ). fcv shedding was determined on day and . statistical analyses included one-way analysis of variance, mann-whitney u, and student's t-test (improvement of clinical signs), fisher's exact test (fcv shedding), and spearman analysis (correlation clinical signs with virus load). clinical signs and general health status improved significantly in both groups. however, while placebo-treated cats had only improved significantly by day , cats receiving antibodies already significantly improved in their 'furtd score' (p = . ) and general health status (p = . ) by day . there was no significant difference in the number of cats shedding fcv and no correlation between viral load and clinical manifestation. administration of antibodies lead to faster improvement of clinical signs in cats with acute viral furtd, but did not influence fcv shedding. no conflicts of interest reported. different viral and bacterial pathogens can be involved in feline upper respiratory tract disease (furtd). although some clinical signs have been associated with certain pathogens, clinical signs can be variable and non-specific. aim of the study was to compare detection rates of feline herpesvirus- (fhv- ), feline calicivirus (fcv),and chlamydophila felis (c. felis) in cats with furtd on different sampling sites, and to correlate test results and clinical signs. swabs of nose, oropharynx, tongue, and conjunctiva were taken from cats with signs of furtd. on all samples, reverse transcription polymerase chain reaction (rt-pcr) was performed for detection of fcv, and polymerase chain reaction (pcr) for detection of fhv- and c. felis. fisher's exact test was used for all comparisons. the level of significance was p < . . pathogens were detected in . % of cats. of these, . % were positive for fhv- , . % for fcv, and . % for c. felis. fcv was isolated significantly more often from oropharynx ( . % of fcv-positive cats) and tongue ( . %) compared to conjunctiva ( . %) (p < . ). there was no significant difference between the sampling sites for detection of fhv- and c. felis. in addition, there was no preferred sampling site in cats with respective clinical signs, including oral ulceration, conjunctivitis, and keratitis. in cats with furtd, the oropharynx can be recommended as the preferred sampling site for detection of fcv, fhv- , and c. felis. based upon clinical signs it cannot be determined which sampling site should be selected for detection of the pathogens. no conflicts of interest reported. pulmonary haemorrhage syndrome (lphs). s. schuller , s. callanan , s. worrall , t. francey , a. schweighauser , j.e. nally . bern university, bern, switzerland, university college dublin, dublin, ireland leptospiral pulmonary haemorrhage syndrome (lphs) is a severe form of leptospirosis, which has been increasingly recognised in humans and many animal species in the past years. patients with lphs may develop rapidly progressive intra-alveolar haemorrhage, leading to high mortality. the pathogenic mechanisms of lphs are poorly understood hampering the application of effective treatment strategies. studies in humans and experimentally infected guinea pigs have demonstrated deposition of immunoglobulin and complement c in lphs lung tissue in the absence of significant numbers of leptospires, suggesting that lphs is, in part, caused by autoimmunity. the aim of this project was to describe the histopathologic features of lphs in dogs and to investigate whether igg and igm deposition is present in affected canine lung tissue. single-step immunohistochemistry (ihc) for dog igg, igm and leptospiral outer membrane vesicles was performed on lung tissues from dogs with lphs, dogs with pulmonary haemorrhage due to other causes and healthy dog lungs. acute intra-alveolar haemorrhage and oedema in the absence of significant inflammatory infiltrates were present in all lphs lung tissues. three ihc staining patterns were observed in lphs lung tissue: alveolar septal wall staining with (igg n = /igm n = ) and without intra-alveolar staining (igg n = /igm n = ) and staining of intra-alveolar fluid only (igg n = /igm n = ). intra-alveolar staining appeared to favour alveolar surfaces in some cases (igg n = /igm n = ). healthy control lungs showed no staining, whereas haemorrhagic lung showed staining of intraalveolar fluid (igg/igm n = )) and occasional, mild and discontinuous staining of alveolar septa (n = ). leptospiral antigens were not detected in any of the tissues. results indicate that histopathologic features of canine lphs are similar to what has been described in other species. ihc demonstrated that alveolar septal deposition of igg/igm is present in most dogs with naturally occurring lphs. while these findings support a role of the humoral immune response in the development of lphs, our findings do not indicate whether autoimmunity is a primary or secondary event in the pathogenesis of lphs. no conflicts of interest reported. leptospirosis, a zoonotic bacterial disease with a worldwide distribution, is a re-emerging disease in humans and dogs. acute renal and hepatic failure are the most frequently reported clinical manifestations of canine leptospirosis. the aim of this study was to describe clinical, laboratory and radiological features, the outcome as well as the distribution of leptospira serogroups in dogs with leptospirosis ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . medical records of dogs diagnosed with leptospirosis were evaluated retrospectively. diagnoses were based on microscopic agglutination testing (mat), blood/urine pcr, and histopathology (levaditi staining). mat-titers ≥ : against non-vaccine and ≥ : against vaccine serovars or a -fold rise of titers within - weeks were considered diagnostic. dogs met the inclusion criteria. in dogs diagnostic mat-titers were present (mainly against serogroups grippotyphosa ( %), australis ( %), and pomona ( %). at initial presentation, the most common clinical signs were lethargy ( %), anorexia ( %), vomitus ( %), a painful abdomen ( %), diarrhea ( %), oliguria ( %), tachypnea ( %), delayed capillary refill time ( %), pale mucous membranes ( %), fever ( %), hypothermia ( %), and icteric mucous membranes ( %). abnormal findings of the cbc included anemia ( %), thrombocytopenia ( %) and leukocytosis ( %). biochemistry abnormalities included increased creatinine concentrations ( %), increased liver enzyme activities ( %), hyperbilirubinemia ( %), hyperphosphatemia ( %), hyponatremia ( %), and hypoalbuminemia ( %). urinalysis often revealed glucosuria ( %) and an elevated urine-protein/creatinine-ratio ( %). radiological pulmonary changes were detected in % of the dogs initially or during the course of disease. dogs died or were euthanized, of them due to "leptospiral pulmonary hemorrhage syndrome". in this study, non-vaccine serogroups were the most common serogroups detected by mat. in the majority of patients renal ( %) and/or hepatic ( %) disease was detected. a pulmonary form of leptospirosis was present in % of the dogs. lung involvement represented a severe complication causing increased mortality depending on the severity of respiratory signs. no conflicts of interest reported. leptospirosis is a zoonotic disease that can affect multiple organs with renal and hepatic involvement being considered to be the most common. the aim of this study was to evaluate a large number of dogs with leptospirosis for cardiac and/or exocrine pancreatic involvement. a total of dogs were diagnosed with leptospirosis based on clinical signs and either microscopic agglutination test, blood/ urine polymerase chain reaction, and/or histopathology. at the time of admission and, in most patients, after an average of two weeks canine pancreatic lipase immunoreactivity (cpli, as measured by spec cpl â ), ultrasensitive cardiac troponin i (ctni), and c-reactive protein (crp) were analyzed. data were analyzed with non-parametric statistics. the level of significance was set at p < . . upon admission, common clinical signs reported included lethargy (n = ), vomiting (n = ), abdominal pain (n = ), dyspnea (n = ), pale mucous membranes (n = ), oliguria (n = ), hypothermia (n = ), and fever (n = ). anemia (n = ), thrombocytopenia (n = ), leukocytosis (n = ), were frequently reported hematology findings. increased concentrations of creatinine (n = / ), phosphorus (n = / ), alt (n = / ), sap (n = / ) and bilirubin (n = / ) were also frequently recorded. crp (median: . mg/l; range: . - . mg/l, reference interval (ri): . - . mg/l), ctni (median: . ng/l; range: . - . ng/l, ri: - . ng/l), and cpli (median: lg/l; range: - lg/l, ri: - lg/l) concentrations were above the upper limit of the reference intervals in / ( %), / ( %), and / ( %) dogs, respectively and serum cpli concentration was above the suggested cut-off value for a diagnosis of pancreatitis in / ( %) dogs. crp and ctni, but not cpli were higher upon admission compared to the re-check measurement (p = . and . , respectively). dogs with increased serum cpli concentrations also showed a higher proportion of dogs with increased serum ctni concentrations (p = . ). there was no statistically significant correlation of cpli concentrations with a history of abdominal pain and/or vomiting. biochemical results were compatible with multiple organ impairment with involvement of kidneys, liver, heart, and exocrine pancreas where at least two organs were affected in / ( %) dogs. forty ( %) of dogs recovered, ( %) died, and ( %) were euthanized. ctni and cpli were higher in non-survivors, but these differences did not reach statistical significance. however, the number of organs affected and outcome were significantly correlated (p = . ). our data suggest that infection with leptospira is characterized by a systemic inflammation with variable multiple organ involvement and damage, often including the heart and also the exocrine pancreas. the study was funded by texas a&m university. the primary author and two co-authors work at the gi laboratory, texa a&m university. canine bartonellosis is increasingly recognized worldwide and may be associated with diverse clinical manifestations. recent evidence suggests that bartonellosis also causes lameness and polyarthritis in dogs. however, pcr amplification of bartonella dna and isolation of bartonella species from canine synovial fluid (sf) samples have rarely been reported. canine leishmaniosis (canl) due to leishmania infantum is a multisystemic disease commonly associated with polyarthritis. based on the hypothesis that concurrent bartonella infection may be a contributing factor for the development of arthritis in dogs with canl, the main objective of this study was to investigate the microbiological and molecular prevalence of bartonella spp. in dogs with naturally-occurring canl, with or without cytologically documented arthritis. from a previous study, dogs with canl were retrospectively studied for bartonella spp. infection. diagnosis of canl was based on compatible clinical and clinicopathological abnormalities, positive serology, and lymph node or bone marrow (bm) cytology. dogs with serological evidence of other vector-borne infections (anaplasmosis, borelliosis, dirofilariosis and ehrlichiosis) and dogs recently vaccinated or medicated were excluded from the study. arthritis defined as a neutrophil percentage in excess of % of nucleated cells in sf cytology was documented in / ( . %) of dogs. a total of archived specimens from dogs, including edta-anticoagulated blood samples, bm and sf aspirates were tested for bartonella spp. dna using a bartonella alpha proteobacteria growth medium (bap-gm) diagnostic platform. eight ( . %) dogs were infected with one or two bartonella species, including candidatus bartonella merieuxii(n = ), b. henselae sa (n = ) and b. rochalimae (n = ). bartonella spp. dna was amplified from bm in dogs and from blood in dogs but was not amplified from any sf sample. overall, ( . %) dogs with and ( . %) dogs without arthritis were infected with a bartonella species. the prevalence of bartonella spp. dna in the dogs with or without arthritis did not differ (v test for independence, p = . the prevalence of giardia in dogs ranges between . % and . %, with a higher prevalence in puppies. however, the risk factors for giardia infection around weaning have been poorly described. the aim of the study was to evaluate risk factors for giardia infection in puppies during the first weeks of life and to determine an impact of this parasite on feces quality. puppies from litters living in a breeding kennel were followed between and weeks of age. each puppy was treated with fenbendazole (panacur â , msd, france, mg/kg, per os, q h) for consecutive days at , , and weeks of age. for each puppy, fecal consistency was evaluated using a -point scale. excretion of enteropathogens was evaluated by qpcr for canine parvovirus type (cpv ), qrt-pcr for canine coronavirus (ccv), coproantigens quantification for giardia (prospect-giardia, remel), and mcmaster flotation technique for any eggs and oocysts. a generalized linear mixed model (proc glimmix) with giardia infection as a binary outcome was used to assess the following effects: breed size, age, and cpv , ccv and isospora ohioensis infections. a linear mixed model (proc mixed) with fecal score as outcome was used to determine the following effects: breed size, age, and giardia, cpv , ccv and i. ohioensis infections. a total of fecal samples were collected; cpv , giardia, i. ohioensis and ccvwere detected in respectively . %, %, . % and . % of the samples. the risk of giardia infection increased with age (odd ratio= . ; %ci= . - . ; p = . ). neither breed, nor cpv , ccv and i. ohioensis infections influenced risk of giardia infection (p = . ; p = . ; p = . ; p = . respectively). giardia infection did not impact feces quality (p = . ), whereas a significant influence of cpv (p < . ), ccv infection (p = . ) and breed size (p < . ) was evidenced. this study underlines that even with an adapted deworming program the eradication of giardia is difficult to obtain in large dog packs. the higher prevalence of giardia in puppies of weeks and older could be linked with the immunity gap during this period. giardia was not associated in our study with an increased risk of diarrhea. the lack of pathogenicity of the parasite per se could be hypothesized, but also an efficacy of the treatment for the prevention of the clinical signs or a local and systemic immunity limiting clinical signs. financial support from royal canin. hepatic encephalopathy (he) can occur in dogs as a complication of primary liver disease or as consequence of congenital portosystemic shunts (pss). the aim of this study was to assess magnetic resonace spectroscopy (mrs) usefulness in he diagnosis. twenty dogs with a presumptive diagnosis of he were enrolled. inclusion criteria were: a clinical examination, a blood cell count and biochemical panel, at least one plasmatic ammonia determination higher than lmol/l, optional hystopatology of the liver, requirement of magnetic resonance imaging (mri) investigation by the clinician. even though mrs represents a non-invasive procedure, informed consent was obtained from dogs'owners. he diagnosis was confirmed in / : / had a pss, / showed hepatic microvascular dysplasia and / had hepatic cirrhosis histologically confirmed. the control-group was made of patients retrieved from our database that underwent mri without showing any abnormalities on brain scans. the mr protocol included t -weighted fast spin-echo, t -weighted sequences, proton density, and radio-frequency pulses gradient-echo. on mri examination cerebral atrophy was evident in all the patients ( / ), ranged from mild to severe. in / patients symmetrical bilateral hyperintensities in the globus pallidus on t -weighted images were evident. mrs was performed using a short te ( ms) and it was defined a volume of interest in the parieto-occipital region by -mm side cube ( cc of volume). glutamine-glutamate (gln), n-acetylaspartate and n-acetyl aspartyl glutamate (naa), choline derivatives (cho), and myo-inositol (ins) were analyzed. comparing spectra from the he affected dogs with those from the control-group decreased values of ins, cho, naa as well as high field mri combined with brain mrs provide accurate and non-invasive diagnosis of canine he. in accordance with human medicine publications, it could be state that mrs has a role in he diagnosis and follow-up with particular mention monitoring. no conflicts of interest reported. diagnosis of hepatobiliary diseases often requires hepatic tissue sampling for histologic evaluation. the laparoscopic technique is a safe method and allows acquisition of tissue by wedge and needle biopsies from different liver lobes in a minimally invasive way.specimens obtained with an -gauge biopsy needle must be interpreted with caution due to considerable variability in tissue involvement with certain disease processes. we hypothesized that needle biopsy specimens would produce findings divergent from those produced by wedge biopsy specimens. the goal of the study was to compare histological findings from two laparoscopic biopsy methods (wedge and needle) and assess which sampling technic can represent the overall disease process. procedure: all dogs included in this prospective study study were suspected diffuse hepatic disease and underwent laparo-scopic hepatic biopsy (wedge and needle - g) between and . all biopsy specimens were examined on the basis of morphologic criteria and a comparison was made between the two types of biopsies procedures according to wsava liver standardization group morphologic criteria. results: twenty-two dogs were included. no complications were reported during the laparoscopic procedure. the median number of portal triads per needle biopsy specimen was (range, to ) compared to (range ; ) with wedge biopsy specimen. the median length of needle biopsy specimens was mm; (range, to mm) and mm for all wedge biopsies. on the basis of biopsy interpretation, the diagnosis was overall similar with the two methods: dogs had vacuolar hepatopathy, acute cholangitis, non-specific acute form in dogs. chronic hepatitis with cirrhosis was found in cases, dogs had diffuse neoplasia and miscellaneous hepatic disorders. the fibrosis was considered to be severe in dogs, moderate in dogs and mild in dogs. no quantitative and qualitative difference was observed between the two types of biopsies specimen. this study demonstrates that the biopsies with a needle length of at least mm brings satisfactory information for the evaluation of most of the inflammatory, vacuolar hepatopathies, fibrosis and diffuse tumoral infiltrations. wedge biopsies allow to examine the largest number of portal triad, more contributory for certain forms of cholangitis affecting larger canals and for a single case, images of peri-hepatitis were counted at the level of the capsule. fibrosis does not seem to be more important in the sub-capsular zone contrary to what is observed in human pathology. no conflicts of interest reported. indocyanine green (icg), a fluorescence dye, is excreted solely by the liver without enterohepatic re-circulation. hence it has been used for decades as an ideal albeit invasive marker of hepatic function and blood flow in cats and dogs. here we evaluated the feasibility of a minimally invasive transcutaneous icg clearance to assess hepatic function instantaneously. transcutaneous icg clearance was performed in healthy research cats and healthy research dogs with normal liver function (bile acid stimulation test, ammonia tolerance test) using a modified device (kidney int : with an excitation wave length of nm and an emission wave length of nm. the devices were placed on different locations (lateral thoracic wall, ventrolateral abdomen, metatarsus and antebrachium) and fixed with a light bandage. to find a suitable dose to reach adequate transcutaneous peak concentrations escalating doses of icg ( . , . and . mg/kg) were injected intravenously with a wash out period of at least h in-between. measurement was continued for hour after injection in awake animals moving freely. the resulting icg disappearance curves were visually inspected to find best location (minimal artifacts, acceptable background noise) and dose. in all animals a dose of . mg/kg was deemed ideal and the thoracic and abdominal wall gave consistent results. half-life of icg clearance was calculated using a one-compartment model. half-lives in cats were . , . and . minutes, respectively; in dogs: . , . and . minutes, respectively. in conclusion, the transcutaneous assessment of icg clearance is feasible in a clinical setting. results are obtained within one hour and can be assessed instantaneously. the procedures are minimally invasive and well tolerated by the animals. given that most patients with a presumed liver problem undergo abdominal ultrasound no further clipping of hair is necessary as the device might be placed in this area. further studies are necessary to obtain reference values in healthy pets and those with various conditions leading to impaired hepatic function. no conflicts of interest reported. gallbladder diseases like gallbladder mucocele and cholecystitis can reduce gallbladder motility and may lead to cholestasis. since impaired gallbladder emptying contributes to sludge and gallstone formation, the evaluation of gallbladder motility requires accurate and appropriate methodology. three-dimensional ( d) ultrasonography has been shown to be accurate and appropriate tool for measurement of gallbladder volume in humans. therefore, we applied this novel technique for the first time to study preprandial and postprandial gallbladder volume in healthy mixed-breed dogs and compared the results to twodimensional ( d) ultrasonography. the dogs were placed in dorsal recumbency to obtain ultrasonographic measurements of the gallbladder. measurements by both d and d ultrasonography were recorded in preprandial state and after ingestion of full-fat milk. the preprandial and postprandial gallbladder volumes determined by d ultrasonography were significantly higher than corresponding volumes by d ultrasonography ( . ae . vs . ae . and . vs . ml/kg, respectively, p < . ). in d ultrasonography, most dogs ( / [ %]) had a preprandial gallbladder volume ≤ . ml/kg. however, in d ultrasonography, / ( %) of dogs had a preprandial gallbladder volume ≥ . ml/kg. gallbladder contraction index was higher in d ultrasonography than d ultrasonography, however, it did not reach statistical significance (p = . ). in conclusion, d ultrasonography showed larger gallbladder volumes than d ultrasonography in healthy dogs. it seems that d ultrasonography is appropriate adjunct device to d ultrasonography to estimate gallbladder volume when d ultrasonography could not detect whole gallbladder volume. more research is needed to determine clinical value of d ultrasonography in canine gallbladder imaging. no conflicts of interest reported. the aim of our study was to compare high-definition oscillometry (hdo) and doppler ultrasonographic measurements with direct blood pressure measurements in conscious dogs. the doppler study was performed by three investigators and by using different sphygmomanometers with different sized cuffs. devices and measurement sites were changed randomly among the investigators. cuffs were wrapped around the antebrachium in the forelimb or around the mid-metatarsus in the hind limb. in addition to the limb sites, cuffs were also placed around the base of the tail in case of the hdo method. cuff sizes were - % of the measured limb circumferences during the doppler measurement. for the hdo method all measurements were performed by the same investigator and the cuffs provided by the manufacturer were used: the smallest cuff was used for the hind limb and tail, and the medium cuff was used for the forelimb measurements. dogs were gently held in lateral recumbent position, measurement were performed on the nondependent limbs. radio-telemetry transducers were implanted to the right femoral artery some months to a year preceding the blood pressure measurements for reasons unrelated to our study. direct blood pressures varied - mmhg and - mmhg during the doppler and hdo measurements, respectively. two-hundred paired simultaneous doppler and direct measurements from dogs and paired simultaneous hdo and direct telemetric measurements from dogs were obtained. at least successful consecutive measurements could be obtained by the same investigator at the same site during the doppler or hdo measurement in and cases, respectively. thus, the mean of these measurements could be calculated similarly to the established everyday clinical practice. bias (mean difference), precision (standard deviation) and limits of agreements were calculated both from the individual paired measurements and from the means of the consecutive measurements using bland-altman spot analysis. systolic measurement performed on the tail with the hdomethod yielded the smallest bias and deviation and the best limits of agreement during this study. both doppler and hdo-measurements performed on the forelimb overestimated, while hind limb measurement underestimated the direct telemetric pressures. results of all three measurement sites by hdo performed better than forelimb or hind limb doppler-measurements, however hdo-measurements were more difficult to obtain and more often resulted with measurement failure compared to the doppler technique. cuff size above % of the measured limb circumference showed better results than smaller cuff sizes during the doppler measurements. no conflicts of interest reported. the doppler technique is considered the most repeatable indirect method to measure systolic arterial pressure (sap) in dogs. however, recent studies emphasized the effect of body position and used limb on sap measurement. the aim of this study was to determine whether a difference existed in sap measured simultaneously in dogs using different limbs, with two doppler units by two different operators. sixty clientowned dogs, admitted to the veterinary hospital for different reason, were enrolled. they were divided in groups based on body size: small breed dogs (< kg); medium breed ( - kg); large breed (> kg). for each dog the anxiety status was recorded. sap was measured via doppler technique when dogs were in right lateral recumbency in a quite environment. right and left forelimb sap and left forelimb and left hindlimb sap were recorded simultaneously, with two identical doppler units equipped with headphones, by two operators. measurement was performed based on the acvim guidelines. five measurements were recorded, the higher and lower values were discarded from the analysis. the relationship of mean sap for each limb with body weight, sex, anxiety status and sap value was evaluated. mean ae sd sap was significantly higher for the right forelimb ( . ae . ) compare to the left forelimb ( . ae . ) on overall population. the difference was significant for large breed dogs, males and dogs with sap ³ mmhg. sap was higher for the left forelimb ( . ae . ) compare to the left hindlimb ( . ae . ) on overall population. the difference was significant for medium and large breed dogs, females, calm animals and dogs with sap ³ mmhg. the mean sap from the left forelimb recorded by two different operators at two different moments, were compared and no difference was evident. in conclusion, sap measurement from different limbs, in dogs in right lateral recumbency, is poorly correlated. measurement of sap from the left forelimb is more repeatable during time and between different operators. sap trend monitoring should be done using the same measurement site for any animal. no conflicts of interest reported. amlodipine has been considered the treatmenf of choice for hypertension in cats for more than a decade. there is, however, an unmet need for a cat-specific formulation. the aim of the study was to assess the efficacy of chewable amlodipine tablets in reducing systolic blood pressure (sbp) in cats diagnosed with hypertension. seventy-seven client-owned cats were included in the study (mean age years). the study was randomised, double-blind, placebo controlled, and consisted of two phases. in the blinded phase, cats received . mg/kg amlodipine once daily for days. if they responded the dose remained the same to day . for non-responders, the dose was increased to . mg/kg. thirty-five cats received placebo following the same protocol. arterial blood pressure was measured using a high definition oscillometry method. at day a responder was defined as a cat showing a decrease of sbp to ≤ mmhg or a decrease from baseline of at least %. after days all cats continued with amlodipine for - months in an open phase with the placebo cats repeating the same dose escalation protocol as in the blinded phase. the responder rate was % in the amlodipine group and % in the placebo group following the dose escalation from day being applied to % and % of cats receiving amlodipine and placebo respectively. cats receiving amlodipine were . ( % ci . to . ) times more likely to be classified as responders when compared to those receiving placebo (logistic regression model, p = . ). from a baseline value of . ae . and . ae . mmhg the mean sbp decreased to . ae . mmhg with amlodipine and to . ae . mmhg with placebo (repeated measures analysis of covariance model, p < . ) by day . the responder rate was not influenced by factors other than amlodipine treatment (e.g. baseline blood pressure, concomitant ace inhibitor therapy, renal disease). there were no differences between the amlodipine and placebo groups in the frequency of adverse events reported during the -day blinded phase. likewise, there were very few changes in the laboratory values over time in either group. the present study is the first large clinical trial to show that amlodipine is clearly superior to placebo in the treatment of cats with hypertension. the chewable amlodipine formulation effectively reduced sbp, had a good palatability and was well tolerated. it can be used concomitantly with ace inhibitors and in cats with renal disease. conflicts of interest: m. huhtinen and j. aspegr en are employees of the sponsor j. elliott has the following information to disclose: amlodipine is the treatment of choice for feline hypertension. limited published data exist on serum concentrations achieved in hypertensive cats. the aim of the study was to assess serum amlodipine concentrations in cats treated with a new formulation of amlodipine and relate these to the blood pressure reduction achieved. seventy-seven client-owned hypertensive cats were enrolled into a randomized, double-blind and placebo controlled study consisting of two phases. in phase one, cats (group a) received . mg/kg amlodipine once daily for days. if they were deemed to have responded (see below) the dose remained the same to day . for non-responders, the dose was increased to . mg/kg. thirty-five cats (group b) received placebo following the same protocol. blood pressure was measured using high definition oscillometry. a responder was defined as a cat showing a decrease of systolic blood pressure (sbp) to ≤ mmhg or a decrease from baseline of ≥ %. following day (phase ), group a continued on amlodipine and group b switched to amlodipine and the dose was adjusted as per phase . both groups were followed for days on amlodipine. blood was collected at days (group a) and (both groups) and serum [amlodipine] measured by liquid chromatography mass spectrometry. the sbp measured on treatment was calculated as percentage of the baseline sbp and plotted against serum [amlodipine] using a sigmoidal emax model (winnonlin software). data are expressed as mean ae se. the serum concentrations of group a cats that remained on . mg/kg were . ae . ng/ml whereas those switched to . mg/kg were . ae . ng/ml. when data from groups a and b were pooled, a sigmoidal relationship between percentage baseline sbp and serum [amlodipine] was found. estimated values of lowest percentage baseline blood pressure on treatment (emax) was . ae . %, with an ec value of . ae . ng/ ml and a slope function of . ae . . the serum concentration required to reduce blood pressure by % was estimated to be ng/ml. the present study related blood pressure reduction to serum [amlodipine] in feline clinical hypertension. the limitations of this study were the limited number of blood samples collected and lack of information relating to the exact timing of blood sampling relative to dosing in some cats. however, these data could be used to define appropriate therapeutic serum [amlodipine] in hypertensive cats. m huhtinen is an employee of the sponsor; j. elliott has the following information to disclose: consultancy: pfizer animal health / zoetis, ceva animal health, boehringer ingelheim, vetoquinol ltd, orion ltd., elanco ltd, idexx ltd, niche generics ltd. triveristas ltd., virbac ltd., advisory board membership: international renal interest society (supported by novartis) european emesis council (sponsored by pfizer animal health -now zoetis) cardiorenal board -vetoquinol ltd. idexx renal advisory board research grants or contracts: vetoquinol ltd, novartis ltd, pfizer animal health ltd (now zoetis), royal canin ltd, boeringher ingelheim ltd, waltham centre for pet nutrition, ceva animal health orion ltd.; l pelligand has the following information to disclose: in receipt of research grant / contract funding from orion ltd., novartis animal health, transpharmation ltd, deltadot ltd; acted as a consultant for: triveritas ltd. and novartis animal health. commercial assays for the measurement of canine and feline pancreatic lipase immunoreactivity (spec cpl â and spec fpl â , respectively; idexx laboratories, westbrook, me, usa) have been available for a few years and have previously been analytically and clinically validated. recently, new commercial assays for the measurement of these parameters have become available, though neither one of these assays have been analytically or clinically validated in the literature. thus, the goal of this study was to compare these newly available assays to the established assays. leftover serum samples from diagnostic submissions to the gi laboratory were collected based on certain parameters (e.g., results throughout the working range of the assay, good quality sample or hemolytic, lipemic, or icteric sample) and were assigned random sample id numbers. the samples were evaluated by spec cpl â or spec fpl â , sent on dry ice to the klinik am hochberg, and one aliquot of each sample was blindly submitted to laboklin for measurement of cpli and fpli by their newly released in-house assay and also to the gi lab at texas a&m university for repeated analysis by spec cpl â and spec fpl â to exclude any effect of shipping. there was no significant difference between serum cpli or fpli concentrations before or after shipping at the gi lab (pvalues for wilcoxon matched-pairs signed rank tests: . and . , respectively). in contrast, there was a significant difference between serum cpli or fpli concentrations between the newly released assays and the previously established assays (pvalues < . and . , respectively). while there was a significant correlation between the newly released and the previously released assays (spearman r: . and . , respectively), this correlation was very poor for assays that supposedly measure the same analyte. also, the interpretation for serum cpli and fpli results between the previously developed assays and the new assays did not agree for many of the samples. finally, both newly developed assays showed some erratic results. in conclusion, the newly released assays for the measurement of cpli and fpli do not agree with previously established and validated assays, provide different interpretations, and show erratic results. thus, further research is needed before these newly released assays could be recommended for clinical use. dr. steiner serves as director and dr. suchodolski serves as associate director of the gastrointestinal laboratory at texas a&m university. dr. steiner also serves as a paid consultant to idexx laboratories, westbrook, me, usa. both the gastrointestinal laboratory and idexx laboratories offer cpli and fpli testing on a fee-for-service basis. digestive health is a main concern for growth, morbidity and mortality in weaning puppies. fecal immunoglobulin a (iga) has been suggested as a useful noninvasive biomarker for mucosal immunity. the purpose of this study was to evaluate the effect of infection with enteropathogens on fecal iga concentrations in puppies and that of physiological factors such as age and breed size. puppies from breeding kennels were included in the study. puppies were between and weeks of age (meanaestandard deviation (sd): . ae . weeks). depending on the mean adult body weight of their respective breed, the puppies were divided into small (if mean adult body weight < kg) or large (> kg) breed puppies. for each puppy, fecal consistency was evaluated using a -point scale and feces were collected for the evaluation of presence of fecal enteropathogens and fecal iga concentrations. the presence of enteropathogens in fecal samples was evaluated by qpcr for canine parvovirus type (cpv ), qrt-pcr for canine coronavirus (ccv), coproantigen quantification for giardia (prospect-giardia, remel), and mcmaster flotation technique for other parasite eggs and oocysts. fecal iga concentrations were measured by an elisa test. statistical analyses were performed using sas software. a linear mixed model (proc mixed) with fecal iga concentration as outcome was used to determine the following effects: enteropathogen infection, breed size, age, and fecal score. the respective influence of litter and breeding kennel as random effects was also determined. data is presented as mean ae sd. small breed dogs represented . % ( / ) of the total number of dogs included. at least one enteropathogen was identified in . % of puppies ( / ). fecal iga concentration was significantly influenced by fecal enteropathogens (p = . ). puppies infected with at least one enteropathogen had significantly lower fecal iga concentrations than puppies without any enteropathogens ( . ae . lg/g vs. . ae . lg/g). breed (p = . ), but not age (p = . ), influenced iga concentration. small breed puppies had significantly higher fecal iga concentrations than large breed puppies ( . ae . lg/g vs. . ae . lg/g). no significant relationship between fecal iga concentration and feces quality was evidenced (p = . ). this study suggests that fecal iga concentration is a promising marker for subclinical infection by at least one enteropathogen and confirms that digestive physiology varies with the breed size. a link between lower digestive immunity and higher susceptibility to enteropathogen infection needs further investigation. conflicts of interest: financial support of royal canin. canine chronic enteropathies (cce) include diet-responsive, antibiotic-responsive, and immunosuppressive-responsive enteropathies (ire). this prospective study was designed to evaluate a commercial hypoallergenic dry diet a containing oligopeptides as the only protein source for the management of dogs with ire and as an alternative to immunosuppressive therapy over a week period. nineteen dogs across france and quebec entered the study. dogs with food or antibiotic-responsive chronic enteropathy, hypoproteinemia, or treated with immunomodulating drugs were excluded from the study. dogs were included in the study after complete clinical, ultrasonographic, endoscopic evaluation and histopathological evaluation of intestinal biopsies showing signs of intestinal inflammation. the owners were instructed to feed exclusively the study diet a . canine inflammatory bowel disease activity index (cibdai) scores, fecal scores as observed by the dog-owners, and body weight were evaluated at baseline, , and weeks after inclusion. dietary treatment was regarded successful if the cibdai score was reduced by at least %. the protocol has been reviewed and accepted by royal canin ethics committee and owners completed an informed consent. results are presented as meanaesd (range). statistical comparisons were performed with a wilcoxon test. thirteen dogs ( intact males, neutered and intact females) completed the trial. seven dogs were excluded ( diagnosed with giardia, s with no histological evidence of inflammation, with hypoadrenocorticism). mean age was . years ae . ( . - . ), mean body weight . kg ae . ( . - . ). cibdai score was . ae . ( - ) at inclusion, was . ae . ( - ) after weeks and was . ae . ( - ) after weeks (p = . and p = . vs inclusion, respectively). fecal scores after [ . ae . ( - )] and weeks [ . ae . ( - ) ] were improved compared to inclusion scores . ae . ( - ) (p = . and p = . , respectively). the low molecular weight poultry feather hydrolyzed proteinbased dry extruded diet a appears to be effective in the management of idiopathic ibd without any concurrent immunosuppressive drug over the week period of this pilot study. these preliminary findings should be confirmed by a prospective, randomized double blind study. feline pancreatitis is the most common exocrine pancreatic disorder with varied mortality. however, there is no available and reliable method to evaluate the severity and prognosis of the disease. ninety-two cats diagnosed as pancreatitis with acute onset of compatible clinical signs and a positive snap â fpl tm test between october and september were enrolled in this study. all cats were divided into survival (n = ) and nonsurvival (n = ) groups. fifty-two parameters including signalments, clinical signs, physical examinations, clinicopathological examinations, diagnostic images, complications and concurrent diseases were analyzed and compared between the two groups. parameters with p ≤ . were considered for further analyses. the mortality in this study was . %. hematocrit, albumin, bun, creatinine, total bilirubin, calcium, phosphorous, body temperature, systolic blood pressure, the body cavity fluids, complications, e.g. systemic inflammatory response syndrome (sirs) and acute renal failure (arf) were found to be significantly associated with disease severity and prognosis, and were selected for constructing the scores. continuous variables outside the reference interval were separated into quartiles to yield quartile-specific odds ratios (ors) for survival. based on the integer value of the or, the scoring system was then developed by incorporating weighting factors assigned to each quartile. a predictive total score was calculated for each cat by summing all weighting factors. the total scores of each cat ranged from to . the severity scores in this study achieved an area under receiver operating characteristic (auroc) of . . the optimal cut-off point for discriminating outcome was . with the sensitivity of . % and specificity of . %, respectively. the mortality was . % with a score ≥ , whereas . % with a score ≤ . there was significant difference (p < . ) between the two groups of the cut-off point. furthermore, the mortality reached to % when the score more than . the severity scoring system of this study provides a reliable and clinical applicable method to predict clinical outcome in cats with pancreatitis. no conflicts of interest reported. convincing evidence for the role of clostridium (c.) perfringens as a primary pathogen in acute haemorrhagic diarrhoea syndrome (ahds) in dogs was recently found. it is suspected that clostridial toxins, especially c. perfringens enterotoxin, play a relevant role in the disease process. however, to date enterotoxigenic c. perfringens strains have only been described in single case reports. thus, the aim of this study was to indentify the specific c. perfringens genotype involved in adhs. small intestinal biopsies were collected with a sterile single-use biopsy forceps from ten dogs with ahds and immediately cultured. in / dogs, clostridial strains were isolated and identified as c. perfringens by mass spectrometry using maldi-tof ms. c. perfringens colonies from each dog were submitted for specific detection of the four major toxin genes (alpha, beta, epsilon, and iota), the enterotoxin gene, and the beta toxin by multiplex pcr. every clostridial isolate was typed as c. perfringens type a based on the detection of the alpha toxin encoding gene. in / isolates, additionally the beta toxin gene was identified, however, none of clostridial strains encoded for the c. perfringens enterotoxin gene. the results of this study suggest that c. perfringens type a is the most important c. perfingens genotype involved in the disease process of dogs with ahds. although c. perfringens enterotoxinhas been associated with intestinal diseases in humans, dogs, horses, pigs, and other animal species, this enterotoxin is most likely not responsible for the intestinal lesions in dogs with ahds. no conflicts of interest reported. p < . ). lack of treatment response was significantly associated with il (il: / , sre: / , fre: / , are: / ; p < . ). anemia, thrombocytopenia, and increased plasma urea were significantly associated with il (anemia: il: / , sre: / , fre: / , are: / , p < . ; thrombocytopenia: il: / , sre: / , fre: / , are: / , p = . ; increased urea: il: / , sre: / , fre: / , are: / , p = . ). hypoalbuminemia (< g/l) and hypocobalaminemia (< pg/ml) occurred significantly more frequently in dogs with sre (hypoalbuminemia: il: / , sre: / , fre: / , are: / , p < . ; hypocobalaminemia: il: / , sre: / , fre: / , are: / , p = . ). results of this study show that elderly and large breed dogs were more frequently affected with il and sre compared to other etiologies and both il and sre were associated with greater disease severity and/or a negative outcome. in comparison, anemia, thrombocytopenia, and increased plasma urea were most frequently detected in il whereas severe hypoalbuminemia and hypocobalaminemia were significantly associated with sre. no conflicts of interest reported. alpha -proteinase inhibitor (a -pi) is a proteinase-resistent protein that can be quantified in fecal, urine, and serum samples from dogs. recently, increased fecal and urinary canine a -pi (ca -pi) concentrations have been described in dogs with gastrointestinal diseases (e.g., inflammatory bowel disease [ibd] , but also in dogs with exocrine pancreatic insufficiency) and in dogs with chronic hepatitis or chronic kidney disease, respectively. decreased serum ca -pi concentrations have been reported in dogs with ibd, protein-losing enteropathy (ple), and hypocobalaminemia. treatment protocols for dogs with ibd and/or ple commonly include corticosteroids, but the effect of corticosteroid therapy on serum ca -pi concentrations have not yet been reported. the aim of this study was to evaluate the effect of hydro-cortisone on serum ca -pi concentrations in healthy dogs. twelve healthy beagle dogs were randomly allocated to a placebo-group (n = ) and to a treatment group (n = ; hydrocortisone-group). the placebo-group received an empty gelatin capsule po q h, whereas the hydrocortisone-group was treated with hydrocortisone at a dose of . mg/kg po q h. serum samples were obtained at baseline and on day , , , , and during treatment as well as day , , , , and post-treatment for all dogs. serum ca -pi concentrations were measured at all time points using an in-house radioimmunoassay. a mann-whitney u test was used to compare the baseline measurements of both groups. the effect of hydrocortisone-treatment on serum ca -pi concentrations was evaluated by comparing ca -pi at baseline and during treatment and between baseline and posttreatment period using a manova. baseline serum ca -pi concentrations did not differ between the hydrocortisone-and the placebo-group (p > . ). serum ca -pi concentrations increased significantly (p = . ) during the treatment period in the hydrocortisone-group (baseline [median in mg/l: , ] ). in contrast, no difference was observed between both groups when comparing serum ca -pi concentrations at baseline and during the post-treatment period (p > . ). this study showed that hydrocortisone-treatment over weeks did affect serum ca -pi concentrations in healthy dogs. whether corticosteroid therapy has any effects on fecal or urine ca -pi concentrations in healthy dogs remains to be determined. the author works at texas a&m university, whose gi lab currently offer a commercial assay for faecal alpha -proteinase inhibitor. canine chronic enteropathy (ce) is a common, but poorly understood syndrome, with variable response to therapy and prognosis. there is a need for novel biomarkers that are specific for intestinal disease and that provide objective measures of disease severity, progression, and prognosis. serum citrulline is a useful biomarker in human intestinal disease as it is specific to the small intestine and indicates globally reduced enterocyte mass and absorptive function in various disease states. it is used to determine, quantitatively, intestinal integrity at the enterocyte level and is not influenced by nutritional or inflammatory status. the aim of this study was to determine whether serum citrulline can be used as a biomarker for ce in dogs. in this retrospective study, computer records from the university of liverpool small animal teaching hospital were used to identify dogs with ce. disease severity was quantified by cibdai. controls were age-and breed-matched dogs without gastrointestinal disease. serum citrulline was measured by ultra-high performance liquid chromatography with tandem mass spectrometry. in dogs with ce, serum citrulline concentration was measured at presentation and at various time points after starting treatment. serum citrulline was measured in dogs with ce and controls. dogs responded to dietary manipulation (food-responsive enteropathy, fre) and responded to antibacterials (antibiotic-responsive diarrhoea, ard), with a further having invasive mucosal bacteria, of which one responded to antibacterials and one was refractory. dogs were diagnosed with idiopathic ibd (on the basis of exclusion of known causes and failure to respond to therapeutic dietary and antibiotic trials), of which responded to immunosuppressive therapy, were refractory, and were lost to follow-up. serum citrulline concentration did not differ between dogs with ce (median . lg/ml, range . - . ) and controls (median . lg/ml, range, . - . , p = . ). there was also no difference in serum citrulline concentration amongst dogs with fre, ard, ibd, and controls (p = . ). serum citrulline did not differ between dogs that responded well, or were refractory to treatment (p = . ), between dogs with and without protein-losing enteropathy (p = . ), or between dogs that survived and that were euthanased because of ce (p = . ). serum citrulline did not correlate with cibdai (r = . ). these findings do not support the use of serum citrulline as a biomarker in determining diagnosis, prognosis, or quantifying severity in dogs with ce. one of the co-authors (marco caldin) has a diagnostic laboratory offering citrulline assays. chronic enteropathy (ce) is a multi-factorial disease, which involves aberrant immune responses to commensal bacteria or dietary antigens. macrophages have an important role in human disease but little information is available in canine intestine. data to date have relied solely on macrophage identification using mac , an antibody directed against calprotectin, which recognizes both macrophages and neutrophils. in this study an alternative antibody for macrophages, am- k, directed against a scavenger receptor (cd ) was used and distribution of both markers was compared. this antigen is of interest as positive cells accumulate in intestine of humans with ce. endoscopic duodenal biopsies were obtained from seven crossbreed dogs. serial histologic sections were stained with mac or am- k. positively-stained cells were counted from random areas from both villous and crypt regions. stained cell localisation was subjectively evaluated and the percentage of positively stained cells from the total nucleated cells per , lm in the villus or crypt was compared between both antibodies using a wilcoxon signed-rank test. mac and am- k did not co-localize on serial sections. there were significantly more am- k positive cells than mac in the crypts ( . % [ - . ] versus . % [ - . ], p = . ). in contrast there was no difference in expression of either markers in the villi ( . % [ - . ] versus . % [ - . ], p = . ). this study reports for the first time the existence of two populations of macrophages in canine intestine. these results in normal dogs will be used to explore further the distribution and function of macrophages in dogs with ce. no conflicts of interest reported. chronic diarrhea and vomiting are common clinical signs in dogs. primary (e.g., inflammatory, infectious, neoplastic, mechanical, or other) and secondary gastrointestinal diseases (e.g., exocrine pancreatic, hepatic, renal, or endocrine disease) are possible underlying causes. the aim of this study was to evaluate the final diagnoses in dogs with chronic diarrhea and/or vomiting and to determine the prevalence of various primary and secondary gastrointestinal diseases in dogs with these gastrointestinal signs. medical records of dogs presented between july and august with chronic diarrhea (d), vomiting (v) or both (diarrhea and vomiting [vd]) were retrospectively reviewed. dogs were included if a minimum work-up (hematology, plasma biochemistry profile, and fecal parasitology) had been performed and if a final diagnosis was recorded ( / ). a primary gastrointestinal disease was recorded in % of the cases ( / ) and included inflammatory diseases ( / , exocrine pancreatic insufficiency, hypoadrenocorticism, polyendocrinopathy, dilated cardiomyopathy, and leukemia in one dog each). in total, % of the dogs were presented with d ( / ) followed by % with vd ( / ), and % with v ( / ). d and vd were significantly more frequent in dogs with primary gastrointestinal disease (d: / , vd: / ), compared to dogs with secondary gastrointestinal disease (d: / ; vd: / ; p = . , chi square test). v was significantly more common in dogs with secondary gastrointestinal disease ( / ) as compared to dogs with primary gastrointestinal disease ( / ; p = . ). in this study, food responsive enteropathy ( %) was the most commonly diagnosed cause of chronic gastrointestinal signs. chronic pancreatitis was the most frequent cause of secondary gastrointestinal disease ( %). diarrhea was significantly associated with primary and vomiting with secondary gastrointestinal disease. no conflicts of interest reported. matrix metalloproteinases (mmps) and are zinc-dependent endopeptidases that contribute to the control of breakdown and reconstitution of extracellular matrix under both normal and pathological conditions. intestinal mucosal levels of mmp- and - have been shown to be increased in animal models and human ibd. to our knowledge, the presense of mmp- and - has not been studied in the intestinal mucosal samples of healthy dogs as well as in canine spontaneous ibd. thus, the main aim of this study was to identify the presence of mmp- and - in the mucosa of the small and large intestines of clinically healthy beagle dogs using gelatin zymography technique. for the study, historical intestinal tissue samples from four different parts of the intestine (duodenum, jejunum, ileum and colon) were used. the samples were taken and snap frozen in liquid nitrogen during necropsy from healthy laboratory beagle dogs after being euthanized when finishing unrelated long-term trials studying canine intestinal microbiota. based on wsava histology standards, recorded findings of all samples were considered insignificant. pro-mmp- and - activities were found in / ( %) and / ( %) of the samples, respectively. among four different parts of the intestine of dogs, the ileum had the highest positivity rates of / ( . %) and / ( . %) for pro-mmp- and - activities, respectively. however, statistical analysis showed no significant difference of pro-mmp- and - activities between the separate parts of the intestine (p > . ). the enzyme activities ranged for pro-mmp- between . and . arbitrary units (au) and for pro-mmp- between . and . au. none of the intestinal samples showed gelatinolytic activity corresponding to the control bands of active mmp- and mmp- . this study showed that pro-mmp- and - could be detected in the intestinal mucosa of healthy dogs using zymography, which seems to be a useful tool to evaluate the role of mmp- and - in the pathogenesis of canine chronic enteropathies, including inflammatory bowel diseases. no conflicts of interest reported. digestive perforation is called spontaneous when it arises in the absence of foreign body ingestion, gastric dilatation and volvulus, external trauma, or previous digestive surgery. in dogs many predisposing factors have been identified, including antiinflammatory administration, severe hepatic or renal disease, stress, shock, gastric hyperacidity, neoplasia and idiopathic inflammatory bowel disease. spontaneous digestive perforation has been uncommonly reported in cats. the objectives of this study were to describe the clinical characteristics of spontaneous digestive perforation in cats, and to compare the frequency of malignant versus non-malignant causes for these perforations. to be included in this study, the perforation had to be spontaneous and confirmed by exploratory surgery. the medical records of cats diagnosed as having spontaneous digestive perforation between and were reviewed. the mean age of cats was . years ( months to years). five cats had concurrent illnesses including viral upper respiratory tract disease, pancreatitis and chronic kidney disease. the most frequently reported signs included anorexia ( %), vomiting ( %), and lethargy ( %). histological examination was performed in cats and diagnosed alimentary lymphoma in % and inflammatory lesions in the other % of them. six cats had received anti-inflammatory within the previous months. half of them were finally diagnosed with lymphoma. five cats with lymphoma received chemotherapy.three cats died early in the postoperative recovery period, cats were euthanized to days after surgery, and cats were still alive at the end of this study. in the absence of pneumoperitoneum, clinical signs and clinicopathological abnormalities are not specific enough to allow differentiation between cats with gastrointestinal ulceration and those with perforation. in most cases, there is no diagnostic test that individually determine whether perforation has occurred or is impending, and clinicians should use of multimodality diagnostic procedures such as radiography, ultrasonography, endoscopy, and abdominal fluid cytopathology to avoid delay in diagnosis of digestive perforation. histological examination of ulceration is essential as lymphoma should be suspected in all cats presented with spontaneous perforation. the link between anti-inflammatory administration and spontaneous perforation in cats is not established. no conflicts of interest reported. campylobacter species are commonly isolated from faeces of dogs and cats with c. upsaliensis (cu) and c. helveticus (ch) being the most frequently isolated. these two species are usually not considered pathogenic in dogs and cats and are closely related to each other and to c. jejuni, the most common cause of bacterial gastroenteritis in humans in the developed world. interestingly, despite their close genetic relationship, in humans cu is considered a pathogen while ch is not. this study aimed to describe whole genomes of cu and ch isolated from dogs and cats and to in silico investigate their pathogenic potential with comparison to several published genomes of c. jejuni and c. coli. genomic dna was extracted from three isolates of each of cu and ch recovered from the faeces of healthy dogs and cats. sequencing was performed using an illumina miseq to generate base paired reads. reads were trimmed for both length and quality. contigs were assembled using the velvet assembler. the concatenated contigs generated for each assembly were quality ranked (by number, size, maximum length and n ) and the three top ranked assemblies were annotated using the prokka annotation tool. ribosomal mlst nucleotide sequences were used as a proxy for the core genome to compare the phylogeny of cu and ch with other species in the campylobacter genus and visualised as a neighbornet using splitstree. annotated draft genomes were clustered using orthomcl and pathogenic traits were investigated in silico using pathogenfinder and viru-lentpred software. the cu and ch draft genomes were~ . mb and . mb in size, and comprised on average and contigs, and on average and predicted genes, respectively. of these cu had on average and ch hypothetical proteins. using orthomcl, a core genome of and genes resulted for cu and ch, respectively. neighbornet trees based on ribosomal mlst nucleotide sequences and the core genome confirmed the close phylogenetic relationship of ch and cu within the campylobacter genus. pathogenfinder predicted all isolates as human pathogens with probabilities of . - . %. both pathogenfinder and virulentpred identified many pathogenic proteins in cu and ch of different functions (e.g. chemotaxis, transporter and motility systems) but considerably fewer than in c. jejuni and c. coli. this study provides many insights into the pathogenic potential of pet-associated emerging campylobacter pathogens and is to our knowledge, the first to report a draft genome of ch. no conflicts of interest reported. there are only few laboratory markers being evaluated for diagnosing and/or monitoring canine chronic enteropathies, including inflammatory bowel disease (ibd). s a belongs to the s /calgranulin-protein family and has been proposed to play a central role in both innate and acquired immune responses. it has been reported to be increased in stool samples, serum and/or intestinal mucosa in human patients with ibd. myeloperoxidase (mpo) is an enzyme found mostly in granulocytes. intestinal mucosal levels of mpo have been shown to be increased in animal models and human ibd. to date, s a and mpo levels in intestinal mucosal samples have been reported neither from healthy dogs nor from dogs suffering from ibd. to start investigating this aspect in dogs, the objective of this study was to evaluate mucosal s a and mpo levels in the small and large intestines by using enzyme-linked immunoassay (elisa) and spectrophotometric methods, respectively. for the study, historical intestinal tissue samples from four different parts of the intestine (duodenum, jejunum, ileum and colon) were used. the samples were taken and snap frozen in liquid nitrogen during necropsy from healthy laboratory beagle dogs after being euthanized when finishing unrelated long-term trials studying canine intestinal microbiota. based on wsava standards the histologic findings of all samples were considered insignificant. s a concentrations were from the highest to the lowest: ileum, . ( . - . ) lg/l; colon, . ( . - . ) lg/l; duodenum, . ( . - . ) lg/l; and jejunum, . ( . - . ) lg/l. the concentration in the ileum was significantly higher than in all other segments (p < . ), and the colonic mucosal concentration was higher than the jejunal (p < . ). the highest mpo activity was found in the ileum ( . [ . - . ] da/min), followed by jejunum ( . [ . - . ] da/min), duodenum ( . [ . - . ] da/min), and colon ( . [ . - . ] da/min). mpo activity was significantly higher in ileal and duodenal than in colonic mucosal samples (p < . ). the jejunal mpo activity was higher than the colonic and duodenal activity (p < . ). this study showed that using elisa and spectrophotometry allow the detection of canine intestinal mucosal s a and mpo, respectively. the levels on s a and mpo seem to differ between certain parts of the intestinal mucosa of healthy dogs. both assays appear to be useful to further evaluate the role of s a and mpo in the pathogenesis of canine chronic enteropathies, including ibd. dr. heilmann, dr. suchodolski, and dr. steiner have a patent pending that includes the canine s a assay used in this study. the authors declare that they have no further conflicts of interest. the aim of the present study was to establish the incidence of innocent cardiac murmurs in a fairly large number of clinically healthy puppies. a second aim was to evaluate a possible correlation between the presence of an innocent cardiac murmur and a lower hematocrit value. puppies of certain breeds are routinely screened for the presence of congenital porto-systemic shunts in the netherlands. breeders bring their nests to our clinic for individual measurement of blood ammonia concentration. in one year time (from february until january ) dogs of different breeds were examined, with of them being cairn terriers. the age of the dogs varied from to days (mean days). while the breeders were waiting for the blood results, the cardiac auscultation was performed by a single board-certified cardiologist (vsz). hematocrit was measured with an automatized hematology analyzer system from the surplus blood sample. cardiac murmur was found in dogs ( %). in all cases this was a soft ( - out of ) systolic murmur, most of the time with a musical character and with the point of maximal intensity on the left hemithorax, compatible with the description of an innocent cardiac murmur. no murmurs were found that could be compatible with a congenital cardiac anomaly. the hematocrit was significantly (p = . ) lower in the group of dogs with a murmur (mean . %, standard deviation . %) compared to the group without a murmur (mean . %, standard deviation . %). multivariate analysis shows that the presence of murmur is correlated with the hematocrit, but not with the age of the dogs. physiologic anemia has been long suspected to be one of the possible causes of innocent cardiac murmurs in young animals and children. however, according to the authors knowledge, no published reports exist that looked for a possible correlation. which other factors contribute to the presence of an innocent murmur is largely unknown. because of a large overlap between the hematocrit values of dogs with and without a cardiac murmur, measuring hematocrit in a particular pup would not help a less experienced first line veterinary practitioner to decide whether a murmur is innocent or the result of a congenital cardiac anomaly. limitation of this study is that no echocardiography was performed to rule out congenital cardiac anomalies as the cause of the murmurs. neither were the dogs re-checked for spontaneously disappearance of the murmur. no conflicts of interest reported. low intensity systolic murmurs, with point of maximal intensity over the outflow tract on the left side of thorax, are not uncommonly heard during cardiac auscultation of apparently healthy siberian husky dogs, often with excellent exercise tolerance. the origin of these murmurs in athletic dogs such as huskies is unlikely to be due to heart disease but more likely due to turbulent blood flow in the outflow tract caused by a large stroke volume and forceful cardiac contractility in early systole. the differentiation of these murmurs from "pathological"significant murmurs can however be problematic in general practice. the aim of the present study was to investigate the prevalence of murmurs in a sample of successfully racing siberian husky dogs and furthermore to study the phonocardiographic characteristics of these murmurs. phonocardiograms and ecgs were recorded in actively racing siberian husky dogs, with normal or excellent exercise tolerance. normal stamina was confirmed by successful racing. phonocardiograms were easy and rapid to record on a pc laptop connected to the meditron stethoscope in ambulant "field"practice. systole was measured as the duration measured from the onset of the first heart sound to the onset of the second heart sound and the murmur duration from the onset the first heart sound to the end of the murmur. the duration of the first heart sound plus the murmur was measured and calculated as a percentage of the duration of systole. cardiac murmurs of grade - were heard in % of dogs examined. phonocardiogram from these dogs revealed early systolic crescendo-decrescendo or decrescendo murmurs with a duration of maximally % into systole. all dogs with murmurs had a silent pause at the end of systole. ecg was normal in all dogs. murmurs in adult athletic dogs should not be regarded as a definite sign of heart disease. physiological flow murmurs of up to % of systole is a common finding in active siberian husky dogs (prevalence % in the examined sample). phonocardiography is a rapid and practical method for differential diagnoses between pathological murmurs and physiological flow murmurs. no conflicts of interest reported. nt-probnp has a degree of overlap with clinically normal animals, particularly those with mild or subclinical heart disease. prior studies have evaluated the sensitivity and specificity of a point-of-care second generation elisa that utilizes snap technology. the snap feline probnp test uses the same biological reagents as the cardiopet probnp test but provides results in minutes. we sought to prospectively validate the assay in a population of clinically normal cats. cats were recruited based upon the absence of a heart murmur, gallop, and/or arrhythmia. all cats received physical examination, non-invasive blood pressure measurement, complete biochemical analysis including a t , urinalysis and echocardiogram. only cats considered free of underlying cardiac or systemic disease were enrolled. sixteen adult cats were enrolled and blood samples were obtained for nt-probnp concentrations at , hr, hr, hr, hr, hr. samples were placed in edta tubes and centrifuged within one hour and split into two tubes for duplicate samples at each time point and stored at - °c. once all samples were collected, they were shipped on dry ice overnight and run in one batch (idexx laboratories) for measurement of nt-probnp concentrations. snap tests were visually evaluated by one blinded reader. comparison of snap assay vs. quantitative elisa revealed a . (auc) degree of correlation between assays, and that a positive snap test result was associated with a nt-probnp concentration of . pmol/l or greater. the average bnp concentration of abnormal cats ( . ae . ) determined by the snap assay was significantly greater than the normal ( . ae . ). this study was funded through idexx and the university of florida college of veterinary medicine resident grant competition. we acquired d echocardiographic cineloops from the left apical -chamber view optimized for the la, and analyzed atrial longitudinal strain (st) and strain rate (sr) in dogs ( healthy dogs and dogs with mmvd - acvim stage b , stage b and stage c). endocardial la ste curves were obtained and peak atrial longitudinal strain (pals), peak atrial contraction strain (pacs), conduit atrial longitudinal strain (cals); pals-pacs) and contraction strain index (csi -pacs/pals* ) were calculated. la sr curves were similarly obtained to determine the peak positive strain rate (srs) during left ventricle systole, the first negative peak strain rate (sre) during early diastole and the second negative peak strain rate (sra) during atrial contraction. for all variables, a mean of measures was used for the statistical analysis. we compared each of these variables between each acvim stage by kruskal-wallis tests and post-hoc pairwise comparisons, with comparison-wise a= . . normal dogs had higher pals and cals than dogs with mmvd (p < . and p = . ); stage c dogs had lower pals, pacs and cals than all other dogs (p < . , p = . and p = . ), but csi did not differ between groups (p = . ). stage c dogs had lower srs (p = . ), higher sre (p = . ) and sra (p = . ) than other dogs. normal dogs had lower sre and sra than dogs with mmvd (p < . ). our data suggest that ste might be useful in assessing la function in dogs with mmvd, and might potentially differentiate dogs with severe subclinical disease from dogs with congestive heart failure. no conflicts of interest reported. sighthounds are athletic dogs and they have been claimed to have larger hearts compared to similar sized breeds. the left ventricle (lv) may enlarge in response to cardiac disease, but also in response to training, so called athlete's heart syndrome, which is a benign condition. to distinguish abnormal echocardiographic measurements from normal, breed-specific reference values are needed. the aim of this study is to establish normal reference ranges for echocardiographic measurements in the saluki breed. the study comprised clinically healthy salukis ( males and females), mean age months (ae sd months), bodyweight (bw) , kg (ae , kg). case history was ascertained and dogs underwent physical examination, complete blood count, serum biochemistry profile, thyroid profile, blood pressure measurement and -min ecg. standard m-mode and d echocardiographic measurements were obtained. dogs with systolic murmur / , and dogs with mitral valve regurgitation (mr) < % (mr color flow jet area/left atrium areax % in apical view) were considered normal. linear regression models were used to establish reference ranges. heart rate (hr) varied from to bpm ( ae bpm). bw was a significant predictor for lv dimensions, i.e. m-mode lv diameter and d volume in diastole (lvidd and lvedv) and systole (lvids and lvesv), and mitral valve end point septal separation (epss). hr was a significant predictor for fs % (fractional shortening). predicted values ( % prediction intervals) were calculated from regression models where mean bw ( , kg) and age ( months), and median hr ( bpm) were used. normal reference ranges were: lvidd , mm ( , - , ), lvids , mm ( , ) , lvedv , ml ( , - , ), lvesv , ml ( , - , ), fs%: , % ( , - , ), ejection fraction ef%: , % ( , - , ), epss , mm ( , - , ), sphericity index , ( , - , ), interventricular septum in diastole , mm ( , - , ) and systole , mm ( , - , ), lv free wall in diastole , mm ( , - , ) and systole , mm ( , - , ), left atrial (la) diameter , mm ( , ) , aortic (ao) diameter , mm ( , - , ) , la/ao , ( , - , ), and aortic and pulmonic flow velocity , m/s ( , ) and , m/s ( , - , ), respectively. this study provides echocardiographic values for normal salukis which can be used as a reference values. no conflicts of interest reported. in mitral valve disease, atrial remodeling is an indicator of evolution and prognosis, the duration of the p wave being considered suggestive of the dilatation of the left atrium. in humans' studies, neurological conditions have a significant impact on cardiac electrophysiology by altering the electrical impulse conductibility. the aim of this study is to examine the duration of the p wave in dogs suffering from mitral valve disease in comparison to dogs diagnosed with different neuropathies without cardiac abnormalities. we analyzed standard electrocardiograms ( min of ecg, on peripheral leads) performed on three polymorphic groups of dogs (different age, weight and breed): group (n = ) healthy dogs, group (n = ) dogs diagnosed with mitral valve disease and group (n = ) dogs suffering from different neuropathies (without any associated or previously diagnosed cardiovascular disease). the duration of the p wave was measured for all dogs (five consecutive p waves without anomalies or artifacts) and reported to the degree of atrial remodeling, assessed by left atrium/ aorta ratio on echocardiography. the interpretation of the ecg and echocardiography was made by the same examiner (md). the results were statistically evaluated in a specialized program (ibm spss vs. ). the p wave recorded average values was . ae . seconds for the mvd group with significant differences between the stages of heart failure (p = . ). no correlations were found between its increase and the dilation of the left atrium (r = . ). there was no statistically significant difference regarding p wave duration when compared dogs of the neuropathy group and those of the mitral valve disease group (the p wave recorded average values = . ae . sec.). both, atrial tissue lesions (as in mitral valve disease) and autonomic nervous system anomalies (secondary to a neurological condition), may change the conductibility of the electrical impulse in the left atrium. the conductibility of the electrical impulse at this level does not seem to be influenced by its actual dilation, but by the impairment of the intra-atrial and inter-atrial conduction pathways. caution must be given when p wave is analyzed in dogs with concurrent cardiologic and neurologic condition. no conflicts of interest reported. newfoundland dogs were prospectively recruited among those undergoing screening for congenital and acquired heart disease. screening includes patient history, physical examination, and systemic arterial pressure measurement by doppler flow meter and transthoracic echocardiography (m-mode, d and echo-doppler). screening is performed on conscious dogs of at least year of age. dogs without historical, clinical, electrocardiographic and echocardiographic signs of cardiovascular disease were included in the study. unpaired, two-tailed student's t-test and linear regression were performed to evaluate the influence of gender, age and body weight (bw) on echocardiographic parameters. echocardiographic measurements were compared to previously reported reference values. the reference limits of echocardiographic parameters in the newfoundland dogs were calculated. forty-six healthy adult newfoundland dogs of both genders ( males and females), to years of age (mean . ae . years), to kg (mean . ae . kg) fulfilled the inclusion criteria. significant but weak correlations were detected between aortic diameter (ao) and age (p = . , r = . ), left atrial to aortic ratio (la/ao) and age (p = . , r = . ), e-point to septum separation (epss) and bw (p = . , r = . ), m-mode left ventricular internal diameter (lvid) in diastole (d) and systole (s) and bw (respectively p = . , r = . and p = . , r = . ), and between ao and bw (p = . , r = . ). none of the echocardiographic measurements was statistically different between males and females. left ventricular internal diameter in diastole, lvids, ao, epss increased with bw, as expected. the aorta appears to become wider with advancing age. a proportion of the studied population had m-mode parameters below the allometric scaling reference range, suggesting that this method can over-estimates m-mode parameters in this breed. these findings stress the importance to report newfoundland breed specific normal ranges for echocardiographic parameters. no conflicts of interest reported. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease usually diagnosed by thoracic ct-scan that mainly affects west highland white terriers (whwt). pulmonary hypertension (ph), a severe co-morbid condition with a challenging diagnosis, may develop in cipf dogs. the ratio between the right pulmonary vein and pulmonary artery (pv/ pa) has been described as an echocardiographic indicator of ph in cipfdogs. this study was intended to investigate whether ct-scan angiography cardiac findings are ) altered in dogs with cipf compared to healthy control dogs and ) correlated with pv/pa measured by echocardiography (pv/pa us ). thoracic cta images from whwt with cipf (group a) and healthy controls from various breeds (group b) were retrospectively reviewed by one observer. all measurements were obtained in transverse post-contrast images displayed in a soft tissue window. pv and pa were measured dorsal to the right atrium, perpendicular to the long axis of these vessels. in addition, pulmonary trunk (pt) was assessed just ventral to the division of pulmonary arteries, perpendicular to its long axis. ascending aorta (ao) was also measured perpendicular to its long axis. transverse reformatted images were obtained to have a view equivalent to the standard chambers-echocardiographic view where right ventricle (rv) and left ventricle (lv) were measured. three ratios were calculated pv/pa ct , pt/ao and rv/lv, compared between groups and correlated with pv/pa us in both bi-dimensional (bd) and m-modes (mm). statistical analyses were performed with xlstat â software. values are given as mean ae sd. statistical significance was set at a p ≤ . . pv/pa ct was lower in group a ( . ae . ) in comparison to group b ( . ae . , p = . ) and correlated with pv/pa us (bd: r = . , p = . ; mm: r = . , p = . ). pt/ao was higher in group a ( . ae . ) compared to group b ( . ae . , p = . ) and correlated only with pv/pa us measured in bd mode (r = - . , p = . ). the rv/lv ratio was increased in group a ( . ae . ) in comparison to group b ( . ae . , p = . ) and a correlation between rv/lv and pv/pa us was found (bd: r = - . , p = . ; mm: r = - . , p = . ). in conclusion, in whwt with cipf, pv/pa ct , pt/ao and rv/lv ratios measured on thoracic cta images are correlated with pv/pa us and may serve in the assessment of ph. no conflicts of interest reported. companion animals presenting to the emergency room in distress need to be assessed rapidly and accurately to implement life-saving therapies. focused cardiac ultrasound (focus) can be a useful adjunct to the physical examination in assessing dyspneic animals in the emergency room. rapid bedside ultrasound evaluations performed by ec are commonly used in human medicine, however feasibility and utility of focus by ec in veterinary medicine has not been fully evaluated. the purpose of this study is to determine the baseline accuracy of focus performed by ec and whether or not a basic training session could improve accuracy compared to evaluation by a cardiology specialist. fifteen ec including boarded emergency-critical care specialists and emergency res-idents performed focus on four animals; a normal cat and dog, and a cat and dog with severe valvular and myocardial heart disease, respectively. ec semi-quantitatively assessed thoracic and echocardiographic parameters including left atrial dimension, left ventricular systolic function and wall thickness, right heart dimension, and presence or absence of pleural or pericardial effusion before and after a structured didactic lecture and hands-on practical session. primary outcome was the level of agreement with examination performed by a cardiologist. level of agreement regarding ec assessment of all parameters improved from . to . after training (p < . ). level of agreement concerning left atrial diameter improved from . to . (p < . ). ec confidence in their overall focus evaluation and findings improved from % to % (p < . ). in summary, ec accuracy and confidence in semi-quantitatively assessing basic cardiac parameters using focus were improved following a simple structured training session. focus might be a valuable tool to rapidly assess simple thoracic and cardiac parameters in the emergency setting. no conflicts of interest reported. obesity is an increasing health problem in dogs. success of weight-loss programs is often limited by compliance issues. the purpose of this study was to determine the effectiveness of a new dietetic weight management food (ndwmf)* in achieving weight loss in overweight/obese, client-owned dogs, under typical household conditions. the objectives were ) to evaluate weight loss parameters in dogs fed a ndwmf* and ) to assess the owner's perception of the dog's quality of life. overweight/obese, otherwise healthy, client-owned dogs (> / body condition score -bcs) were enrolled in the study (n = ). initial veterinary evaluation included physical examination, nutritional assessment, determination of ideal body weight (ibw), and development of weightloss feeding guidelines. daily energy requirement (der) for weight loss was calculated as der = x ibw kg . . initial and follow-up evaluations (monthly for months) encompassed determination of body weight, bcs, body fat index (bfi), muscle condition score (mcs), and feeding practices. quality of life assessment by owners included dog's level of energy, happiness, appetite, begging behavior, flatulence, stool volume, and fecal score. statistical analysis comprised scatterplots, regression analysis, summary statistics as appropriate for the type of analyses performed (continuous or categorical variables, distribution), and a mixed model anova to assess changes over time (with statistical significance at p < . ). ninety four percent of the dogs lost weight (n = ) with an average weight loss of . % (sem, . %) over months and an average weekly weight-loss rate of . % (sem, . %). the mean duration of weight loss was days (sem, . days) with an average of days (sem, . days) between rechecks. thirty nine percent of dogs achieved ibw ( . , ci: . - . ). fifty five percent of dogs ate more calories from ndwmf* than the recommended der for weight loss (median fed above der= %) and % of these dogs ( . , ci: . - . ) still lost weight. thirty six percent of dogs received treats. bcs and bfi decreased significantly over time compared to baseline. owners perceived a significant increase in energy and happiness in the dogs that lost weight without changes in appetite or begging behavior. in conclusion, this clinical study confirmed the effectiveness of the ndwmf* in achieving weight loss in overweight/obese client-owned dogs in spite of higher than recommended caloric intake. owners reported significant improvements in dog's quality of life without negative side effects. * haptoglobin is a moderate acute phase protein in cats. as a part of the innate immune system its concentration rises within - hours after tissue damage. aim of the study was to validate an elisa which was recently developed for the measurement of feline haptoglobin and to compare it with a commonly used spectrophotometric assay. the concentration of haptoglobin was measured in healthy and sick cats using a sandwich-elisa (tecomedical group, rheinbach, germany). the validation included the detection of intra-assay and inter-assay variation, dilution linearity, spike recovery and lower detection limit. a spectrophotometric assay (tridelta development ltd, maynooth, ireland) was used as a reference method. all samples were measured in duplicate. statistical analysis was performed using ibm â spss â statistics (ibm corporation â ) and included descriptive statistics, spearman correlation (rs) and coefficients of variation (cv). the coefficients of variation were . %, . % and . % for intra-assay variability and . %, . % and . % for inter-assay variability. the ratio of observed to expected dilutional parallelism of serum samples diluted times ranged from to %. the ratio of observed to expected spike recovery of serum samples ranged from % to %. the lower detection limit was . mg/ml. the correlation between the assays was significantly strong (rs = . , p < . ). the recently available sandwich-elisa provides a high accuracy and precision and can therefore be used for the measurement of feline haptoglobin. the rd and th author (m. hennies and c. wienen) work for the company tecomedical group that developed the elisa which was evaluated in the study. they provided the kits and they helped with performing the tests, but they did not have any influence on the results and the interpretation of the data. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease that mainly occurs in the west highland white terrier (whwt) breed. the cipf diagnosis commonly relies on thoracic high-resolution computed tomography (hrct) findings and ultimately on histopathology. as those tests are not easily performed in practice, identification of measurable markers of fibrosis, that might help to diagnose and/or monitor the course of cipf, is helpful. vegf is an angiogenic regulator involved in a variety of physiological and pathological processes. in human ipf, serum vegf concentration has been shown to be higher in ipf patients compared to healthy volunteers and may reflect the severity of the lung disease. the aims of the present study were ( ) to investigate the potential role of vegf as a peripheral blood biomarker in cipf; and ( ) to investigate possible breed-related differences in basal vegf concentration, that might explain the high predisposition of the whwt breed for cipf.therefore, vegf was determined by elisa (canine vegf quantikine elisa kit, r&d systems) in the serum of whwt with cipf confirmed by hrct and/or histopathology (median age years, range - ), healthy whwt ( , - ), and healthy dogs of other breeds, including: scottish terrier (st) ( , - ), jack russell terrier (jrt) ( , - ), maltese ( , - ), king charles spaniel (kcs) ( , - ), labrador retriever (lr) ( , - ) and malinois belgian shepherd ( , - ). health status was based on clinical examination, serum biochemistry and haematology in all healthy dogs and a thoracic hrct was performed in / healthy whwt. the khi² test with the threshold % was used for the statistical analysis (xlstat â software). eight cipf whwt ( %) have serum vegf concentrations above the kit detection limit ( . pg/ml) compared to whwt ( . %) in the group of healthy dogs (p = . ). concerning inter-breed differences in healthy dogs, most values obtained were below the kit detection limit with only kcs ( %), jrt ( %), lr ( %) and st ( %) having vegf serum levels above . pg/ml (p = . ). results of the present study show that ( ) vegf might be an interesting blood biomarker for cipf; ( ) canine vegf quantikine elisa kit is not appropriate for measurement of serum vegf levels in healthy canine populations. no conflicts of interest reported. the total protein (tp) concentration and cell count of pleural and abdominal fluid is used to differentiate a transudate from an exudate. tp can be measured by automated wet chemistry analyser or more easily using a refractometer. the aim of this study was to assess if refractometer values of tp are useful for this purpose. retrospectively samples from canine pleural and abdominal effusions in which tp concentration was measured both with a refractometer as well using pentra (abx horiba, montpellier) were included. samples were collected into heparinized tubes and analysed within hours. bland-altman diagrams were created and correlation between both measurements was calculated by spearman s nonparametric correlation. over a -months period, pleural and abdominal effusion samples were analysed with both techniques. median (range) tp concentrations in pleural effusion measured by refractormeter or by pentra was ( - ) g/l and ( - ) g/l, respectively. median (range) tp concentrations in abdominal effusions measured by refractometer or pentra was ( - ) g/l and ( - ) g/l, respectively. tp measurement between refractometer and pentra values were significantly correlated in pleural (r = . , p < . ) and abdominal (r = . , p < . ) effusion. the bland-altman graph showed a bias in the thorax and abdomen of . and . . the refractometer is an acceptable, rapid and efficient method for determination of total protein concentration in pleural and abdominal effusions in dogs to differentiate transudates from exudates. no conflicts of interest reported. coagulation factor vii (fvii) deficiency has been reported in beagles since the 's. deficient dogs show a mild hemorrhagic tendency, but often remain asymptomatic and are incidentally discovered by an isolated prolonged prothrombin time due to < % plasma fvii activity. factor vii deficiency occurs commonly in beagles, alaskan klee kais and scottish deerhounds. in these breeds it is caused by a single missense mutation (c. g>a, p.gly glu) in the second epidermal growth factorlike domain of fvii, which drastically reduces the secretion and activation of fvii. research beagles were also commonly affected which may have pharmaco-toxicologically affected studies but specific dna screening programs have been established. we report here on the discovery of fvii deficiency in welsh springer spaniels (wss) in finland based upon a novel screening panel for~ known mutations underlying inherited disorders in different canine breeds (www.mydogdna.com). among wss initially tested, were heterozygously ( %), and homozygously affected for the same fvii mutation, which was confirmed by sequencing in all dogs. in order to determine whether the mutation causes fvii deficiency also in this breed, we recruited littermates and their mother. none of these wss had shown an increased hemorrhagic tendency, but affecteds bled excessively following blood collection. we found that the homozygous affected dogs of the litter exhibited markedly prolonged prothrombin time but normal partial thromboplastin time. they also had drastically reduced fvii activities but normal to high fviii and fix activities compared to their littermate controls. the heterozygous carriers tested did not show any prolongations in their prothrombin time, but had half normal fvii activity. in conclusion, we document here the presence of fvii deficiency in wss based upon dna and coagulation activity testing. the common gly glu mutation must have arisen prior to the separation of the very different fvii deficient breeds. there is no knowledge of an advantage of the heterozygote state. while there is only a mild hemorrhagic tendency, bleeding dogs could be treated with fresh frozen or cryo-poor plasma or human recombinant fviia. this preliminary study indicates a high carrier frequency in wss. screening by new platform dna methods for this and other ancestral defects is helpful to detect additional hereditary diseases and genetic predispositions in different breeds, while other mutations are new and restricted to one or related breeds. authors are affiliated with genetic disease screening test laboratory. remarkably little has been published on haematological and serum biochemical phenotypes of the domestic dog. information on the signalment and complete blood cell count of all dogs with normal red and white blood cell parameters judged by existing reference intervals was extracted from a veterinary database; similar information was collected from all dogs with normal serum biochemical profiles, considering all parameters other than glucose as inclusion criteria. normal haematological profiles were available for dogs, of which also had machine platelet concentrations within the reference interval; normal serum biochemical profiles were available from dogs, of which also had accompanying normal serum glucose concentrations. for the haematological data, pure breeds plus a mixed breed control group were represented by or more dogs, while for the serum biochemical data, pure breeds plus a mixed breed control group were represented by or more individuals. all measured haematological parameters except mean corpuscular haemoglobin concentration (mchc), and all serum biochemical analytes except sodium, chloride and glucose, varied with age. concentrations of white blood cells (wbcs), neutrophils, monocytes, lymphocytes, eosinophils and platelets, but not red blood cell parameters, all varied with sex, as did total protein, globulin, potassium, chloride, creatinine, cholesterol, total bilirubin, and activities of alanine aminotransferase (alt), creatine kinase (ck), amylase and lipase. neutering status had an impact on haemoglobin concentration, mean corpuscular haemoglobin (mch), mchc, and concentrations of wbcs, neutrophils, monocytes, lymphocytes and platelets, as well as all serum biochemical analytes except albumin, sodium, calcium, urea and glucose. principal component analysis (pca) of haematological data revealed pure breeds with distinctive phenotypes, while pca of serum biochemical data revealed over pure breeds with distinctive phenotypes. furthermore, all haematological parameters except mchc and all serum biochemical analytes except urea and glucose showed significant differences between specific individual breeds and the mixed breed group. twenty-nine breeds had distinctive haematological phenotypes and breeds had distinctive serum biochemical phenotypes when assessed in this way. tentative breed-specific reference intervals were generated for breeds with a distinctive phenotype identified by comparative analysis. this study represents the first large-scale analysis of haematological and serum biochemical phenotypes in the dog and underlines the important potential of this species in the elucidation of genetic determinants of haematological and biochemical traits, triangulating phenotype, breed and genetic predisposition, as well as the urgent need for breed-specific reference intervals in clinical practice. the author has received funding from bbsrc, petplan charitable trust, and cruk), but none of thesegrants were for this study. for each cat aged years or older, irrespective of their suspected thyroid status, presented to eight veterinary practices in portugal, the veterinarian and the pet owner had to complete a questionnaire and the veterinarian had to take a venous blood sample (into a plain tube) from the cat, after obtaining signed owner consent. the veterinary questionnaire included history, attitude, activity, heart rate and thyroid palpation. cats aged < years and those diagnosed previously with hyperthyroidism were excluded. blood samples were centrifuged and the serum harvested and stored frozen until collection by the laboratory within days of sampling. total t was measured using a chemiluminescent method (immulite , siemens). cats were classified as hyperthyroid, equivocal or euthyroid based on a total t concentration of > nmol/l, - nmol/l or < nmol/l, respectively. repeat measurement of total t after - weeks was recommended for all equivocal cases. the individual cat was the statistical unit. descriptive statistics was used to summarise the data and associations between different clinical signs analysed using chi-square, fisher's exact test or the mann-whitney u test. the level of significance was set at . . thirty cats were excluded from the prevalence analysis because they were aged < years (between and years, n = ) or their age was not stated (n = , four of these cats were hyperthyroid). by the end of february , samples had been submitted from cats that met the inclusion criteria. based on the thyroid hormone analysis, there were / ( %) hyperthyroid, ( %) equivocal and ( %) euthyroid cats. very few follow-up blood samples were taken. hyperthyroidism appears to be not uncommon in portuguese cats. getting owners to return for follow-up blood sampling appears to be problematic. under-reporting of hyperthyroidism appears to be a significant problem in portugal, as has been reported for some other countries. thyroid palpation should form part of routine physical examinations, especially of middle aged and older cats. older cats in portugal should be screened for hyperthyroidism even in the absence of a detectable thyroid nodule. both authors are employees of msd animal health. msd animal health funded the study. msd animal health has an approved veterinary medicinal product for the treatment of feline hyperthyroidism. this product is available commercially in some eu markets but not in portugal. diabetes mellitus is one of the most commonly encountered endocrinopathies in cats and its prevalence has increased in the past. similar to human type diabetes, feline diabetes is associated with comparable lesions occurring in the pancreatic islets, namely islet amyloidosis and beta-cell loss. studying the pathophysiology of feline diabetes and the molecular mechanisms through which glucose metabolism is disturbed is largely hampered by the lack of a method for the isolation of pure pancreatic islets. the aim of this project was to improve a previously established method for the isolation of pancreatic islets; in particular enhancing the purity of isolated islets in this species. cats that died or were euthanized due to severe illness other than pancreatitis or other pancreatic disease were enrolled. pancreata were perfused post-mortem with ml collagenase type iv ( . mg/ml) through the pancreatic duct. the perfused organ was then digested for ', ' and ' at °c in a water-bath and purified using a filtration method. islet cell viability and purity were determined by thiazolyl blue tetrazolium bromide (mtt assay) and dithizone staining, respectively. perfusing the pancreas through the pancreatic duct allowed collagenase to access the islets using anatomical structures and to improve islet yield compared to previously established protocols in this species. the digestion time of ' provided the best islet yield. after digestion, feline pancreatic islets remained satisfactorily viable for days in the culture system following regular media changes. the current study has successfully optimized the isolation, purification and culture maintenance of feline islets. the successful yield and viability of islets isolated through the suggested protocol may provide promising potential as a source of islets for diabetes research in cats. no conflicts of interest reported. nesidioblastosis describes a syndrome of acquired hyperinsulinaemia and associated hypoglycaemia secondary to focal or diffuse (non-neoplastic) beta cell hyperplasia within the pancreas. beta cell dysregulation is thought to occur secondary to pancreatic injury. this syndrome has been reported in humans with increasing frequency, but it has not previously been described in domestic pets. a six year old, de-sexed female british shorthair cat presented with acute onset weakness and mental dullness. upon initial presentation the cat was mildly hyperglycaemic ( . mmol/l; . - . mmol/l). over the following hours the cat developed central blindness, tremors, intermittent seizures and opisthotonus. repeat blood sampling revealed a marked hypoglycaemia ( . mmol/l). an insulin level (performed on serum obtained while the cat was hypoglycaemic) was inappropriately elevated ( pmol/l; reference range - pmol/l). an intravenous bolus of % glucose resulted in rapid resolution of all clinical signs and mild transient hyperglycaemia ( . mmol/l). despite frequent feeding, the hypoglycaemia ( . mmol/l) recurred, so an intravenous glucose continuous rate infusion was commenced. an abdominal ultrasound was unremarkable, although three cranial mesenteric lymph nodes were noted to be prominent ( mm in width). an exploratory laparotomy revealed a firm and erythematous left limb of the pancreas. the body and right limb of the pancreas appeared grossly normal. following surgical resection of the left limb of the pancreas, the cat returned to a euglycaemic state after a brief rebound hyperglycaemia. histopathology revealed pancreatic fibrosis with marked multifocal micronodular hyperplasia of exocrine and endocrine cells, mild lymphoplasmacytic inflammation and ductular ectasia. synaptophysin immunohistochemistry confirmed nodular beta cell hyperplasia. mild granulomatous lymphadenitis and hydropic change within hepatocytes was also noted. the cat recovered uneventfully without any further intervention. it gained weight and remained euglycaemic over the following six months. while beta cell hyperplasia has been reported as an incidental histopathological finding in euglycaemic young beagles, this is the first reported case of clinically significant hypoglycaemia secondary to nesidioblastosis in a domestic pet. while this condition is rare, nesidioblastosis is being increasingly recognised in the human field and it is an important differential to consider when investigating hypoglycaemia as it cannot be differentiated from insulinoma without histopathological evaluation. age of onset may provide a clue to this non-neoplastic disease, as this cat was much younger than all previously reported cases of feline insulinoma (all > years of age at diagnosis). while recurrence has been reported in humans, a favourable outcome is anticipated following partial pancreatectomy. no conflicts of interest reported. hyperthyroidism is the most common feline endocrinopathy of geriatric cats worldwide. nonetheless, data concerning the accurate prevalence of feline hyperthyroidism (fh) is scarce; and apparently exhibit geographical variation, which can be an important instrument in investigating risk factors through the analysis of exposure to different factors in areas of high and low prevalence. in europe, fh is considered more frequent in the northern than in the southern countries. the aims of this study were to determine the occurrence of fh in a region of portugal, to characterize clinical presentation and potential risk factors. during an -month period, geriatric cats ≥ eight years from aveiro (central region of portugal) were selected. cats were excluded if presented in shock or moribund, or in treatment with drugs that might affect total t (tt ) serum concentration. the tt concentration was determined through chemiluminescence (immulite â , siemens), and diagnosis of fh established if tt serum concentration ≥ . lg/dl (reference values . - . lg/dl) associated with compatible clinical signs. information on age, gender, breed, weight, housing conditions (indoor vs outdoor), use of external parasiticides, food (dry vs canned food and flavor), use of litter box, environment, clinical signs and laboratory data was collected. all owners gave informed consent. population studied included males ( . %) and females ( . %), mainly domestic short-haired cats ( . %). ages ranged from eight to years old ( . + /- . ). eight ( . %) cats were diagnosed with hyperthyroidism. if only cats ≥ years of age were considered (n = ), prevalence raised to . %. hyperthyroid population comprised four males and four females, ranging from to years old ( . + /- . ). increasing age (p = . ), polyphagia (p˂ . ), weight loss associated with increased (p˂ . ) or normal appetite (p˂ . ), presence of thyroid uni or bilateral nodules (p˂ . ), vomiting (p = . ) and hyperactivity (p = . ) were significantly associated with hyperthyroidism in the geriatric population studied. environment was also significantly associated with development of fh (p˂ . ), with cats from urban or semi-rural areas at higher risk of developing the disease than cats living in a rural environment. no other significant associations were found between hyperthyroidism and other factors analyzed. in our knowledge, no epidemiologic studies on fh have been performed in portugal, a country where the occurrence is believed to be low, but in which the population of pet cats, the feline geriatric population and the clinical cases diagnosed have been increasing. conflicts of interest: the study was partially supported by laborat orio segalab s.a. and dechra veterinary products. canine diabetes mellitus (cdm) has been proposed to be a spontaneous animal model of human autoimmune diabetes, and comparative research can be undertaken to investigate the interaction between genetic and environmental factors. most epidemiological studies of cdm have been performed in northern european and north american populations. our aim was to evaluate the epidemiology and clinical features of the diabetic dog population from the canary islands, with special focus on immune-mediated disease. dogs attending our veterinary teaching hospital were included from january to january . previously diagnosed and new cases were considered. prevalence was calculated as number of cdm/total number of dogs attending the hospital and incidence as newly diagnosed cases divided by the same value per year. anti-insulin antibodies were assessed by elisa. genotyping for dog leukocyte antigen (dla) and measurement of canine anti-gad and anti-ia antibodies by radio-immunoprecipitation assay were performed in dogs with suspected immune-mediated diabetes. twenty-nine dogs with cdm were identified from a mean population of ( - ) dogs per year (mean prevalence . % and mean incidence cases per year per , ). age at diagnosis was . years (range: . - y). most dogs were not neutered ( % females; % males). nine breeds were represented, including poodle ( %) and andalusian wine-cellar rathunting dog ( %). seasonality was observed in the diagnosis with peaks in december and march-april. diabetes was classified as dioestrus diabetes ( %), idiopathic/immune-mediated ( %), iatrogenic ( %) and secondary to pancreatitis ( %) or other endocrine disorders ( %). insulin-treated dogs were negative for anti-insulin antibodies (n = ). from the suspected immune-mediated cases (n = ), autoantibody reactivity was shown in two cases (anti-gad , n = ; anti-ia , n = ). no previously described, diabetes-risk dla-types were identified. although age, prevalence and incidence did not differ from previous studies, the high proportion of entire females likely explained the high frequency of dioestrus diabetes. the andalusian wine-cellar rat-hunting dog was identified as a high-risk breed for cdm. most of the dla-types seen have not been previously described, but at least two have been associated with increased risk of autoimmunity in dogs. further population-based studies are needed in different regions, to assess the heterogeneous nature of this disease. no conflicts of interest reported. the cortisol-dehydroepiandrosterone (dhea)-ratio is widely used in human medicine as a marker for stress however it is not clear whether it could also help in distinguishing hyperadrenocorticism (hac) from other diseases which might have a negative impact on the outcome of a dexamethasone low dose test. therefore the aim of the study was to evaluate the cortisol-dhea-ratio as an additional diagnostic marker for hac in dogs. to achieve this aim, a reference range of this ratio depending on the sex should be evaluated in healthy dogs and compared with dogs having a hac. in healthy dogs (age: - . years) and in dogs with hac (age: . - . years) of different breeds the plasma concentration of cortisol (immulite system, siemens healthcare diagnostics) and dhea (beckman coulter) was measured and the ratio was calculated. all dogs were patients of the small animal clinic except five of the healthy dogs which were recruited from the institute of pharmacology, toxicology and pharmacy of the university. with these data the cortisol-dhea-ratio was calculated for male dogs (healthy dogs n = ; dogs with hac n = ), neutered males (healthy dogs n = ; dogs with hac n = ), female dogs (healthy dogs n = ; dogs with hac n = ) and spayed females (healthy dogs n = ; dogs with hac n = ). the statistical analysis was performed with sigma stat. the plasma cortisol-dhea-ratio of healthy male dogs was the lowest ratio of all sexual categories (mean average . ae ) and it differed significantly to all other sexes (neutered males = ae , p = . ; females = ae , p < . and spayed females ( ae . , p = . ). the cortisol-dhea-ratio showed no significant difference between male and female dogs with hac. spayed females with hac had significantly higher cortisol-dhea-ratios ( ae ) than healthy spayed females (p = . ) but no significant differences were found in other sexual categories. this preliminary data indicates that the cortisol-dhea-ratio might not be a very promising tool for the diagnosis of hac. in addition, the significant gender-dependency of this parameter has to be considered and may generally limit its clinical usefulness. this study is financially supported by the bruns-stiftung. no conflicts of interest reported. hyperthyroidism is common in older cats. the aim of this study was to assess the prevalence of feline hyperthyroidism and potential intrinsic risk factors in a hospital population in southern germany. total thyroxine (t ) was prospectively measured by enzyme immunoassay (eia) in sera of cats older than years that were presented to the clinic of small animal medicine. a standardized physical examination was performed, and body condition score (bcs) and thyroid palpation score (tps) were assessed. association between signalement, bcs and tps was analyzed by student s unpaired t-test, chi-square, and mann-whitney test. level of significance was set at . . fifty nine cats were diagnosed with hyperthyroidism leading to a prevalence of . % (ci . - . ). hyperthyroid cats were older than non-hyperthyroid cats (p < . ) and more often female (p = . , odds ratio . ). domestic short or long hair cats were more often affected than pedigrees (p = . ). hyperthyroid cats had higher tps (p < . ) and lower weight than non-hyperthyroid cats (p < . ) although bcs was not different (p = . ). in ( . %) cats, the elevated t was an incidental finding. in of those, the disease was confirmed later (the others were dead due to unrelated diseases). in patients, hyperthyroidism was considered a differential diagnosis and was confirmed in ( . %) cats although in cats additional diagnostic means were necessary. older female domestic cats are predisposed to hyperthyroidism which is frequently diagnosed after the initial clinical suspect. in a few affected cats an elevated t is not present or can precede clinical signs. conflicts of interest: the study was partially funded by msd intervet. the main endocrinopathy affecting both humans and pet felines is diabetes mellitus. accurate diagnosis is the most important aspect in the future outcome of the disease. a computer based decision support system (dss) is targeted on assisting clinicians with one or more steps of the diagnostic process. the novelty of our dss emerges from the possibility of assisting both clinical and paraclinical diagnosis stages of diabetes mellitus and all common combination of disorders associated with this endocrinopathy. the motivation behind the development of such system is the desire to maximize the reliability of clinical decisions. the design of our feline diabetes mellitus dss emerges from the syndrome of polyuria-polydipsia, with the possibility of spotting the accompanying pathologies. fuzzy logic is used for dealing with knowledge representation and uncertainty. the fuzzy rules proposed to represent this knowledge emerge from anamnesis, clinician's input, clinical and paraclinical description, and confirmation diagnostic tests. clinical signs such as polyuriapolydipsia, persistent hyperglycemia, polyphagia, weight fluctuations, administration of drugs with a diabetogenic potential, were considered decisive in the pattern of diagnosis establishment. registered medical records of cats, males and females, whit ages from to years old, were analyzed in order to validate the dss. using matlab software, the dss was implemented and tested. for any case with polyuria-polydipsia the system provides, via a friendly graphical user interface, the diagnosis with the highest probability. the set of diagnoses which can be generated by the dss consists in: a) diabetes mellitus; b) diabetes mellitus induced by (b. ) hypersomathotropism, (b. ) hyperthyroidism, (b. ) hyperadrenocorticism and (b. ) diabetogenic medication; c) diabetes mellitus in association with (c. ) chronic kidney failure and (c. ) heart failure; d) ketoacidodic diabetes mellitus; e) pancreatitis. the dss was applied with success on all cases, revealing the following diagnoses / no of cases: (a) - , (b. ) - , (b. ) - , (b. ) - , (c. ) - , (c. ) - , (d) - . an adequate treatment protocol requires an accurate and complete diagnosis. advanced computational systems accompany clinicians in their decision making, leaving a reduced space for medical errors and superfluous, expensive and time consuming tests. future work will be targeted on exploring the possibilities of combining the dss with an artificial neural network model for diabetes mellitus. this can be the foundation of a complete case oriented management system for feline diabetes mellitus and associated disorders. no conflicts of interest reported. activins are cytokines belonging to the transforming growth factor (tgf)-b superfamily. it is thought that activins may be the key intermediary in tgf-b mediated fibrotic response. activin a has been suggested to participate in the pathogenesis of human idiopathic pulmonary fibrosis (ipf), but studies regarding the role of activin b are still spares. canine ipf (cipf) is a chronic, incurable interstitial lung disease occurring particularly in west highland white terriers (whwts). during the disease course, acute exacerbations (aes), with poor prognosis, can occur. histopathologically aes of cipf are featured by diffuse alveolar damage, which is also a key feature in acute respiratory distress syndrome (ards). our objective was to study the expression of activin a and b by immunohistochemistry in the lung tissue of cipf whwts (n = ), cipf whwts with concurrent ae (n = ), and dogs of various breeds with ards (n = ), and to compare these findings to healthy whwts (n = ). in addition, western blot analysis of activin b from bronchoalveolar lavage fluid (balf) of cipf whwts (n = ) and healthy whwts (n = ) was conducted. we demonstrated that activin b, but not activin a, is strongly expressed in the altered alveolar epithelium in lungs of diseased whwts as well as in ards lungs. furthermore, activin b was detected in balf of cipf whwts, most notably in samples from dogs with ae, but not in balf of healthy whwts. this novel finding suggests that activin b participates in the pathophysiology of cipf and might act as a potential marker of alveolar epithelial damage. no conflicts of interest reported. dogs of the breed nova scotia duck tolling retriever (nsdtr) are affected by several immune-mediated diseases, in particular steroid-responsive meningitis-arteritis (srma) and an immune-mediated rheumatic disease (imrd). imrd is a systemic lupus erythematosus-related disease characterized by chronic stiffness and pain in several joints. the aim of this study was to investigate the morbidity in nsdtrs and to test the hypothesis that nsdtrs are predisposed to srma and imrd. insurance data from a swedish insurance company (agria insurance company, stockholm, sweden) from - was used for the study. approximately one third of swedish dogs are insured by agria and the insurance database is a validated tool for epidemiological studies. assessment of morbidity was based on veterinary care events. disease diagnoses were grouped in both general and specific disease categories. individual diagnoses that were likely to represent imrd were combined. morbidity was defined as incidence rates and presented as number of cases per dog years at risk (dyar). relative risk (rr) for nsdtrs compared to other breeds combined was calculated. the study included dogs, were nsdtrs. the most common general causes of veterinary care for nsdtrs were injuries followed by gastrointestinal and musculoskeletal disorders with significant increased risk (rrs between . and . ) for nsdtrs compared to other breeds. the highest relative risk for nsdtrs was for systemic lupus erythematosus (rr . ). compared to other breeds, nsdtrs had an increased risk for srma (rr . ) and imrd (rr . ) with an incidence rate of . cases per dyar for srma and . cases per dyar for imrd. the incidence rate for srma and imrd in nsdtrs were also compared to dogs of other retriever breeds. the comparison revealed that nsdtrs also had a significant increased risk for both srma (rr . ) and imrd (rr . ) when compared to other retrievers only. this study is the first to investigate the morbidity for imrd in nsdtrs, which is important for further research and breeding practice. for several reasons the incidence rates might be underestimated and exact numbers should be interpreted with caution. however underestimation of incidence rates should not differ between dogs of different breeds, therefore not affecting the risk calculations. it can be concluded that nsdtrs are predisposed to the diseases srma and imrd with an increased risk compared to other breeds and to other retrievers. brenda n. bonnett consults with agria insurance company on various projects. agria insurance company has also funded work leading to the development of the insurance data base that my study was based on. canine infectious respiratory disease (cird) is a multifactorial contagious disease caused by respiratory viruses and selected bacterial pathogens. cird has been shown to be a predisposing factor in the development of bacterial pneumonia (bp) in dogs housed in dense populations such as kennels and rehoming centers. the aim of this study was to determine the prevalence of viral co-infection and to assess its effects on disease severity in household dogs diagnosed with bp. a prospective cross-sectional observational study was conducted and dogs diagnosed with bp caused by opportunistic bacteria were included. dogs with chronic (> days) tracheobronchitis caused by bordetella bronchiseptica were included as controls for virus analysis. diagnosis was confirmed by thorough clinical examinations as well as with cytological and bacterial analysis of bronchoalveolar lavage (bal) or transtracheal wash (ttw) samples. canine parainfluenssavirus (cpiv), canine adenovirus, canine herpesvirus, canine distempervirus, canine respiratory coronavirus (crcov) and canine pneumovirus were analysed in bal or ttw samples using rt-pcr assay. cpiv was detected in / ( %) and crcov in / ( %) respiratory samples in dogs with bp. respiratory viruses were not detected in dogs with chronic tracheobronchitis. there were no significant differences in the duration of hospitalization (p = . ) or arterial pao at presentation (p = . ) between bp dogs with and without a viral co-infection. these results indicate that co-infections with respiratory viruses are common also in household dogs with bp. additionally, viral co-infections did not cause a more severe course of bp. the author's researcjh is financially supported by the finnish foundation of veterinary r and the finnish veterinary foundation. esvim-p- causes of canine anemia in taiwan: a five-year retrospective survey. e.c.y. lin , p.c. liu , l.l. chueh , b.l. su . graduate institute of veterinary medicine, national taiwan university, taipei, taiwan, institute of veterinary clinical sciences, national taiwan university, taipei, taiwan anemia is a common hematologic disorder in dogs, however, few data are available regarding epidemiology and causes in taiwan. to investigate the causes of anemia, anemic cases (pcv< %) collected between january and december at national taiwan university veterinary hospital (ntuvh) were analyzed. most dogs ( . %, n = ) presented with a mild form ( %≦pcv< %), which was followed by a moderate form ( %≦pcv< %; . %, n = ) and a severe form (pcv< %; . %, n = ). among the dogs with identifiable causes, . % ( dogs) were induced by single cause, whereas . % ( dogs) by multiple causes. neoplasia-related anemia (n = ), infectious pathogens-related anemia (n = ), renal disease-related anemia (n = ) and post-surgery/ traumarelated anemia (n = ) account for . , . , . and . % of single-cause cases, respectively. furthermore, of them ( . %) presented with severe anemia. severe anemia primarily resulted from infectious disease-related anemia ( . %), followed by imha ( . %), and tumor-related anemia ( . %). of the infectious disease-related severe anemic dogs, the most common diagnosed pathogen was babesia gibsoni ( . %, n = ), followed by ehrlichia canis ( . %, n = )and babesia canis ( . %, n = ). taken together, tumors, infectious diseases, and renal failure are the most frequently causes of canine anemia in taiwan, furthermore, b. gibsoni appeared to be the most important infectious pathogen causing severe anemia which may be associated with the climate in this geographical area. no conflicts of interest reported. bordetella bronchiseptica (bb) is one of the primary causative agents of canine infectious respiratory disease (cird). this contagious disease, commonly seen in young dogs, is often self-limiting, although a wide range of respiratory signs can be found, from mild illness to severe pneumonia leading to death. although mycoplasma cynos (m. cynos) was recently identified as an emerging and possibly lethal pathogen in cird , the role of m. canis and m. cynos as primary respiratory pathogens still remains unclear. detection of these bacteria is now improved by quantitative polymerase chain reaction (qpcr). in dogs with cird due to bb, the frequency of co-infection with mycoplasma spp, in par-ticularm. cynos, and their possible role in the severity of the clinical signs are unknown. the aim of the present study was to investigate the presence of m. canis and m. cynos in a population of dog infected with bb, compared with other populations: healthy dogs and dogs with bacterial bronchopneumonia where bb was not involved (bbp). therefore, bb, m. canis and m. cynos were detected by qpcr in the bronchoalveolar lavage fluid (balf) sample in dogs with bb (mean age = . y, mean bw = . kg), dogs with bbp ( . y, . kg), and healthy dogs ( . y, . kg). bordetellosis was diagnosed based on clinical findings together with demonstration of pleiomorphic cocci/coccobacilli adhering to the cilia of the epithelial cells on cytospin balf preparations, and positive qpcr on balf. a clinical severity index (csi to ) was assigned based on clinical signs (cough - , dyspnea - , lethargy - , fever - ), thoracic radiographic pattern ( - ), and balf score ( - ). bbp was diagnosed based on clinical findings, balf cytology and culture. m. canis was indifferently detected in healthy ( / , %), bbp ( / , %) and bb dogs ( / , %) while m. cynos tended to be more frequently detected in bb group ( / , %) than in healthy ( / , %) and bbp dogs ( / , %) (khi² test, p = , ). in bb dogs, no correlation could be detected between csi and presence of m. cynos (khi² test p = , ). in conclusion, the present data suggest that, in cird, coinfection with bb and m. cynos is frequent, but is not correlated with clinical disease severity. further studies are required to investigate whether coinfection of bb and m. cynos deserves specific therapeutic considerations. no conflicts of interest reported. canine idiopathic eosinophilic bronchopneumopathy (ebp) is a disease characterized by eosinophilic infiltration of the lung and bronchial mucosa in young adults. aetiology remains unclear although immunologic hypersensitivity is clearly suspected, while inciting antigens are generally unidentified. in humans as in cats, infections with mycoplasma spp. have been discussed as potential triggers in inflammatory bronchial disease , . bordetella bronchiseptica (bb) is a recognized pathogen agent of canine infectious tracheobronchitis. detection of bb and mycoplasma spp, especially mycoplasma cynos (m. cynos), and their potential role of in canine inflammatory bronchitis, have not been investigated. the aim of the present study was to investigate the frequency of bb, mycoplasma canis (m. canis) and m. cynos in canine ebp. therefore, presence of bb, m. canis and m. cynos were retrospectively assessed by quantitative polymerase chain reaction (qpcr) in bronchoalveolar lavage fluid (balf) samples from dogs with ebp (mean age = . y, mean body weight = . kg) as well as in dogs with aspecific chronic bronchitis ( . y, . kg). based on clinical signs, a clinical severity score (css, - ) was assigned each ebp dog. although all balf culture and cytology were negative for this bacteria, bb was more frequently detected by qpcr in ebp dogs ( / , %) than in cb dogs ( %) (khi² test, p = , ). presence of bb in ebp dogs was independant of age but significantly associated with css (khi² test, p = , ). results of qpcrwere positive for m. canis and m. cynos in ( %) and ( %) ebp dogs and in ( %) and ( %) cb dogs, respectively. there was no difference between the groups for any of the organisms. any relation between age or css and presence of m. spp in ebp dogs was observed. in conclusion, m. canis and m. cynos do not seem to be predominantly involved in the pathogenesis of canine ebp. however, bb is more frequently detected in balf from ebp dogs than from dogs with aspecific cb and its presence is associated with clinical severity. whether bb is able to trigger eosinophilic inflammation or is only more easily collected in an inflamed environment is unclear. but ebp dogs could potentially act as bb carriers and source of infection. therefore, bb should be systematically searched for in canine ebp cases and treated accordingly. no conflicts of interest reported. distal renal distal renal tubular acidosis (drta) was recently reported in three dogs with imha. the purpose of this study was to explore the hypothesis that drta is an underdiagnosed concurrent disorder in dogs with imha. we report the clinical presentation and outcome of three dogs where the combination of imha and drta was strongly suspected. the medical records of dogs diagnosed with imha at the university of edinburgh hospital for small animals between january and may were reviewed to identify cases where venous blood gas analysis and urinalysis had also been carried out. for the purpose of this retrospective study imha was defined by the presence of anaemia with pcv < %, and one or more of the following criteria; a positive slide agglutination test, positive coombs' test or moderate to marked spherocytosis. the criteria for diagnosis of drta included moderate to marked hyperchloremic metabolic acidosis with a normal anion gap; urine ph (> . ) in the face of metabolic acidosis; hypokalaemia (< . mmol/l). fifty-seven records were evaluated, with cases being excluded due to insufficient clinical information, including inability to determine urinary ph due to the severity of pigmenturia in four cases. of the cases where there was sufficient clinical data to assess the likelihood of drta only one case fulfilled all the criteria; two cases fulfilled all but one of the criteria and drta was strongly suspected based on clinical progression and persistence of urine ph > in the face of severe metabolic acidosis. of the three cases where concurrent imha and drta was suspected, two survived to discharge; one was still alive at the time of writing ( months after discharge) and the other was euthanased months after discharge following the development of multiple joint effusions and skin lesions suggestive of sle. venous blood gas analysis and assessment of urine ph should be considered in all cases of imha to exclude the possibility of concurrent drta, particularly where persistent hypokalaemia is detected. prospective evaluation of a larger cohort of imha cases is required to determine the actual incidence of concurrent drta. no conflicts of interest reported. persistent renal proteinuria is considered an early marker of chronic kidney disease (ckd) and it is listed among the initiation factors and progression factors according to kdoqi guidelines. nevertheless, few data are available about the prevalence of proteinuria in cats affected with ckd, in which it is assumed that nephropathy is mainly characterized by tubulointerstitial damage. the aim of this study is to determine the prevalence of proteinuria in cats affected with ckd and to valuate the relations between urine protein to creatinine ratio (upc) or iris substaging by proteinuria, towards purebred, sex, age, haematology, biochemistry and urinalysis. wilcoxon test, linear regression and chi-square test were used for the statistical analysis. data from cats were considered. non-renal proteinuria was an exclusion criterion. proteinuric cats (upc> . ) were . % in ckd cats, while . % could be substaged as bordeline proteinuric ( . ) in cats, proteinuria tends to increase with aging (p < . ) and with worsening of the nephropathy (p = . ). proteinuria was related to the anaemic state in ckd cats: upc significantly increases with rbc count, hb, ht and mch decreasing (p < . and p = . respectively). proteinuria tends to increase with wbc count (p = . ) and neutrophils increasing (p = . ), while tends to decrease with lymphocytes increasing (p = . ). furthermore, upc significantly increases in presence of an inflammatory serum protein electrophoretic pattern. upc tends to increase with phosphorus and alp increasing (p < . and p = . respectively); while the role of phosphorus in ckd is well known, the increase of alp is questionable: it has been hypothesized that higher alp levels in ckd could be related to b-alp increase due to bone remodelling in secondary renal hyperparathyroidism. considering urine parameters, upc increases when urinary specific gravity and ph decrease (probably related to worsening of ckd and development of a metabolic acidosis) and when glicosuria is present, regardless of the cause. furthermore, proteinuria increases in presence of rbc in urinary sediment and in samples where casts were observed, in particularly when rbc casts (considered always pathological and indicative of glomerular damage) were present. upc values assessed in proteinuric cats and data analysis suggest the need of deepen the analytical variability of upc and the opportunity to reconsider the intervals of substaging by proteinuria in cats. no conflicts of interest reported. the aim of this retrospective study was to evaluate: a) the relations between urine culture results and urinalysis parameters; b) the results of the antimicrobial susceptibility tests. urine samples were collected by cystocentesis from dogs and cats, whose diagnostic workup included a differential diagnosis of uti: all samples underwent a complete urinalysis, upc ratio assessment and urine culture. infected vs sterile results were related to urine physical, chemical parameters and observations from urinary sediment analysis. statistical analysis was performed using jmp . (sas institute inc.). a p value < , was considered significant. urine culture resulted positive in dogs ( %) and cats ( %). the presence of uti was significantly related to urine physical properties (color and turbidity), usg and leukocyturia: infections tended to be more frequent in urine samples characterized by a light yellow color, cloudy or sub-limpid aspect and low usg. nevertheless, urine was limpid in % of infected samples, and a normal usg was found in . % of dog's uti but only in . % of cats. although leukocyturia tends to become higher in infected samples both in dogs and cats (p < . ), in . % of infected sediments wbc count was normal. haematuria detected by dipstick was significantly related to uti in dogs but not in cats, nevertheless the rbc count in sediment was not related to infection in both species: rbc count was normal in . % of infected feline samples and in . % of canine samples. no significant relation between presence or absence of uti and albuminuria, bilirubinuria, glycosuria was detected, while upc tends to become significantly higher in dogs. although the chi-square test showed a significant relation between infection and the detection of bacteria in urinary sediment, a pseudobacteriuria was found in . % of samples; furthermore bacteria weren't observed in . % of infected samples (usg< . ). e. coli was isolated in the majority of samples ( , %), compared to other species: staphylococcus( . %), proteus ( . %) and streptococcus. ( . %). the urinalysis pitfalls and the high antibiotic resistance verified towards the most widely used molecules (penicillins, cephalosporins, quinolones) strongly indicates the importance to perform antimicrobials susceptibility tests to avoid the risk of failure associated with the use or abuse of empiric therapies in utis. no conflicts of interest reported. azotemia in dogs with chronic heart failure may reflect impaired renal function not only because of inadequate renal perfusion, but also due to organic renal injury. impaired renal function is observed in % of dogs with heart failure. altered renal hemodynamics due to decreased cardiac output results in renal hypoperfusion, and resultant elevation of blood urea nitrogen and creatinine, defined as azotemia. azotemia is a prognostic factor in dogs with mitral regurgitation, therefore, preservation and/or restoration of renal function is thought to improve prognosis. medical treatment for heart failure, however, includes angiotensin converting enzyme inhibitors and loop diuretics, which has been shown to increase the risk of developing azotemia. we hypothesized that mitral valve repair surgery ameliorates renal function by improvement of systemic hemodynamics. the change in renal function in dogs with mitral regurgitation was assessed by evaluating time-dependent changes in glomelular filtration rate by inulin clearance before and after cardiac surgery. eighteen dogs with severe mitral regurgitation with azotemia (plasma urea nitrogen level > mg/dl, plasma creatinine level > . mg/dl) were included in this study. the glomerular filtration rate in all dogs were evaluated by determining inulin clearance before and months after surgery. serum atrial natriuretic peptide level, plasma nt-pro brain natriuretic peptide level, plasma urea nitrogen concentration, and plasma creatinine concentration were measured at each time point as well as during the initial staging of heart failure based on the international small animal cardiac health council (isachc). left atrial/aorta ratio by echocardiography and vertebral heart size by thoracic radiographs were also measured. glomerular filtration rate significantly increased months after surgery ( . ml/min/m [ . - . ], . ml/min/kg [ . - . ]) compared to before surgery ( . ml/min/m [ . - . ], . ml/min/kg [ . - . ]) (p < . ). the isachc stage of heart failure was improved at months after surgery compared to before surgery. in addition, serum atrial natriuretic peptide level, plasma nt-pro brain natriuretic peptide level, plasma urea nitrogen concentration, la/ao and vhs significantly decreased after surgery (p < . ). the use of diuretics decreased after mitral valve repair surgery and consequently, a decrease in plasma urea nitrogen and creatinine levels were observed. therefore, this suggests that the main cause of azotemia in dogs with mitral regurgitation may be due to inadequate renal blood flow and exacerbation by the use of diuretics. no conflicts of interest reported. glomerular filtration rate (gfr) is generally considered to be the gold standard measurement of kidney function. gfr can be calculated by measuring serum iohexol clearance using concentrations at , and hours following a bolus injection. for validation, serum samples were spiked at low ( . mg/ ml), medium ( . mg/ml) and high ( . mg/ml) iohexol concentrations. they were analysed, along with standard calibration curves ( concentrations ranging from . to . mg/ml), using deltadot's label-free high performance capillary electrophoresis (hpce) system. data were analysed using deltadot's general separation transform (gst). clinical and spiked serum samples were also sent for analysis by mass spectrometry (ms) at a reference laboratory ( samples for comparison). concentrations obtained by hpce and ms were compared in a bland altman plot. gfr for clinical samples was calculated from the measured iohexol concentrations using the method reported by bexfield ( ) . a validated method was produced, with a lower limit of detection of . mg/ml and an lower limit of quantification of . mg/ml. the upper limit of quantification was . mg/ml. the standard curve had excellent linearity (r = . ). maximum inaccuracy was less than . % of the true value, except at lloq, where it was within . %. average within day variability was less than . % at all levels, while between day variability was less than . %, except at lloq, where it was less than . %. agreement between the results obtained by measurement with hpce and ms was good (bias . %, lower and upper limits of agreement of - . and . %, respectively). method specificity was confirmed by the absence of matrix effect in six serum specimen obtained from clinical dogs. clinical samples were analysed and gfr reported with a day turnaround time. in conclusion, hpce provides an accurate and precise method for measuring iohexol in canine serum. conflicts of interest: l pelligand has the following information to disclose: in receipt of research grant / contract funding from orion ltd., novartis animal health, transpharmation ltd, deltadot ltd; acted as a consultant for: triveritas ltd. and novartis animal health; s williams is an employee of the rvc and works in collaboration with deltadot ltd; j. elliott has the following information to disclose: consultancy: pfizer animal health / zoetis, ceva animal health, boehringer ingelheim, vetoquinol ltd, orion ltd., elanco ltd, idexx ltd, niche generics ltd. triveristas ltd., virbac ltd., advisory board membership: international renal interest society (supported by novartis) european emesis council (sponsored by pfizer animal health -now zoetis) cardiorenal board -vetoquinol ltd. idexx renal advisory board research grants or contracts: vetoquinol ltd, novartis ltd, pfizer animal health ltd (now zoetis), royal canin ltd, boeringher ingelheim ltd, waltham centre for pet nutrition, ceva animal health orion ltd. esvonc-p- cystic pancreatic neoplasia in cats. c.m. borschensky , k. steiger , a. staudacher , m. schlitter , i. esposito , h. aupperle . laboklin gmbh&co. kg, bad kissingen, germany, tu m€ unchen, institute of pathology, m€ unchen, germany, veterinary clinic dr. staudacher, aachen, germany pancreatic neoplasms in the cat mostly exhibit a solid growth pattern and are diagnosed as carcinomas. in contrast, only few reports about cystic pancreatic lesions exist. until now, only benign cystic pancreatic lesions are described in the literature. according to the histological pattern, they have been termed as cysts, (acinar) cystadenoma or pseudocysts. in man, cystic pancreatic neoplasms are classified according to the localisation (intra-/extraductal), growth pattern and differentiation (mucinous, (tubulo)papillary, serous, acinar). the aim of this study was to characterise feline pancreatic neoplasms in more detail, based on the human classification system with a special view on cystic lesions. pancreatic masses sent to laboklin from domestic cats ( - years) were investigated routinely macroscopically and by histological methods (h.&e. stain). the neoplasms showed a cystic (n = ) or solid (n = ) pattern. cystic pancreatic tumors were up to cm in diameter and were classified as benign variants in five and malignant variants in three cases. based on the human classification system, they were classified as tubulopapillary (n = ), acinar (n = ) and mixed (n = ) adenomas and mixed carcinomas (n = ), respectively. solid pancreatic nodules were diagnosed as carcinomas with a tubular (n = ) or acinar (n = ) differentiation pattern. in summary, the gross structure (solid versus cystic) seems to be of prognostic relevance. in contrast to solid tumors, cystic pancreatic lesions in the cat behave benign in a higher percentage of cases, resulting in a better prognosis. therefore, surgical excision of these cystic masses can be recommended. with respect to the human classification system, three different subtypes of cystic pancreatic neoplasms were detected in the cat that have not been described before in veterinary medicine: tubulopapillary, acinar and mixed. to best of our knowledge, this is the first report of cystic adenocarcinomas in feline pancreas. further corresponding clinical and histological investigations are needed for a better diagnostic (ultrasound, mri) and prognostic characterisation of cystic lesions in feline pancreas. no conflicts of interest reported. the immunohistochemical detection of cyclooxygenase- (cox- ) expression in canine mast cell tumours was recently described by our team (prada et al., ) . however its prognostic value needs to be established. the aim of the present work was study the prognostic value of cox- expression by investigating the relationship with several clinical and pathological variables including the overall survival (os) time. we included dogs with mast cell tumours ( grade i; grade ii and grade iii). cox- immunohistochemical expression was carried out by a streptavidin-biotin method. for the cox- immunoreactivity evaluation were considered the number of positive cells (cox- extension), the intensity and the score of cox- . the following clinical and pathological features were considered: animal age, sex, tumour anatomical location, tumour size, skin ulceration, histological grade, histological safety margins and number of mitosis. cox- expression was correlated with the clinical and pathological data and with the overall survival. cox- intensity was statistical significantly associated with skin ulceration (p = , ); histological grade (p = , ) and absence of histological safety margins (p = , ), high mitotic number (p = , ) and with overall survival (p = , ). both cox- extension and cox- immunohistochemical score present no statistical relationship with the variables considered neither with the overall survival. our results suggest that cox- have an important role in dog mast cell tumours progression and could constitute a promising therapeutic target in this neoplasia. however, our study also demonstrated that in mcts, is the cox- intensity that has the prognostic value, not the number of cox- positive cells (cox- extension) and not the cox- immunohistochemical score. consequently, cox- intensity should be elected for evaluating the cox- positivity in mcts immunohistochemical studies. merial provided financial support for immunohistochemical analayis. the research centres has also received financial suppoprt from cecav, ceca and citab. dogs which were radiated for a subcutaneous sarcoma between and were included. medical records were reviewed and patient characteristics, treatment protocols, adjuvant therapies and outcome were analysed. follow-up information was obtained from medical records and by phone conversations with veterinarians or pet owners. thirty-two dogs were included into this study. mean age was years and mean body-weight was kg. male dogs were slightly overrepresented ( . %). curative intent radiotherapy was applied in dogs and palliative intent in dogs with a mean total dose of and . gray, respectively. in dogs microscopic disease was radiated. five dogs received liposomal doxorubicin concomitantly with radiotherapy, two received adjuvant doxorubicin and one intralesional cisplatin. overall median survival time was days with curative and days with palliative treatment. overall median survival time in dogs with macroscopic disease was days and in patients with microscopic disease it was not reached. radiotherapy was generally accepted as new treatment modality by pet owners and referring veterinarians. comparable to the literature, best outcome was achieved for dogs radiated with microscopic disease conflicts of interest: no conflicts of interest reported. oncept â , is indicated for the treatment of stage ii or iii oral melanoma after local control with survival times significantly increased following vaccination. a similar improvement in survival times has also been reported with digit melanoma. medical records of dogs diagnosed with melanoma between march and december were retrospectively evaluated. inclusion criteria were a histopathological diagnosis of melanoma, surgical excision of the tumour, and vaccination using the oncept â vaccine. dogs met the inclusion criteria. nificant renal involvement in dogs with cvl. this result indicates staffordshire terrier ( ), bouvier, giant schnauzer, maltese, irish setter, kerry blue terrier, golden retriever, scottish terrier, and great dane ( of each). dogs had stage ii digit melanoma with an equal sex distribution and a median age of . years (range - ). currently still alive and one dead, the latter following surgery for resection of a rib osteosarcoma the median survival time of the dogs still alive is . months (range - ) versus . months (range - ) for the dogs that have died. none of the dogs showed any adverse effect to the vaccine infected macrophages can cause injury in different organs, including the kidney. cvl is known as a common cause of glomerulonephritis. thus, this study aimed to investigate and characterize the renal lesions in dogs seropositive for leishmania sp. in brazil. this project was approved by the animal ethics committee of uece, brazil. twenty adult dogs seropositive for cvl from center for zoonosis control were randomly selected for this experiment. cvl was diagnosed by immunofluorescence and elisa. urine and blood sampling and kidney harvesting were performed immediately after euthanasia that the glomerulonephritis is a common sequelae related to leishmaniasis infection. even dogs in stage of ckd showed significant renal histopathological changes. animals infected with leishmania sp. may have severe renal damage and risk of progressive chronic kidney disease even when no increase of creatinine levels or proteinuria is detected.conflicts of interest:the authors received funding to pay the phd scholarship of one student (conselho nacional de pesquisa e desenvolvimento -cnpq-brazil) and received a research grant from fundac ßão cearense de apoio ao desenvolvimento cient ıfico e tecnol ogico -funcap. anaplasma phagocytophilum, the causative agent of canine granulocytic anaplasmosis, is an obligatory intracellular bacterium transmitted by ixodes ticks. transmission via blood transfusion has rarely been described in human medicine and once in a dog. in the berlin/brandenburg area the seroprevalence rate in dogs was % regardless of health status.the aim of this study was to evaluate pcr screening results for a. phagocytophilum in canine blood donors between - in order to estimate the risk of transfusion-transmitted infection. edta blood samples from dogs were submitted for a. phagocytophilum real-time pcr testing (targeting the msp gene). altogether dogs were tested up to times. clinical and laboratory data were examined before each donation. statistical analysis was performed using spss . .the pcr test was positive for of the samples. none of the dogs tested pcr positive more than once. positive results were most often detected in june ( ), may ( ), and july ( ), but also in five other months. in three dogs a mild increased in rectal temperature (≥ , °c) was documented. mild laboratory abnormalities were noted in dogs: thrombocytopenia ( ), leukocytosis ( ), leukopenia ( ), anemia ( ) and hyperproteinemia ( / ); four dogs had more than one abnormality. there was no significant difference between the pcr negative and positive blood samples in regard to laboratory abnormalities.altogether, . % of blood samples from healthy canine blood donors were pcr positive for a. phagocytophilum. therefore, blood donors should be screened by pcr in endemic areas all year round. no conflicts of interest reported. the study population consisted of cats, including control cats recruited from veterinary practices across the country. among the disease cats, cats presented urtd, cats had conjunctivitis and cats suffered chronic gingivostomatitis, many of them presenting more than one clinical sign. pcr for the above-mentioned pathogens was performed from pooled conjunctival and oropharyngeal swabs for each cat. a questionnaire regarding signalment (age, breed, sex, neuter status), environment (indoor, number of cats in household) and vaccination history was obtained. data was analysed by multivariable logistic regression with alpha equal to < . .the prevalence for the four pathogens has been previously reported in detail. briefly, the prevalence among the four groups (including controls) ranged from to % for fhv- , - % for fcv, - % for chlamydophila felis and - % for mycoplasma felis.in the univariate analysis, age, neutering status, being purebred, indoor keeping, number of cats in the household and body weight were variably associated with the different groups of disease and the presence of the pathogens. in the multivariable analysis, only the following factors remained significant. in the multivariable analysis, only the following factors remained significant: urtd was significantly associated with positive results for fhv- , chlamydophila felis and mycoplasma felis (in addition to being male and not castrated); conjunctivitis was significantly associated to positive results for fhv- and chlamydophila felis (in addition to being young, not castrated and purebred) and cgs was significantly associated to positive results for fcv (in addition to being young, male and purebred). not being properly vaccinated was a significant risk factor only when all three groups were analyzed together. the number of cats in the household was an independent risk factor for detecting each of the pathogens studied. the age was also a significant factor in cats with fcv and chlamydophila felis, being older cats predisposed to fcv and younger cats predisposed to chlamydophila felis.the present study describes important epidemiological data for cats presenting urtd, conjunctivitis and/or cgs, and emphasizes the complex interrelationships occurring among the different pathogens. our results also support the role of fcv in cats with chronic gingivostomatitis.conflicts of interest:the study was funded and designed by merial laboratories. reports of methicillin-resistant staphylococci (mrs) in animals have become more frequent in last years. various studies have demonstrated the transmission of mrsa between animals and humans in daily contact with animals, however there is only limited data so far available on the transmission of methicillinresistant coagulase-negative staphylococci between animals and humans. the objective of this study was to investigate the frequency of methicillin-resistant staphylococci (mrs) carriage in healthy veterinarians, veterinary nurses, veterinary assistants, veterinary students and farm workers from several veterinary hospitals, clinics and farms.nasal swabs were collected from veterinarians ( small animal veterinarians and pig veterinarians), veterinary nurses, veterinary students, veterinary assistants and farm workers. mrs were screened on brilliance tm mrsa agar (oxoid) or chromid tm mrsa (biom erieux). after - h of incubation at °c, suspected colonies on both media were subcultured onto blood agar plates. species identification was obtained by species-specific pcr. methicillin-resistance was confirmed by pcr amplification of the meca gene. mrsa isolates were characterized by mlst.thirty-nine mrs were identified in humans ( veterinarians, veterinary nurses, veterinary students, veterinary assistants and farm workers). the mrs isolates were identified as staphylococcus aureus (mrsa, n = ), s. epidermidis (mrse, n = ), s. pseudintermedius (mrsp, n = ), s. haemolyticus (mrsh, n = ) and mrs coagulase-negative staphylococci. the frequency of colonization by mrs was similar in both small animals and pigs veterinarians (ae %). one veterinary student was colonized simultaneously with an mrse and an mrsh. the predominant st in humans in contact with small animals was st and in humans in contact with pigs was st .in our study the frequency of colonization by mrsa was high, but the frequency of mrse should not be underestimated. mrsa isolates in this work belonged mainly to the st lineage which is the most frequent in small animals and humans in europe. humans in daily contact with animals can become colonized by mrs of animal origin and thus are important keys for infection control programs in veterinary hospitals and farms.conflicts of interest: dr pomba currently receives research funding from the government and national programmes (fundac ßão para a ciência e a tecnologia). in the past, she has occasionally received research support or honoraria for lectures from pharmaceutical companies including zoetis and atral cipan. she is vice-chair of the antimicrobial working party (awp) and member of the antimicrobial advice ad hoc expert group (ameg) of the european medicines agency (ema). , species not determined [ ]) did not. all cats underwent antibiotic treatment (doxycycline or a fluoroquinolone); cats received blood transfusions and/or oxyglobin â . three cats were euthanatized within days due to concurrent disease (fiv, pancreatitis/cholangitis) or financial constraints, one cat due to persistent anemia after weeks. four cats were lost to follow-up. the remaining cats underwent follow-up for a period of - weeks (median ). hemoplasma pcr analysis was conducted - times on blood samples at variable time points from of the follow-up cats. the first negative pcr in cases occurred after (cmhm, during antibiotic treatment), (cmhm, during antibiotic treatment), (cmhm, during antibiotic treatment) and (mhf, after completion of antibiotics) weeks. one cat remained pcr positive (cmhm) at , , and (all during antibiotic treatment) weeks, and another cat (cmhm) was pcr positive at weeks. reactivation of the hemoplasma species (documented by hemolysis and positive pcr) occurred in cats (both cmhm) and times, respectively, up to weeks after initial presentation. reactivation was suspected (no pcr testing available) in additional cases (cmhm [ ], mhf [ ]). four of the follow-up cats were euthanatized after - weeks (median ) due to concurrent disease (cardiomyopathy, immune-mediated thrombocytopenia, postoperative complications, diabetes mellitus). infection with hemoplasmas is often chronic, can reactivate months later and is rarely the reason for euthanasia. no conflicts of interest reported. canine distemper (cd) is a worldwide occurring infectious disease caused by a morbillivirus of the family paramyxoviridae. cdv infection can result in a systemic infection. dogs presented with neurologic signs revealed the terminal stage of the disease and usually failed to therapy. additional passive immunotherapy is hypothesized to be beneficial in the early stage of cdv infection. porcine anti-cdv antibody subunit f (ab') [f(ab') ] was produced by animal technology laboratories, agriculture technology research institute. eighteen cdvnaturally infected dogs showing respiratory signs but no neurological signs were treated with the combination of f(ab') and supportive therapy (group ). group included dogs in a similar clinical signs (without neurological signs) that received only supportive therapy. the survival rate was . % ( / ) in group and . % ( / ) in group , respectively, with a significant difference between the two groups (p < . ). the progressive rates of developing neurological signs during therapy of group and group were . and . %, respectively. there was no significant difference between the two groups. the survival rates of dogs developing neurological signs during therapy were % ( / ) in group and . % ( / ) in group , respectively, with a significant difference between the two groups. in conclusion, additional administration of porcine anti-cdv antibody subunit f(ab') before developing of neurological signs could decrease the mortality and furthermore reduce the rate of developing neurological signs. no conflicts of interest reported. key: cord- - fld authors: al‐ani, aysha h.; prentice, ralley e.; rentsch, clarissa a.; johnson, doug; ardalan, zaid; heerasing, neel; garg, mayur; campbell, sian; sasadeusz, joe; macrae, finlay a.; ng, siew c.; rubin, david t.; christensen, britt title: review article: prevention, diagnosis and management of covid‐ in the ibd patient date: - - journal: aliment pharmacol ther doi: . /apt. sha: doc_id: cord_uid: fld background: the current covid‐ pandemic, caused by sars‐cov‐ , has emerged as a public health emergency. all nations are seriously challenged as the virus spreads rapidly across the globe with no regard for borders. the primary management of ibd involves treating uncontrolled inflammation with most patients requiring immune‐based therapies. however, these therapies may weaken the immune system and potentially place ibd patients at increased risk of infections and infectious complications including those from covid‐ . aim: to summarise the scale of the covid‐ pandemic, review unique concerns regarding ibd management and infection risk during the pandemic and assess covid‐ management options and drug interactions in the ibd population. methods: a literature review on ibd, sars‐cov‐ and covid‐ was undertaken and relevant literature was summarised and critically examined. results: ibd patients do not appear to be more susceptible to sars‐cov‐ infection and there is no evidence of an association between ibd therapies and increased risk of covid‐ . ibd medication adherence should be encouraged to prevent disease flare but where possible high‐dose systemic corticosteroids should be avoided. patients should exercise social distancing, optimise co‐morbidities and be up to date with influenza and pneumococcal vaccines. if a patient develops covid‐ , immune suppressing medications should be withheld until infection resolution and if trial medications for covid‐ are being considered, potential drug interactions should be checked. conclusion: ibd patient management presents a challenge in the current covid‐ pandemic. the primary focus should remain on keeping bowel inflammation controlled and encouraging medication adherence. in december , reports of a novel coronavirus, since named sars-cov- , emerged from wuhan, central hubei province, china. - the virus causes the disease covid- , which manifests as a severe acute respiratory illness that can be complicated by acute respiratory distress syndrome (ards), multiorgan failure and even death. following rapid spread of the virus across the globe, the world health organisation (who) declared covid- a pandemic on march . there are currently almost million confirmed cases across more than countries with a total death count greater than at the time of writing. as the pandemic expands, there has been increasing concern regarding the impact of covid- on patients with ibd. the primary management of ibd involves treating uncontrolled inflammation with a significant number of patients requiring immune-based therapies. in the last decade, there has been a considerable expansion of the therapeutic armamentarium for patients with ibd to include immunomodulators, tnf antagonists, non-tnftargeted biologics and targeted small molecule therapies. however, these therapies, in addition to malnutrition which can complicate ibd, may weaken the immune system and potentially place ibd patients at increased risk of infections and infectious complications. consequently, there is a concern that ibd patients are at greater risk of developing covid- and at increased risk of progressing to a more severe clinical course or even death compared to the general population. in addition, if an ibd patient develops covid- , there is a lack of guidance on medication management and concern regarding drug interactions if trial medications are utilised to treat therefore the aim of this review is to summarise the evidence and discuss in detail the data regarding the risks of developing covid- , strategies that can be implemented to reduce these risks and issues surrounding the treatment of covid- , including potential drug interactions and ibd medication management, in the ibd patient cohort. coronaviruses (of the family coronaviridae) are a group of related single-stranded, positive sense, enveloped rna viruses. they are the largest known rna viruses, ranging from to kilobases in size. they are named after their appearance under electron micros- syndrome coronavirus (sars-cov- ). these viruses are all known to cause respiratory symptoms ranging broadly in severity, both between the different viruses and in different hosts infected with the same virus. most spread easily and result in relatively mild illness in immunocompetent patients, with certain strains being responsible for almost % of the common cold. other coronaviruses (covs), including the sars-cov and the mers-cov, have previously emerged as epidemics with significant mortality and socioeconomic impact. compared to sars-cov- , mers-cov causes a much more severe illness, with a case-fatality rate (cfr) of up to %, but appears to have a lower person-to-person transmission, limiting its global impact. there are still new cases of mers being reported today. similarly, the sars-cov outbreak in - had a high cfr ( . %), but its reduced infectivity compared with sars-cov- lessened its overall impact. this outbreak appears to have been contained. sars-cov- is the first pandemic coronavirus. therefore, it poses a threat of uncertain dimensions and represents uncharted territory for the public and global healthcare systems alike. , sars-cov- , the virus previously known as novel -coronavirus, causes the disease covid- . it was first discovered following the reports of an outbreak of pneumonia in china, with initial infections linked to a single seafood market. , genetic studies have shown its genetic sequence is % similar to sars-cov, and % similar to bat-sl-covzc and bat-sl-covzxc , leading to the postulation that bats served as reservoir host for the new coronavirus' progenitor. human transmission is thought to have been facilitated by an animal intermediate host, currently hypothesised to be a pangolin. human-to-human transmission is believed to be predominantly via direct contact, exhaled droplets and fomites from an infected individual. sars-cov- can also be detected in saliva, urine and the gastrointestinal tract in both biopsy specimens and stools, although their role in transmission remains unclear. [ ] [ ] [ ] data suggest it is more easily transmitted than seasonal influenza based on a basic reproduction number (r -the expected number of cases directly generated by one case in the population) of - . . entry of coronaviruses into human target cells requires spike (s) protein binding to a cellular receptor followed by s protein priming by host proteases to facilitate cell entry. sars-cov- human-to-human transmission is enabled by the interaction of the sars-cov- s-protein with the human angiotensin-converting enzyme (ace ) receptor. , transmission occurs when the virus enters the nose, mouth or eyes, and potentially the digestive system and attaches to cells that produce ace . ace are found in multiple organs and are highly expressed in lung at cells, enterocytes of the small intestine and colon. , once the virus is attached to ace it uses the host serine protease tmprss for s priming allowing fusion of viral and cellular membranes and viral entry into the cell. once a person develops initial symptoms of covid- , respiratory viral shedding occurs for a median of days in survivors and is sustained until death in nonsurvivors. of note, prolonged shedding has been demonstrated with other coronaviruses in immunocompromised patients and in immunocompromised animals with mers-cov. asymptomatic people are also potential sources of sars-cov- as transmission may occur in the pre-clinical period ( - days) or from an oligosymptomatic individual. , furthermore the virus has been detected in up to % of stool specimens and can remain positive for viral rna following negative respiratory samples in more than % of sars-cov- patients. , , these characteristics may have implications regarding isolation and decision-making for ibd patients who are infected with sars-cov- although there are no evidence-based guidelines on this currently. the major clinical manifestations of covid- are fever ( % on admission and % during admission), dry cough ( %), shortness of breath ( %), diarrhoea/vomiting/abdominal pain ( %), generalised myalgia/arthralgia ( %), headache ( %), malaise and bilateral interstitial pneumonia. , , covid- pneumonia manifests with chest ct imaging abnormalities, even in asymptomatic patients, with rapid evolution from focal unilateral to diffuse bilateral ground-glass opacities that progress to or co-exist with consolidations within - weeks. while the mechanism of covid- induced gastrointestinal symptoms remains to be clarified, enteric symptoms may occur due to malabsorption secondary to direct damage of the invaded enterocyte in the setting of a viral infection. furthermore, there is a recent case report of a possible sars-cov- gastrointestinal infection causing acute haemorrhagic colitis and signalling covid- disease. it is not clear if gastrointestinal symptom rates secondary to covid- differ in patients with ibd but if an ibd patients presents with worsening gastrointestinal symptoms, covid- infection should be considered as a differential. the median age of an infected individual is years old and the majority of infected individuals range from to years. fifty-one percent of reported cases is male. healthcare workers may be at increased risk of infection secondary to close contact with infected patients and exposure to aerosolised virus from medical equipment and procedures including endoscopy. in china healthcare workers made up . % of covid- patients and italy have reported that % of its responding healthcare work force is being infected. , current data suggest that children under the age of years have a low infection rate ( . %), with a large proportion being identified through contact tracing in adults rather than through symptomatology. the median incubation period for sars-cov- is - days, with most patients having symptom onset before days, but there have been cases with longer incubation. a mean interval of . - . days between illness onset and hospitalisation has been documented highlighting the challenge in the identification and isolation of individuals at an early stage of disease. in recovered cases, the median time from initial symptoms to discharge from hospital was days and in those that succumbed the average time to death was . days. overall cfr of covid- are estimated to be approximately %- %, rising to more than % in patients aged years and over. current who data surmise that most cases of covid- have mild to moderate disease ( %) with . % experiencing severe disease defined by the following signs and symptoms within - hours: shortness of breath; tachypnoea > breaths per minute; hypoxia < %; hypoxaemia with pao /fio ratio < and/or pulmonary infiltrates > % of the lung field. critical infections occur in . risk factors for contracting covid- have not been fully elucidated. healthcare professionals are at greater risk secondary to increased exposure and it is thought that smoking may exacerbate contraction. for all patients with covid- , severity and mortality is associated with age (median age years for critically ill vs years in the overall patient population) and underlying cardiovascular disease, hypertension, chronic pulmonary disease, diabetes and cancer. , , severe covid- is less common in children with reports suggesting approximately % develop severe disease and . % become critical. the crude fatality rate is higher in males ( . %) vs females ( . %) and smokers. , for ibd patients, the greatest risk factor for general immunosuppression and infections remains pharmacological. , nutritional status, co-morbidities, age and disease activity also contribute. , furthermore, low vitamin d levels are prevalent in ibd patients and may be associated with an increased risk and/or severity of influenza and covid- . , vitamin d increases anti-inflammatory cytokines and lowers viral replication, which in turn may reduce pro-inflammatory cytokines that propagate lung injury and ards. [ ] [ ] [ ] therefore assessment of vitamin d and supplementation when it is low is reasonable. as gastrointestinal tract permeability may be increased in ibd a diagnosis of covid- should be considered in patients who display fever, cough, anosmia, nausea, vomiting and diarrhoea. of note, a subgroup of patients may present with mild disease marked by the presence of diarrhoea initially and these patients often experience delayed diagnosis compared to those that present with respiratory symptoms. biochemical markers of covid- include lymphopenia, thrombocytopenia and leukopenia as well as elevated c-reactive protein (crp), which may also correlate with severe disease. less commonly, there may also be elevations in alanine aminotransferase (alt), aspartate aminotransferase (ast), creatine kinase (ck) and d-dimer. recommendations on whom to test for covid- will vary depending on the local guidelines, availability of testing kits and regional transmission dynamics. in high-risk settings where an outbreak has occurred, all patients with clinical features consistent with covid- should be considered for testing. in non-outbreak areas, a case of covid- should be suspected and tested for if a patient has clinical features suggestive of covid- and fits epidemiologic criteria for example international travel or contact with a confirmed covid- case. a patient is also considered a suspect case of covid- regardless of their history if they are admitted to hospital with acute respiratory illness or unexplained fever. diagnosis of covid- is via nucleic acid testing of nasopharyngeal and oropharyngeal swabs. serology testing is currently not widely available. it is important to ensure that routine local protocols for testing in acute pneumonia/pneumonitis are also followed, such as bacterial cultures, urinary antigen testing, acute and convalescent serology and respiratory virus panels. at this stage, faecal sampling is not standard; however, this may be indicated in those with gastrointestinal symptoms. a positive test for covid- must be immediately notified to the relevant communicable diseases agency. despite a lack of evidence demonstrating any increased susceptibility to covid- , (table ). however, theoretically, there may also be some beneficial effects seen with certain immunosuppressive medications, given the cause of death in covid- is a cytokine storm resulting in ards. overall, apart from corticosteroids, the risks of ibd therapies, particularly -asas and biologics, appear limited, and the importance of careful discussion regarding the risks versus benefits of medications with patients and among medical practitioners cannot be overstated. inappropriate cessation of effective agents due to unjustified fear of adverse events may lead to ibd relapse, which when requiring the use of steroids or hospitalisation may inadvertently strain medical resources and increase the risk of covid- exposure and infection. sulfasalazine and the -asa medications are used as first-line induction and maintenance treatment for uc. -asas have very mild immunosuppressive activity and they are often well tolerated with minimal side effects. there are no reports of these medications being associated with an increased risk of infection and studies of large cohorts evaluating the safety profile of -asas do not demonstrate an increased risk of serious or opportunistic infections. sulfasalazine and -asa • treatment with -asa therapy should continue without concern for increased risk of contracting or developing severe covid- . • if a patient is in contact with someone who has covid- or develops covid- , treatment with -asa should continue. corticosteroids systemically reduce inflammation by turning off multiple inflammatory genes. in patients with covid- , a systemic inflammatory response syndrome can perpetuate lung injury with persistent inflammation resulting in ongoing pulmonary damage even subsequent to viral suppression. in sars, corticosteroid treatment was of no clinical benefit in regard to infection resolution but was associated with an increased risk of psychosis, delayed clearance of viral rna, diabetes and avascular necrosis. finally, in a systematic review on corticosteroid treatment in influenza patients, corticosteroids were associated with an increased length of stay in intensive care, an increased rate of secondary bacterial and fungal infections and an increase in mortality compared to those who did not receive corticosteroids. however there is limited evidence that they increase the risk of either upper respiratory tract infections or pulmonary infections. , in addition, mercaptopurine has been shown to inhibit one of the proteases essential to viral maturation of mers-cov in vitro, although no further animal-based models exist to suggest clinical efficacy. methotrexate has variably been demonstrated to increase the risk of infections in patients with inflammatory diseases. reassuringly, a recent systematic review reassuringly found that in the nonrheumatoid arthritis inflammatory disease population, there was not an sulfasalazine and -asa • treatment with -asa therapy should continue without concern for increased risk of contracting or developing severe covid- . • if a patient is in contact with someone who has covid- or develops covid- , treatment with -asa should continue. increased risk of infection with methotrexate ( . , % ci . - . ). this applied similarly to respiratory infections specifically. anti-tnf agents have been shown to increase the risk of upper and lower respiratory tract infections, as well as serious and opportunistic pulmonary infections. , , compared with anti-tnf monotherapy, risk of serious infections increases with combination of anti-tnf and an immunosuppressive agent or most significantly in combination with corticosteroids. in a study that did not separate mono-or combination-anti-tnf therapies, the risk of pneumonia was only slightly increased in patients receiving a tnf antagonist of note, anti-tnf therapy has been employed in the management of severe sepsis, with a meta-analysis demonstrating reduced overall mortality when used in severe sepsis before shock, and improved survival at days in patients with shock or high baseline levels of il- . there are no data on the incidence and severity of sars or covid- in patients on anti-tnf medications. anti-tnf agents were utilised in the initial phase of the sars epidemic, although the overall evidence for efficacy is lacking. the main non-tnf antagonist biologics for ibd include the anti-il / biologic ustekinumab and the anti-integrin therapy, vedolizumab. reassuringly, the risk of severe respiratory tract infections, severe infections in general or opportunistic infections does not appear to be increased in long-term follow-up studies of ustekinumab in both ibd and psoriasis. [ ] [ ] [ ] similar data have also been reported for vedolizumab. [ ] [ ] [ ] [ ] there is a theoretical concern of an increased risk of respiratory infections, and hence covid- , with vedolizumab treatment as vedolizumab binds the t-cell integrin receptor α β , inhibiting its binding to madcam present on the small intestinal endothelium and vasculature. these receptors are also expressed in the t cells occupying the nares, although to a far lesser extent when compared to the intestine. interestingly, baricitinib, a jak inhibitor used in rheumatoid arthritis, has a potential therapeutic role in the management of sars-cov- through both its anti-inflammatory action and numbassociated kinase (nak) inhibition. tofacitinib has no affinity for this particular kinase. naks are involved in the function of clathrin, which mediates endocytosis of sars-cov- into cells resulting in infection. due to the anti-inflammatory effects of the jak inhibitors, it has also been speculated that they may be able to combat the elevated levels of cytokines observed in covid- which may potentially further decrease the severity of the infection. if a patient is exposed to or gets infected with covid- , the withholding of immunosuppressive medications should be considered where possible. it is important to note that many ibd medications may take months to be eliminated from the body so the utility of withholding these medications in the short term is likely to be limited. (see table ). the response to covid- is rapidly evolving and gastroenterologists should regularly review local, institutional and international recommendations. despite limited data, patients with ibd are theoretically at increased risk of covid- due to proximity to medical facilities and immunosuppression. there are therefore several recommendations that clinics and patients can implement to reduce risks. nutrition should be optimised as malnutrition can disrupt the innate immune response, including complement and mucosal secretory antibody formation, and is associated with increased infection risk in ibd patients. smoking has been associated with worse outcomes and increased mortality in covid- and therefore patients should be encouraged to quit smoking. immunisation: immunisation of patients should be strongly encouraged to reduce preventable co-infection with other viruses: patients should receive pneumococcus (pcv and ppsv ) and influenza (quadrivalent inactivated vaccine) vaccination. therapies are summarised in table . where interactions exist, it is possible for patients to cease their ibd medications and continue on trial medications. remdesivir is a prodrug of the adenosine nucleotide analogue gs- chloroquine is a widely used anti-malarial and autoimmune disease drug. it has a broad spectrum anti-viral action that is exerted chloroquine is not accessible in australia and it has been suggested that hydroxychloroquine could be an effective alternative given that it is an identical molecule to chloroquine apart from the addition of a hydroxyl group. favipiravir, a guanine analogue, effectively inhibits the rnadependent polymerase of rna viruses. this anti-viral effect has efficacy on influenza (approved use), ebola, yellow fever, chikungunya, norovirus and enterovirus. activity against covid- has been reported. it is currently being tested in japan and has no significant drug interactions. in a recent study it has been reported to improve clinical 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the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? chloroquine and hydroxychloroquine as available weapons to fight covid- chloroquine for the novel coronavirus sars-cov- hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial therapeutic options for the novel coronavirus ( -ncov) faviiravir versus arbidol for covid- : a randomized clinical trial searc hproj en.aspx?title = covid - &of fic ialna me=&subje ctid=&secon dar yi d=&appli er=&study leade r=&ethic alcom mitte esanc tion=&spons or=&study ailme nt=&study ailme ntcod e=&-study type= &study stage = &study desig n= &minst udyex ecute time=&maxst udyex ecute time=&recru itmen tstat us= &gende r= &agree tosig n=&secsp onsor =&regno =&regst atus= &count ry=&provi nce=&city=&insti tutio n=&insti tutio nleve l=&measu re=&inter code=&sourc eofsp ends=&creat eyear = &isupl oadrf =&wheth erpub lic=&btngo =btn&verif ycode =&page= protecting 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bowel disease comparative effectiveness of azathioprine in crohn's disease and ulcerative colitis: prospective, long-term, follow-up study of patients pharmacokinetics and immune reconstitution following discontinuation of thiopurine analogues: implications for drug withdrawal strategies subcutaneous absorption contributes to observed interindividual variability in adalimumab serum concentrations in crohn's disease: a prospective multicentre study clinical pharmacokinetics and pharmacodynamics of monoclonal antibodies approved to treat rheumatoid arthritis clinical pharmacokinetics and use of infliximab infliximab pharmacokinetics in inflammatory bowel disease patients clinical pharmacokinetics and pharmacodynamics of infliximab in the treatment of inflammatory bowel disease understanding the mechanisms of action of methotrexate: implications for the treatment of rheumatoid arthritis pharmacokinetics of lopinavir/ritonavir crushed versus whole tablets in children review article: the pharmacokinetics and pharmacodynamics of drugs used in inflammatory bowel disease treatment review article: prevention, diagnosis and management of covid- in the ibd patient key: cord- -i nvm authors: schlabitz, franziska; teich, niels; michl, patrick; walldorf, jens title: inflammatory bowel disease and covid- —“preventive” sick certificates as a frequent coping strategy in the face of the pandemic date: - - journal: inflamm bowel dis doi: . /ibd/izaa sha: doc_id: cord_uid: i nvm nan we read with great interest the article by occhipinti and pastorelli. the authors report their experiences and challenges in managing patients with inflammatory bowel disease (ibd) during the covid- pandemic. we complement this important information by presenting a survey conducted among german patients with ibd regarding their personal evaluation of their work-related risk of contracting sars-cov- . in europe-as in many other regions of the world-people are now facing contact restrictions because of the covid- -pandemic. thus, returning to work becomes an increasingly important issue-also from a socioeconomic point of view. considering the viewpoint of patients with ibd is very important for the patients' health care management in the next phase of the pandemic. an online questionnaire was completed by patients, who were recruited via social media channels of ibd self help groups (for details see table ). of the participants, . % feared a sars-cov- infection and . % were worried about going to work because of a potentially increased infection risk. half of the participants ( . %) believed that ibd predisposed them to a higher risk of contracting sars-cov- compared with their colleagues who did not have ibd. consequently, . % of the survey participants received a "preventive" sick certificate for a duration of more than days from their general practitioner even if they did not show any symptoms of an infection. of the participants being treated using azathioprine/ -mercaptopurine, . % reported using this strategy, as did . % of those treated using antitumor necrosis factor α treatment, % of those treated using prednisolone, . % of those treated using ustekinumab, . % of those treated using budesonide, and . % of those treated using vedolizumab. liberally issuing preventive sick certificates may be an exceptional strategy used for individual patients experiencing stressful life circumstances. however, during the covid- pandemic, the survey participants have used this coping strategy frequently. they also reported that they have delayed the decision on whether it is safe to go to work because of divergent or ambiguous opinions of their physicians as well as expert opinions from other sources (internet sources, media). clearly, the ability to work is very important to most patients with ibd. still, returning to work is often hampered by uncertainties because of the pandemic. therefore, straightforward recommendations stating clearly who is at risk and who should avoid which type of work are very important for managing the daily life of our patients. in addition, public financial support for patients who cannot go to work must be considered. author contributions: franziska schlabitz: study concept and design, acquisition of data, administration, analysis and interpretation of data, writing the manuscript. niels teich: acquisition of data, analysis, critical revision of the manuscript for important intellectual content. patrick michl: analysis, critical revision of the manuscript for important intellectual content, study supervision and resources. jens walldorf: study concept and design, acquisition of data, analysis and interpretation of data, writing of the manuscript, statistical analysis, study supervision and administration. conflicts of interest: niels teich has served as a speaker, a consultant, and/or an advisory board member for abbvie, amgen, falk foundation, janssen, msd, norgine, takeda, tillotts, and vifor and has received research funding from ferring arzneimittel gmbh. jens walldorf has served as a speaker, a consultant, and/or an advisory board member for abbvie, janssen, msd, and takeda. challenges in the care of ibd patients during the covid- pandemic: report from a "red zone" area in northern italy coronavirus and ibd reporting database. secure-ibd database public data update key: cord- -g b u yf authors: taxonera, carlos; alba, cristina; olivares, david; martin, maría; ventero, alejandro; cañas, mercedes title: innovation in ibd care during the covid- pandemic: results of a cross-sectional survey on patient-reported experience measures date: - - journal: inflamm bowel dis doi: . /ibd/izaa sha: doc_id: cord_uid: g b u yf background: the coronavirus (covid- ) pandemic is a clinical situation that could be used as prototype for implementation of new systems of care. methods: this was a single-center, cross-sectional study. we evaluated the feasibility of a strategy based on the conversion of face-to-face visits to telephone consultations to manage ibd outpatients during the covid- pandemic. a -item telephone survey ( closed questions and a -point numeric description scale) was conducted to evaluate satisfaction of patients with telephone consultations. results: between march and april , , % of the scheduled face-to-face visits could be converted to telephone consultations, and we resolved an additional urgent consultations by telephone. the rate of ibd-related hospitalization and visits to the emergency department decreased by % and %, respectively, compared with rates in the same period the previous year. the -item survey was conducted in outpatients. in closed questions, patients reported a very high degree of satisfaction with telephone consultations, with no differences between scheduled (n = ) and urgent consultations (n = ; p = ns). the overall satisfaction rating with the telephone consultation evaluated with the numerical description scale was % and % for scheduled and urgent consultations, respectively (p < . ). less than % of patients would have preferred a face-to-face visit to the telephone consultation at the time. conclusions: a strategy based on the conversion of face-to-face visits to telephone consultations was able to guarantee a minimum standard quality of care during the covid- pandemic. patients reported a very high degree of satisfaction with telephone consultations. the world health organization recently declared the outbreak of coronavirus (covid- ), caused by infection with novel severe acute respiratory syndrome coronavirus (sars-cov- ), as a pandemic of international concern. health facilities in the worst affected regions are struggling, resulting in extensive reallocation of hospital resources to help manage covid- patients. as a consequence, rapidly evolving national and international statements regarding covid- recommended avoiding in-person care for treatment of patients with no suspicion of infection; not only would this alleviate the burden on hospital resources but it would also eliminate the risk of infection in the clinic. , the safety and management of patients with inflammatory bowel disease (ibd) during the pandemic are of particular concern, - given that management often requires the use of immune-modifying therapies, some of which have welldescribed risks for severe viral infections. , although several studies offer guidance on gold-standard strategies for improved care of ibd patients in western countries, outcomes of such strategies have not been evaluated. , the aim of this study was to assess the feasibility of a strategy based on the conversion of face-to-face visits to remote telephone consultations to improve care of patients with ibd during the covid- pandemic, and to evaluate satisfaction of patients with telephone consultations. this was a single-center, cross-sectional study. the eligible population included patients with an established diagnosis of ibd followed at an ibd referral unit in the madrid region of spain (ibd unit of hospital clínico san carlos, madrid). baseline demographic and clinical doi: . /ibd/izaa published online august characteristics, in addition to treatments for ibd, were extracted from the prospectively maintained database eneida (supplementary table ). an appointed committee, which included ibd staff, ibd nurses, data manager, and administrative staff, developed a strategy to promote the use of telehealth care facilities. face-to-face scheduled visits in the outpatient clinic were converted to telephone consultations at the same time and on the same day as the scheduled visit. patients were notified of the change by the administrative staff. in addition, the existing nurse advice telephone line was reinforced to receive calls from patients seeking medical attention or health information during the pandemic. inflammatory bowel disease nurses triaged incoming calls and decided if the case required an urgent remote consultation by staff or if the patient was only requesting health information in the pandemic situation. these ibd nurses advised all patients to maintain their current treatment regimens to avoid relapse due to nonadherence and to strictly follow the general health recommendations to prevent covid- , according to international or national guidance. we discouraged ibd patients from coming to the emergency department without prior indication from ibd staff, and hospitalizations were avoided if possible. during virtual consultations, ibd staff and nurses questioned patients for suggestive symptoms or for a confirmed diagnosis of covid- , and cases were recorded. scheduled or urgent telephone consultations where made by physicians of the ibd unit. clinical data including patientreported outcomes were noted using the harvey-bradshaw index (hbi) for crohn's disease (cd) and the partial mayo score (pms) for ulcerative colitis (uc); blood or fecal test results were taken from the medical chart when available. based on this information, physicians gave indications about therapy and follow-up procedures. changes in treatments were introduced in the single prescription module that allows patients to collect their medications at pharmacies. in general, ongoing maintenance therapy was unchanged to avoid severe ibd flares. in patients diagnosed with covid- , immunosuppressants or biologic agents were temporarily discontinued according to guidance. [ ] [ ] [ ] endoscopic disease assessment was limited to absolutely essential cases, including the most urgent suspected new ibd cases. biologics clinic visits were maintained to administer iv drugs with rigorous measures to avoid cross-contamination. the day before scheduled iv dosing, patients were called by the ibd nurse and questioned for symptoms suggestive of covid- and/or close contacts with infected cases in the previous weeks. infusion date was delayed when necessary. health care workers and patients used surgical masks and latex gloves, and a -meter distance between chairs was maintained at the biologics clinic. an ibd staff member attended the biologics clinic daily, and a face-to-face visit was made during infusion. subcutaneous administration of biologics or small molecules was maintained, ensuring the home administration of these therapies. to evaluate patient-reported experience measures (prems) with telephone consultations, we developed a cross-sectional -item telephone survey that was conducted by an ibd staff member or nurse between and weeks after the initial call. to minimize nonresponse rates, we kept the survey short and focused on closed questions and a last question with a -point numeric description scale, where represents the least satisfaction and the most satisfaction, to allow for conversion of subjective answers to quantitative data ( fig. ) . to validate the survey instrument, the initial content was first analyzed by ibd nurses and ibd staff members and corrected as necessary. we then performed a pilot evaluation in a random sample of ibd patients to validate the survey questionnaire before full implementation. to assess intra-individual agreement, we repeated the survey at least days apart in a random sample of patients. the survey was conducted in consecutive patients who had a telephone visit each day to complete a sample size of % of the remote consultations that day, and so on. the study was approved by the clinical research ethics committee of the hospital universitario clínico san carlos, madrid, spain (c.i. / -e_epa od, march , ). verbal informed consent was obtained from all surveyed patients. study variables were summarized descriptively using numbers and percentages for discrete variables and mean and standard deviation (sd) or median and interquartile range (iqr) as appropriate for continuous variables. responses for each questionnaire item for scheduled and urgent telephone consultations were compared. we evaluated test-retest reliability using the cohen kappa for closed questions and intraclass correlation coefficient (icc) for numeric description scale. the health care activities carried out in the ibd unit and hospital facilities between march and april , , are summarized in table . of the scheduled face-to-face visits in this period, % could be converted to remote telephone consultations, and we resolved an additional urgent consultations by telephone. in the study period, we performed and face-to-face care visits at the biologics clinic and at the ibd unit, respectively. during the study period, only patients required hospitalization, and required a visit to the emergency department for their ibd, which represents a reduction of % and % in the rate of ibd-related hospitalization and visits to the emergency department, respectively, compared with the rates in the same period of the previous year. inflammatory bowel disease nurses provided remote health advice to patients. the -item telephone survey was evaluated in outpatients. surveys from patients were excluded because they were not included in the eneida database, and patients ( %) did not provide informed consent. table shows the baseline characteristics of patients and changes in treatment and reason during telephone consultations. during scheduled telephone consultations, a significantly higher percentage of patients were in remission. during urgent consultations, a higher percentage of patients required discontinuation of immunosuppressants or biologics due to diagnosis or high suspicion of covid- , needed corticosteroid courses or mesalazine initiation or escalation to treat flares, or received antibiotics for enteral infections. in the closed questions, patients reported a very high degree of satisfaction with and acceptance of telephone consultations, with no differences between scheduled (n = ) and urgent consultations (n = ; fig. ). less than % of patients would have preferred a face-to-face visit to the telephone visit at the time. the global satisfaction rating with the telephone consultation evaluated with the numerical scale was % and % for scheduled and urgent consultations, respectively (p < . ). test-retest reliability of the survey questionnaire was perfect for closed questions (cohen's kappa ), and excellent for the numeric description scale (icc . ; % ci, . - . ). the covid- pandemic is a clinical situation that could be used as prototype for implementation of a telehealth consultation system for evaluation of high-risk populations like ibd patients. although an increasing number of studies have recommended strategies to reorganize ibd units in western countries during the pandemic, , the outcomes of such procedures have only been briefly evaluated in one study. here, we report the outcomes and patient perception of a strategy based on the conversion of face-to-face visits to remote telephone consultations to improve care of outpatients with ibd during the covid- pandemic. the conversion of outpatient follow-up visits to virtual consultations was feasible, with % of consultations resolved by telephone or email, avoiding travel to in-person care sites, and with a very low rate of missed visits. during remote consultations, we were able to escalate treatment due to ibd flares, avoiding face-to-face clinic visits. in accordance with guidance, we temporarily discontinued immune-modifying therapies in patients with diagnosis or strong clinical suspicion of covid- . [ ] [ ] [ ] during the study period, very few patients required hospitalization or emergency department visits related to ibd, which was important in the days when hospitals' health facilities were overwhelmed. twelve patients were diagnosed with covid- . of these, were hospitalized, and self-isolated at home. all covid- cases were community-acquired rather than linked to visits to hospital facilities. the biologics clinic was maintained following the strict protocol to avoid cross contamination, and no cases of covid- were detected. a recent study reported that implementation of virtual clinics, drug home delivery, and ibd networking was able to maintain acceptable standards of care for ibd patients. the outcomes of the new system of care implemented during the pandemic helped us determine what kind of patients could be attended and what problems could be resolved without a face-to-face consultation and supported efforts to reorganize the activities of the ibd clinic during the de-escalation period. some surveys have assessed health care providers' perceptions of the implementation of new care systems during the pandemic, but studies that evaluate patient-reported indicators for assessing health system performance are lacking. as part of this study, we administered for the first time a survey evaluating consumer perceptions and preferences with the new system of care. patient experience is one important measure of the quality and efficiency of health care, and the use of prems is recommended. outpatients reported a very high degree of satisfaction with telephone consultations, with no differences between scheduled and urgent consultations, and very few patients would have preferred a face-to-face visit at the time. home patient management is a well-accepted approach by patients with ibd, as evidenced by the high adherence to home therapies or recommendations from physicians. telemedicine has also been associated with a reduction in face-to-face visits and hospitalizations and could be a valid alternative to improve the quality of ibd patient care. [ ] [ ] [ ] these studies were performed before the covid- outbreak, and we believe that results of our study confirmed the validity of telemedicine to manage patients with ibd while still maintaining quality standards of care. results of a global telemedicine survey among gastroenterologists by the international organization for the study of inflammatory bowel disease (ioibd) demonstrated the shift from face-to face clinics to remote telemedicine during covid- , with telephone consultations increasing the most, currently accounting for over half of all ibd visits. a survey among ibd gastroenterologists in spain reproduces the global results, reflecting the rapid structural changes in the ibd units to guarantee virtual, non-face-to-face consultations. our study has some limitations. the reduction in visits to the emergency department and hospitalization may not be attributable to telehealth intervention. during the pandemic, avoidance of hospital health facilities was a conscious choice by patients (and providers to some degree) due to fear of contracting covid- . furthermore, a period of month may be insufficient to assess the impact on medical resource usage. we were not able to evaluate provision of telemedicine by video consultation due to the lack of appropriate equipment and technological skills at our outpatient clinics. the surveys were carried out by staff from the ibd unit and not by independent surveyors, which could have biased the responses of the patients. in conclusion, the reorganization of the ibd clinic following a strategy of switching face-to-face visits to remote telephone consultations when possible was able to guarantee a minimum standard quality of care to our patients during the covid- pandemic. the implementation of the new care system could have contributed to a reduction in emergency department visits and hospitalization when the pandemic was at its worst. considering the high degree of satisfaction in all prems with remote consultations, once the pandemic is over, health care providers could consider applying some of the principles of telemedicine permanently. doing so, always in accordance with patient preferences, would represent a deeper commitment to patient-centered care. supplementary data is available at inflammatory bowel diseases online. healthcare facilities: managing operations during the covid- pandemic virtually perfect? telemedicine for covid- aga clinical practice update on management of inflammatory bowel disease during the covid- pandemic: expert commentary british society of gastroenterology guidance for management of inflammatory bowel disease during the covid- pandemic increased incidence of systemic serious viral infections in patients with inflammatory bowel disease associates with active disease and use of thiopurines opportunistic infections with anti-tumor necrosis factor-α therapy in inflammatory bowel disease: meta-analysis of randomized controlled trials inflammatory bowel disease care in the covid- pandemic era: the humanitas, milan experience challenges in the care of ibd patients during the covid- pandemic: report from a "red zone" area in northern italy maintaining the quality standards of care for inflammatory bowel disease patients during the covid- pandemic novel coronavirus disease (covid- ) in patients with inflammatory bowel diseases feasibility and acceptance of a home telemanagement system in patients with inflammatory bowel disease: a -month pilot study telemedicine for management of inflammatory bowel disease (myibdcoach): a pragmatic, multicentre, randomised controlled trial a randomized controlled trial of telemedicine for patients with inflammatory bowel disease (tele-ibd) delivering high value inflammatory bowel disease care through telemedicine visits innovation in ibd care during the covid- pandemic: results of a global telemedicine survey by the international organization for the study of inflammatory bowel disease management of covid- pandemic in spanish inflammatory bowel disease units: results from a national survey the authors thank dr. g. morley for reviewing the english manuscript. key: cord- -bayabroa authors: brenner, erica j.; pigneur, bénédicte; focht, gili; zhang, xian; ungaro, ryan c.; colombel, jean-frederic; turner, dan; kappelman, michael d.; ruemmele, frank m. title: benign evolution of sars-cov infections in children with inflammatory bowel disease: results from two international databases date: - - journal: clin gastroenterol hepatol doi: . /j.cgh. . . sha: doc_id: cord_uid: bayabroa nan the coronavirus disease (covid- ) pandemic caused by the highly infectious severe acute respiratory syndrome coronavirus (sars-cov ) presents most often with mild clinical symptoms, but the severe forms are of major concern. sars-cov enters human cells via the angiotensin-converting enzyme (ace ) receptor, expressed on epithelial and endothelial cells. since highest ace expression is in the terminal ileum and colon, further upregulated during inflammation, and many covid- patients experience gastrointestinal symptoms, longitudinal data are necessary determine whether inflammatory bowel diseases (ibd) patients are at risk for severe or complicated covid- . a recent analysis in ibd patients from the secure-ibd registry revealed older age, steroid medication and comorbidities as risk factors for severe evolution, while the same study showed that the ibd patients less years had only mild disease courses. the purpose of this report is to describe the disease course of covid- in an expanded sample of pediatric ibd patients from two international databases. bowel disease (secure-ibd) and the covid- database of the paediatric ibd porto group of the european society for paediatric gastroenterology hepatology and nutrition (espghan) were created in march with the aim to monitor outcomes of covid- occurring in ibd patients. in this analysis, we included all subjects ≤ years of age from the secure-ibd and porto group databases through october st , . we used descriptive statistics to summarize the demographic and disease characteristics of the study population, both overall and stratified by hospitalization status (hospitalized versus outpatient only), and performed bivariate comparisons (see supplemental methods). we collected covid- cases in pibd patients from countries ( table ). the most common ibd treatment was tnf antagonist monotherapy ( %), followed by sulfasalazine/mesalamine ( %). most patients ( %) had no comorbidities other than ibd. there were no deaths in the study population, and fourteen children ( %) were hospitalized, of whom only two ( %) required mechanical ventilation. the two children requiring mechanical ventilation were on sulfasalazine/mesalamine and developed a multi-system inflammatory syndrome and concomitant secondary infection, respectively, with favorable evolution. characteristics of the fourteen hospitalized patients are provided in supplemental table . factors associated with hospitalization included comorbid conditions other than ibd ( % hospitalized vs % not; p value < . ), moderate/severe ibd disease activity ( % vs %; p value < . overall), gastrointestinal symptoms ( % vs %, p value < . ), sulfasalazine/mesalamine use ( % vs %; p value . ), and steroid use ( % vs %, p value . ). tnf antagonist monotherapy was associated with a decreased likelihood of hospitalization ( % vs %; p value < . ) ( table ). sulfasalazine/mesalamine use remained a risk factor after adjusting for disease activity (aor . , % confidence interval . - . ) we analyzed children and adolescents ≤ years with pibd who developed covid- . the % hospitalization rate found here is markedly less than - % hospitalization reported in adult ibd patients. , our data are in line with other reports indicating that children are at low risk for complicated covid- . reported cases likely under-represent the actual case burden, since most paediatric covid- manifestations are mild or asymptomatic and sars-cov testing is not indicated. a case series of , children found that . % of cases were asymptomatic or mild, and only . % were severe. additionally,mild cases may be under-reported. thus, the low observed hospitalization rate is likely an overestimation of the true hospitalization rate. the findings that sulfasalazine/mesalamine and steroid use were associated with increased hospitalization risk and that tnf antagonist monotherapy was associated with decreased risk parallel those reported in adult ibd patients. other risk factors for hospitalization included other comorbid conditions, moderate/severe ibd disease activity, and gastrointestinal symptoms. the six-year-old colitis patient requiring icu care in this series is in line with the recent reports of multi-systemic inflammatory (kawasaki-like) syndrome temporarily related to sars-cov- infection in children. our patient had a favourable evolution with steroid medication, while a recently reported -year-old boy with cd also developing a kawasakilike syndrome had an immediate improvement with infliximab medication. the other iculevel patient developed a secondary infection that required multiple antimicrobial agents. in conclusion, our data suggest pibd patients have a relatively low risk of severe covid- , even when receiving biologic and/or other immune suppressive therapies for their ibd. this finding may reassure parents of children with ibd who are debating the safety of sending their children back to school in the fall. these data support earlier guidance from the pediatric porto group to continue maintenance ibd treatment for pibd throughout the current pandemic. j o u r n a l p r e -p r o o f ( ) ( ) ( ) ( case details were reported directly by providers using case report forms created in redcap (research electronic data capture), a secure, web-based electronic data capture tool, as has been previously described. , we removed duplicate reports known to have been entered into both databases. additionally, we double checked for duplicate reports by identifying records with matching age, sex, ibd disease type, and country. potential duplicates were manually reviewed, and true duplicates were removed. incomplete reports with missing outcome data were excluded from analysis. disease activity was assessed by physician global assessment (pga), and comorbid conditions were defined as any chronic condition beyond ibd as determined by the reporter. we summarized continuous variables using means and standard deviations. we expressed categorical variables as number of participants and proportions. we performed bivariate comparisons by hospitalization status using the fisher's exact test for each categorical variable and the t-test for continuous variables. we listed demographic and disease characteristics for all cases that required hospitalization. a novel coronavirus from patients with pneumonia in china quantitative mrna expression profiling of ace , a novel homologue of angiotensin converting enzyme but not tnf antagonists, are associated with adverse covid- outcomes in patients with inflammatory bowel diseases: results from an international registry novel coronavirus disease (covid- ) in patients with inflammatory bowel diseases epidemiology of covid- among children in china kawasaki-like multisystem inflammatory syndrome in children during the covid- pandemic in paris, france: prospective observational study pediatric crohn's disease and multisystem inflammatory syndrome in children (mis-c) and covid- treated with infliximab ) from the paediatric ibd porto group of european society of paediatric gastroenterology, hepatology, and nutrition but not tnf antagonists, are associated with adverse covid- outcomes in patients with inflammatory bowel diseases: results from an international registry ) from the paediatric ibd porto group of european society of paediatric gastroenterology, hepatology, and nutrition we performed a post-hoc logistic regression evaluating the association between mesalamine/sulfasalazine and hospitalization with adjustment for ibd disease activity by pga. we reported the adjusted odds ratio and % confidence interval.the databases were constructed and maintained according to the local ethics instructions/committees. j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f key: cord- - jwwmoys authors: d'amico, ferdinando; rahier, jean-françois; leone, salvo; peyrin-biroulet, laurent; danese, silvio title: views of patients with inflammatory bowel disease on the covid- pandemic: a global survey date: - - journal: lancet gastroenterol hepatol doi: . /s - ( ) - sha: doc_id: cord_uid: jwwmoys nan patients with chronic diseases have experienced substantial changes to the routine management of their conditions during the coronavirus disease pandemic. , although insights into the management of patients with inflammatory bowel disease (ibd) during the outbreak have been described, little attention has been paid to the patients' point of view. therefore, we did an anonymous web survey with the support of the european federation of crohn's and ulcerative colitis associations (efcca) between march and april , , to investigate the concerns, fears, and behaviours of patients with ibd during the early phase of the covid- pandemic. the questionnaire was initially developed in english through the cooperation of ibd specialists and patient association representatives, focusing on the most frequent questions asked by patients during daily clinical practice. subsequently, the questionnaire was translated into ten languages (italian, dutch, french, spanish, greek, polish, portuguese, croatian, bulgarian, and slovenian) by volunteer native speakers. patients with ibd were invited to participate in the survey via the efcca. we had responses from participants from countries worldwide (appendix the results of this survey highlight that a gap between doctors and patients still exists. there is an urgent need to improve physician-patient communication and to provide clear and specific recommendations in a period of substantial confusion for people with chronic diseases. the european crohn's and colitis organisation, the british society of gastroenterology, and the international organization for the study of inflammatory bowel diseases have responded to this request, providing practical guidelines for the management of patients with ibd during the pandemic, focusing not only on drug treatment but also on advice for daily life (eg, social distancing, use of masks, and travel avoidance). [ ] [ ] [ ] in patients with ibd who do not have symptoms suggestive of covid- , see online for appendix immunosuppressive and biological drugs should not be discontinued as a preventive strategy, since there is no evidence to date to suggest that there is an increased risk of sars-cov- infection with these therapies. [ ] [ ] [ ] in addition, patients are recommended to stay at home, avoid travel, respect the rules of social distancing (at least m between one person and another), pay close attention to hand hygiene, and use protective masks outside the home. [ ] [ ] [ ] in this context, patient associations are a key link between doctors and patients and should be increasingly involved in patient management. close cooperation could allow greater patient compliance with the recommendations of health-care providers and could also help to establish long-lasting, trusting relationships. applied molecular transport, ose immunotherapeutics, enthera, and theravance; grants from abbvie, msd, and takeda; and stock options from clinical trials mobile application. sd has served as a speaker, consultant, and advisory board member for schering-plough european federation of crohn's and ulcerative colitis associations clinical characteristics of coronavirus disease in china clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china prevention of covid- in patients with inflammatory bowel disease in wuhan, china european crohn's and colitis organisation british society of gastroenterology guidance for management of inflammatory bowel disease during the covid- pandemic international organization for the study of inflammatory bowel disease. ioibd update on covid- for patients with crohn's disease and ulcerative colitis key: cord- -qeo qn d authors: fiorino, gionata; peyrin-biroulet, laurent; danese, silvio title: protecting patients with ibd during the covid- pandemic date: - - journal: lancet gastroenterol hepatol doi: . /s - ( ) - sha: doc_id: cord_uid: qeo qn d nan ibds are immune-mediated diseases, which usually require treatment with corticosteroids, immunomodulators, or monoclonal antibodies to induce and maintain clinical and endoscopic remission. the use of these agents can increase the risk of opportunistic infections, but not that of serious infections. therefore, adoption of adequate measures to prevent and protect patients is an essential part of the quality standards of care in ibd. an and colleagues stopped biologics (infliximab infusions) and immunosuppressive treatments for all patients with ibd. this decision is challenging. indeed, a systematic review showed that the risk of hospital admission (odds ratio · , % ci · - · ) and surgery ( · , · - · ) is significantly reduced by use of biologics for patients with ibd. the probability of relapse after stopping effective immunomodulators or biological therapy is about % and is associated with an increased need for steroids, and risk of hospital admission and surgery. some considerations are needed. first, sars-cov- infection should be considered as a serious rather than an opportunistic infection, as the risk of infection is not related to concomitant immunosuppression. second, severe covid- might be associated with cytokine storm and is possibly related to a hyper-immune response in addition to virus-related damage. third, around % of patients who relapse because of withdrawal of effective therapies will require hospital admission against a backdrop of overwhelmed hospital capacity. thus, the risk and benefits of continuing or stopping biologics should be carefully balanced and should not be assumed to be a general rule for all patients with ibd, especially given the length of time the pandemic is likely to last. in conclusion, protection of patients with ibd from covid- is crucial and strongly advisable. whether stopping or adapting therapies will have substantial positive benefits for patients with ibd requires further, longer-term data from different countries. vandoeuvreles prevention of covid- in patients with inflammatory bowel disease in wuhan, china inflammatory bowel disease care in the covid- pandemic era: the humanitas, milan experience management of ibd during the covid- outbreak: resetting clinical priorities systematic review with meta-analysis: comparative efficacy of immunosuppressants and biologics for reducing hospitalisation and surgery in crohn's disease and ulcerative colitis systematic review of effects of withdrawal of immunomodulators or biologic agents from patients with inflammatory bowel disease key: cord- -befymalm authors: sultan, keith; mone, anjali; durbin, laura; khuwaja, samreen; swaminath, arun title: review of inflammatory bowel disease and covid- date: - - journal: world j gastroenterol doi: . /wjg.v .i . sha: doc_id: cord_uid: befymalm the first cases of a novel corona virus infection were reported in wuhan china in december of , followed by the declaration of an international pandemic by the world health organization in march . early reports of the virus, now known as severe acute respiratory syndrome coronavirus , and its clinical disease coronavirus disease (covid- ), has shown higher rates of morbidity and mortality in the elderly and those with pre-existing medical conditions. of particular concern is the safety of those with compromised immune systems. inflammatory bowel disease (ibd) is itself caused by a disordered immune response, with the most effective medical therapies being immune suppressing or modifying. as such, the risk of covid- , virus related outcomes, and appropriate management of ibd patients during the global pandemic is of immediate concern to gastroenterologists worldwide. there has been a rapid accumulation of clinical data and expert opinion on the topic. this review will highlight the latest source information on clinical observation/outcomes of the ibd population and provide a concise summary of the most up to date perspectives on ibd management in the age of covid- . the first cases of a novel coronavirus infection were reported in wuhan, china in december of [ ] . since that time the virus has spread to all continents except antarctica, with the world health organization declaring a global pandemic on march , . as with other, similar coronaviruses, such as those associated with severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers), the primary manifestations of active infection are respiratory. patients typically develop fever, cough, and shortness of breath, with a significant minority progressing to severe lung injury requiring the use of supplemental oxygen, and the need for mechanical ventilation with a high associated mortality rate [ ] [ ] [ ] . since its identification, the virus has been assigned the formal nomenclature of severe acute respiratory syndrome coronavirus (sars-cov- ), with the associated clinical illness designated as novel coronavirus ( -ncov) or coronavirus disease (covid- ) [ ] . as was observed during the initial outbreak in china, and then even more dramatically in italy, those at highest risk were noted to be the elderly and those with preexisting medical conditions, particularly cardiovascular, respiratory, endocrine, and oncologic [ , ] . as with most communicable infectious diseases, particular concern has also been raised for the safety of those with coexisting immune mediated disease, and/or those on immune compromising therapies. for the gastroenterology community (providers, patients and caregivers) this has obviously sparked particular concern for those individuals with inflammatory bowel disease (ibd). ibd is regarded as a disease of immune dysregulation, and with the exception of some limited use of diet, antibiotic and topical anti-inflammatory therapies, the vast majority of effective ibd medications for moderate to severe disease are immune suppressing/ modifying [ ] [ ] [ ] . while ibd itself is not regarded to increase non-gastrointestinal (gi) infectious disease risk [ ] , there is ample evidence demonstrating an increased risk of non-gi, opportunistic infections associated with ibd therapies [ ] [ ] [ ] [ ] [ ] . given the need for clinical evidence and expert guidance, the past weeks have seen the rapid growth of information specifically geared towards answering the core questions faced by ibd patients and providers. this work falls into two main categories, each of which we will briefly review: ( ) clinical observation of the ibd patient experience during the covid- pandemic; and ( ) expert opinion on the management of ibd in an environment of covid- . though there is currently no evidence of sars-cov- exacerbating underlying ibd, it is now well recognized that many patients with covid- will develop gi complaints. the sars-cov- invades human cells by interactions with angiotensin-converting enzyme (ace ). the ace receptor is found in different tissues throughout the body, including those of enterocytes [ ] [ ] [ ] . studies have shown the presence of sars-october , volume issue cov- in stool with persistence of viral shedding in the stool even after the resolution of respiratory complaints [ , ] . notably, recent basic scientific evidence has observed an up-regulation of ace in the inflamed mucosa of ibd patients, suggesting how ibd patients might be at increased risk for covid- [ , ] . however, it is also worth noting that a soluble form of ace circulating in the blood is also up-regulated in ibd, which may provide an alternate binding site for sars-cov- that could limit viral binding to cell surfaces [ ] . further studies on viral load and viral dynamics are required to clarify the clinical significance of these findings. cheung et al [ ] in their systematic review and meta-analysis of studies ( from china) including covid- patients, demonstrated a pooled prevalence of all gastrointestinal symptoms of . % [ % confidence interval (ci): . - . ] from china, and . in studies from all other countries. the most common complaint was anorexia . % ( %ci: . - . ), followed by nausea/vomiting . % ( %ci: . - . ), diarrhea . % ( %ci: . - . ), and abdominal pain/discomfort . % ( %ci: . - . ). it is unknown however how many of these patients had a prior ibd diagnosis or other gi condition. goyal et al [ ] in a more recent analysis of consecutive patients admitted to new york city hospitals also showed gi complaints were prominent, including diarrhea ( . %) and nausea with vomiting ( . %). despite these high rates of gi complaints, mao et al [ ] in their report of covid- 's impact on those with preexisting gi conditions, noted that there had been no reports of ibd patients infected with sars-cov- in the ibd elite union, a consortium of the seven largest chinese ibd referral centers, caring for over patients. the authors also reported that there had been no cases of ibd/sars-cov- infected patients in the three largest tertiary ibd centers in wuhan (tongji hospital, union hospital, and zhongnan hospital) at the time their manuscript was prepared, march , . while these results are encouraging, the methodology of case identification/reporting, and thus the true rate of ibd/sars-cov- , remains unclear. also, as rates of ibd and utilization of ibd medication may differ in china from those in other countries, these results may not be applicable to other populations. low rates of ibd/sars-cov- have also been reported in lombardy, italy, the next major covid- hot spot. norsa et al [ ] acknowledging that the pandemic is still ongoing, reported on their region's experience (including their ibd center) up to the time of publication. at that time they observed the highest rates reported in the world: cases of covid- out of a population of . million. of the ibd patients followed at their center ( % pediatric, % on immunomodulators (imm), and % on biologics) there had been no cases of covid- reported. based on wuhan population modeling, the authors had anticipated ibd infected patients by that time point. the authors do acknowledge that their results are not definitive, as only patients with severe symptoms and/or those receiving a nasopharyngeal swab were counted. the case reporting methodology, which was at least partially dependent upon patient self-reporting, again may have been biased towards an underestimate of true cases. more recently, case series and observational cohort data has emerged reporting on identified ibd/covid- patients. rodriguez-lago et al [ ] reported on cases of ibd ( hospitalized) with confirmed positive tests for sars-cov- from sites in the basque country (spain), median age years, % male, % crohn's disease (cd), with % on immune therapy, % biologic, and % systemic corticosteroids. two deaths ( %) were reported, including an years old male, on mesalamine, with prostate adenocarcinoma, and a years old male on mesalamine and methotrexate. taxonera et al [ ] reporting from the madrid region of spain, observed ibd cases with laboratory confirmed covid- from ibd patients followed in their database. their patients' mean age was years, with % female, and . % cd. seven patients ( . %) were on immune and/or biologic therapy. there was no reporting of rates of corticosteroid use. eight patients required hospitalization, required mechanical ventilation and died; a years old male with uc and a years old female with uc, neither of whom was receiving immune or biologic therapy. the authors additionally compared their findings in the ibd cohort to the observed rates and mortality of covid- in the general population of madrid. they found a significantly lower risk of covid- for ibd [odds ratio (or) . , %ci: . - . ; p < ], with no significant difference in the case fatality rate for covid- for ibd patients of . % vs . % for the general population (or . , %ci: . - . , p = . ). an additional ibd patients with covid- have been reported by the combined centers of nancy university hospital in france and humanitas, milan, italy [ ] from their combined cohorts of over ibd patients, they identified patients who tested positive for covid- via routine tele-medicine and infusion center visits. thirteen patients were on immune and/or biologic therapy, and there was no mention of corticosteroid use. five patients required hospitalization, but no deaths were reported. the authors observed an incidence of covid- positive ibd patients in the cohort of . , which was similar to the current cumulative incidence of . in france and italy at that time. to date, the largest national case reporting has come from a combined ibd referral centers in italy, affiliated with the italian group for the study of inflammatory bowel disease (ig-ibd) [ ] . patients either had laboratory testing confirming sars-cov- or a known infected contact and a combination of suspicious clinical complaints and/or lung ct findings of covid- . in total patients were described, median age years, . % female, cd, of whom % were on thiopurines, % anti-tnf, % vedolizumab, % ustekinumab and % systemic corticosteroids. additionally, % of patients ( % of cd and % of uc) were determined to have active disease based upon chart abstraction of the harvey-bradshaw index for cd and partial mayo score for uc. overall patients ( %) had covid- related pneumonia, ( %) were hospitalized, ( %) required mechanical ventilation, and ( %) died. important observations included a significant association between active ibd and covid- related pneumonia (or . , , p = . ), and active ibd and covid- related death (or . , %ci: . - . , p = . ). there was no association between either corticosteroid use or anti-tnf use and covid- related death. age > years was the strongest predictor of covid- related death (or . , %ci . - . , p = . ). also, in keeping with the observed low rates of clinically significant disease in the young, low rates have also been reported from a sample of the pediatric ibd (pibd) centers (mostly in europe), part of the porto group of the european society of pediatric gastroenterology, hepatology and nutrition (espghan) [ ] . a voluntary reporting system was constructed to include those with virologically confirmed sars-cov- , as well as cases with strong clinical suspicion in those without access to testing. reporting required a -d follow-up to ensure documentation of disease severity. the chinese pediatric centers ( % from wuhan) reported confirmed or suspected cases of covid- , none in the ibd patients. the south korean cohort reported no cases of covid- out of the children with ibd followed at four tertiary care centers. reporting from a combined centers in europe, canada, and israel up through march , resulted in a total of cases of pibd and covid- , all with mild disease despite ongoing treatment with immunomodulators, corticosteroids and/or biologics. notably, despite reporting no cases of ibd patients contracting covid- at the chinese centers, the crisis created by covid- resulted in delays of scheduled infusions. there were pibd patients scheduled to receive infliximab during the pandemic. of these, ( %) had their infusions delayed, resulting in disease exacerbations and hospitalizations. in an attempt to keep up with the pace of the pandemic and the need for updated data, the surveillance epidemiology of coronavirus under research exclusion (secure-ibd) database has been established [ ] . it is an international, pediatric and adult database to monitor and report on outcomes of covid- occurring in ibd patients. the database is open to reporting by ibd clinicians, both pediatric and adult, worldwide. reporters are encouraged to include both symptomatic and asymptomatic patients. de-identified data points collected for analysis include age, gender, country of origin, ibd disease type and ibd medication use. the database is tracking rates of hospitalizations, icu admission, need for mechanical ventilation and mortality. at the time of this manuscript's submission, the first published reports from the database have become available. currently "in press," the authors report cases from countries (median age years, % men). the primary outcome of interest was severe covid- , defined as a composite of icu admission, ventilator use, and/or death. thirty seven patients ( %) had severe covid- (as determined by physician global assessment), ( %) were hospitalized, and patients died ( % case fatality rate). age-standardized mortality ratios for ibd patients were . ( %ci: . - . ), . ( %ci: . - . ), and . ( %ci: . - . ) relative to data from china, italy, and the us, respectively. on multivariable analysis, risk factors for severe covid- among ibd patients included increasing age [adjusted or (aor) . , %ci: . - . ], ≥ comorbidities (aor . , %ci: . - . ), systemic corticosteroids (aor . , %ci: . - . ), and sulfasalazine or -aminosalicylate use (aor . , %ci: . - . ). tnf antagonist treatment was not associated with severe covid- (aor . , %ci: . - . ). of note, only cases of covid- were reported in the age range of - years, and patients in the range - years. only pediatric patients required hospitalization; none required icu or ventilator support. october , volume issue in the weeks and months since the initial outbreak, several gi professional societies and patient support organizations have developed recommendations for the management of ibd in the era of covid- [ , [ ] [ ] [ ] [ ] . expert opinion has focused on several core questions: ( ) are ibd patients at greater risk for contracting covid- ? ( ) how should ibd be managed in an environment of covid- ? and ( ) how should ibd patients with known or suspected covid- be treated? as acknowledged by the authors, much more data is still needed, with the current recommendations drawing heavily upon ibd experience with other infections, and with the mechanisms and the accumulated clinical experience with different ibd therapies. the current consensus is that ibd itself is not a risk factor for covid- , but that the risk lies mainly with the use of ibd medications, including corticosteroids, immunomodulators and biologic therapies. while there are active clinical trials using immune therapies to treat the inflammatory storm typical of severe covid- , none of the drugs involved are those currently approved for ibd management, and the results of these trials all are still pending. none of the society statements recommend discontinuing -asa/mesalamine therapies. all of the recommendations support continuity of ibd therapy as long as the patient has not acquired sars-cov- or developed covid- , and all of the groups that address endoscopy/surgery suggest postponing any nonurgent procedures. tables and summarize some key points related to disease management from the recommendations. for detailed clinical management scenarios, we recommend referring to the treatment algorithm provided in the aga practice update or to the expert consensus statements provided by the ioibd. just a few months ago patients with ibd and their providers entered a new and uncertain world dominated daily by the specter of covid- . added to the significant concerns of the general public, facing a highly communicable and sometimes fatal illness, the ibd community carries the additional concerns of a high-risk group. while ibd is characterized by an innate immune dysfunction, there fortunately is no evidence yet to suggest a higher risk for a severe clinical course of covid- conferred by ibd alone. while it is too early to say whether the therapies used for ibd, currently centered around immune suppression/modification, place patients at higher risk of infection itself or severe outcomes of infection, we are hopeful that the rapid accumulation of collaborative data from around the world will begin to provide answers. while the rapidity of data collection is impressive, there remains a significant risk of bias in the cases submitted to "real time" registries that may prevent their generalization to specific populations. it is also not clear whether "risks" of a severe outcome from covid- infection in this population is modified by country specific variables, such as severity of lockdowns, access to care, access to ventilators, threshold for admission to hospitals based on availability of beds, availability of covid pcr testing, all of which vary by locality and cannot be adjusted for in the final analysis. this leaves a knowledge gap for concentrated data from a single location that minimizes the risk of bias during data collection and variability in outcomes resulting from country specific health care resources. just as we are increasingly in a world where many of our patients can receive expert care without the risks of leaving their own home, so too does the almost real time collection and analysis of data from around the world offer the promise of rapidly providing answers to those most urgent questions raised by the worldwide ibd community. covid- : gastrointestinal manifestations and potential fecal-oral transmission prolonged presence of sars-cov- viral rna in faecal samples are patients with inflammatory bowel disease at increased risk for covid- infection? imbalance of the renin-angiotensin system may contribute to inflammation and fibrosis in ibd: a novel therapeutic target? upregulation of circulating components of the alternative renin-angiotensin system in inflammatory bowel disease: a pilot study gastrointestinal manifestations of sars-cov- infection and virus load in fecal samples from a hong kong cohort: systematic review and meta-analysis clinical characteristics of covid- in new york city chinese society of ibd, chinese elite ibd union chinese ibd quality care evaluation center committee. implications of covid- for patients with pre-existing digestive diseases uneventful course in patients with inflammatory bowel disease during the severe acute respiratory syndrome coronavirus outbreak in northern italy characteristics and prognosis of patients with inflammatory bowel disease during the sars-cov- pandemic in the basque country (spain) novel coronavirus disease (covid- ) in patients with inflammatory bowel diseases incidence and patterns of covid- among inflammatory bowel disease patients from the nancy and milan cohorts italian group for the study of inflammatory bowel disease (ig-ibd). outcomes of covid- in patients with ibd in italy: an ig-ibd study paediatric ibd porto group of espghan. covid- and paediatric inflammatory bowel diseases: global experience and provisional guidance british society of gastroenterology guidance for management of inflammatory bowel disease during the covid- pandemic aga clinical practice update on management of inflammatory bowel disease during the covid- pandemic: expert commentary international organization for the study of inflammatory bowel disease. management of patients with crohn's disease and ulcerative colitis during the coronavirus disease- pandemic: results of an international meeting key: cord- - qcdnd y authors: barberio, brigida; zingone, fabiana; bertani, lorenzo; savarino, edoardo title: the adherence to infusible biologic therapies in inflammatory bowel disease patients during covid- pandemic: is it really a problem? date: - - journal: gastroenterology doi: . /j.gastro. . . sha: doc_id: cord_uid: qcdnd y nan we read with great interest the nationwide retrospective study by khan et al. a evaluating the adherence to infusible biologic therapies, defined as receiving an infusion within ten weeks of the prior infusion, in patients with inflammatory bowel disease (ibd) during the current coronavirus disease (covid- ) pandemic. the authors compared the adherence to infusion therapy between a cohort of ibd subjects, who received infusible biologic therapies during covid- , and another cohort of ibd patients who underwent the same therapies in . they found that the adherence decreased from . % in to . % during the covid- crisis, thus recording a drop in the weekly number of infusions in their center since late march . finally, this exploratory analysis also confirmed a significant association between nonadherence to biologics infusion and the subsequent risk of corticosteroid requirement. in order to evaluate the impact of covid- and the following lockdown on the routine activities of our gastroenterology unit, we collected data from all accesses for infusible biologic therapies to our infusion center, between the th of january and the th of february (pre-lockdown) and between the th of march and the th of april (after the lockdown and covid- breakout). in contrast with khan and coworkers we did not observe a particular reduction of activity compared to the pre-lockdown period ( vs ) and at the same time we did not observe an increase of corticosteroid need or hospitalization among our ibd patients. surely, this pandemic with the extraordinary measures to contain the viral spread captured the public attention and generated misconceptions and fears. to that end, a recent survey, conducted on a german cohort of ibd patients to investigate their perception of the emergency and their medication compliance, demonstrated that the fear was more pronounced in patients taking immunosuppressants. in particular, they were concerned about interactions between medication and covid- . nevertheless, . % of patients adhered to their medication schedule. therefore, the rapid countermeasures adopted at many ibd centers, including ours, such as the implementation of telemedicine and distance education allowed to reassure ibd patients about the lack of risk related to covid- infection. , for instance, at our center, one week before the infusion, patients were contacted by email to reassure about the lack of risk in moving to the hospital and the benefit of continuing medical therapy. moreover, the day before the infusion each patient was contacted by phone in order to confirm the appointment and to explain the measures adopted to reduce the risks for all the patients entering to the hospital (i.e. triage for potential covid infection). finally, a mobile phone number was provided to the patients in case of doubts or concerns. these experiences teach that a greater contact and dialogue with ibd patients is important to reassure them and also to provide the correct information and psychological support. we should explain to our patients with chronic diseases why it is important that they adhere to therapies which are relative safe and manageable despite the covid- pandemic. adherence of infusible biologics during the time of covid- among patients with inflammatory bowel disease: a nationwide va cohort study no need of transforming gastroenterology units to covid units at the time of sars-cov infection-a single-center analysis from northern italy. digestive and liver disease knowledge and perceptions of covid- among the general public in the united states and the united kingdom: a cross-sectional online survey pandemic-the patients 'perspective medical and gastroenterological education during the covid- outbreak safety of drugs during previous and current coronavirus pandemics: lessons for ibd. journal of crohn's and colitis key: cord- -a ie fs authors: nan title: digestive system, liver, and abdominal cavity date: - - journal: the cat doi: . /b - - - - . - sha: doc_id: cord_uid: a ie fs nan these complex pathways highlight the need to consider the whole cat and not just the cat's gastrointestinal vomiting can be defined as the ejection of part or all of the contents of the stomach and/or upper intestine through the mouth, usually in a series of involuntary spasmodic movements. the disturbances in gastrointestinal (gi) motility are coordinated with respiratory and abdominal muscle contractions and mediated by the central nervous system (cns). vomiting begins with retching, a series of brief negative intrathoracic pressure pulses that coincide with positive abdominal contractions. these pressure changes occur as a result of repeated herniations of the abdominal esophagus and cardiac portion of the stomach into the esophagus. during retching, food freely moves back and forth in the esophagus, which is now dilated because of the ingesta. ultimately, the diaphragm rapidly moves cranially, resulting in positive intrathoracic pressure that leads to expulsion of these contents. vomiting is such an active process that it seems to involve the whole cat, and so it is little wonder that it concerns owners so much. since vomiting is mediated by the cns with input and influence from just about anywhere in the body, it is important to summarize this physiology so it can be appreciated when managing clinical cases. vomiting results from stimulation of the "vomiting center," which is located in the brainstem; there are four main pathways that stimulate the vomiting center, and these are summarized below and in figure (though not all older cats have grown out of this habit). some extragastrointestinal problems, such as hyperthyroidism and renal disease are more likely to occur in older cats. most texts and references instruct clinicians to distinguish between vomiting and regurgitation, with the latter noted as being quite passive. , , in practice, it can be hard to make this distinction, because it is the author's experience that cats with esophageal disease can have quite forceful, spasmodic movements when ejecting ingesta by regurgitation-although it is also possible for regurgitation to be a passive process. given that the physiology of vomiting, as described above, results in ingesta being forced to and then evacuated from the esophagus, it is hardly surprising that it can resemble regurgitation. fortunately, regurgitation and esophageal disease do vary from vomiting in other ways! vomiting . blood and urine testing . imaging (radiography, ultrasonography) . biopsy samples . treat and manage underlying problem the decision to proceed to steps and is based on the assumption that the prior steps have narrowed down the underlying cause as gastrointestinal, pancreatic, or hepatic in origin. the important aspects of the clinical history are given in chronic kidney disease. the author has found that some cats with dental disease can gorge their food, resulting in vomiting; so, paying attention to the state of the teeth and gums is important. of course, some cats have multiple problems, and correction of dental disease may not resolve vomiting if there is another process. in the examination, it is also important to note consequences of both the underlying process and the vomiting itself; these include the demeanor of the cat, hydration status, and abdominal pain. the physical examination findings, together with the clinical history, help determine the next appropriate steps. well cats that are not continually vomiting and are appropriately hydrated, with no other specific signs, may be treated as outpatients by fasting them for hours, then returning to food with a bland diet, such as plain cooked chicken or commercial, low-residue prescription diets designed for this purpose. follow-up is important to ensure signs do not progress. cats with nonspecific signs may require supportive care with subcutaneous or intravenous fluids and perhaps analgesia (with opioids). if clinical signs do not resolve, the pursuit of a specific diagnosis should be attempted. the practitioner must ask the following important questions: • are ancillary tests appropriate? • is supportive care necessary? • are any medications required? routine serum/plasma biochemistries, hematology, urinalysis, and total thyroxine (t ) (for older cats) testing is not only important to distinguish primary from secondary gastrointestinal disease but to look for consequences of vomiting that may need to be addressed, such as hydration status and electrolyte abnormalities. careful interpretations should be made. severe azotemia, even with hyperphosphatemia, can occur as a result of primary gastrointestinal disease, and the distinction from renal disease usually requires an assessment of urine specific gravity. cobalamin, folate, feline trypsin-like immunoreactivity (ftli), and feline pancreatic lipase immunoreactivity (fpli) tests are useful markers of intestinal and pancreatic disease, , , , but it is important to note that they mostly do not give a precise diagnosis. more detail about the utility of these tests is noted below in the section approach to the cat with diarrhea. is usually preceded by the cat licking its lips, salivating, or making attempts to swallow. regurgitated ingesta is often in a tubelike structure and if undigested can be covered with frothy saliva. partially digested food suggests vomitus, and the presence of bile or digested blood confirms this. it is important to determine if the cat vomits regularly. many owners have seen their cats vomit on a regular basis with no evidence of the cat being unwell, and this is noted frequently in the veterinary literature. , hairballs can cause gastric irritation, and it may be that eating quickly also stimulates the peripheral sensory receptors that contribute to vomiting. if a cat does vomit regularly, it is important to assess if the cat is presenting for a change in the vomiting pattern (e.g., frequency or timing in relation to eating) and if the cat is unwell in any way, such as anorexia or weight loss. the pattern of vomiting is important in all cases, because cats presenting with acute gastritis usually have a sudden onset of frequent vomiting compared with those with chronic disease processes that may vomit every few days. the timing in relation to eating can be helpful, because the stomach should empty by to hours after a meal; so, vomiting longer than hours after a meal can suggest motility or retention disorders. the description of the vomitus can be helpful. if bile is present, the pylorus is not obstructed; the presence of blood (digested or fresh) indicates ulceration. hair in the vomitus can indicate hairball gastritis, and the possibility of trichobezoar obstruction should be considered. access to foreign bodies or toxins is an important aspect of the clinical history. has the cat been seen playing with an insect, mouse, or other prey? are there any medications unaccounted for (e.g., a dropped aspirin tablet)? are lilies present in the house? vomiting is the major sign of gastric disease, but given the number of potential organ systems that can be involved, a thorough physical examination should be undertaken. because linear foreign bodies are a common cause of vomiting, all cats presenting for anorexia or vomiting should have the underside of the tongue evaluated for the presence of string caught there. applying gentle pressure with a thumb in the intermandibular space to elevate the tongue is an effective way to visualize lesions or foreign bodies in the sublingual area (see . a thorough examination may reveal specific signs, such as a palpable thyroid nodule and tachycardia in the case of hyperthyroidism or palpably small kidneys with and analgesia, many cats recover uneventfully. one survey assessed that % of cats undergoing exploratory laparotomy survived the hospitalization, and although complications occurred in % of cats, these were more likely to be associated with the underlying disease process and not surgery or anesthesia. laparoscopy is not readily available in all veterinary clinics. this alternative is less invasive and allows exploration of the abdomen but not as thoroughly as with laparotomy. organs are usually exteriorized for biopsy. there is the possibility of anesthetic complications associated with insufflating the abdomen. endoscopy is the least invasive procedure and is the only alternative that allows examination of the intestinal lumen. this option limits the parts of the gastrointestinal tract that can be biopsied; it does not allow examination or sampling of any other part of the gastrointestinal tract and does not enable full-thickness biopsy samples. one study found that, of cats investigated for gastrointestinal disease, of cats ( %) had no pathology recognized proximal to the jejunum (i.e., the effective length of diagnostic endoscopes would have precluded diagnosis), and other organs were affected in of cats with inflammatory bowel diseases and of cats with intestinal small cell lymphoma. careful case selection for endoscopy from survey ultrasonography can reduce the number of missed diagnoses from endoscopy, but the possibility still remains. the quality of endoscopically obtained biopsy samples varies greatly with the skill of the endoscopist. it has been stated that "it is exceedingly easy to take inadequate tissue samples with a flexible endoscope." in an assessment of endoscopically obtained biopsy samples, two laboratories were compared, one that received samples from any practitioner and the other that received samples only from practitioners trained to take, mount, and submit endoscopy samples. all slides were reviewed by three pathologists who found that, of samples from the first laboratory, % of the slides were considered inadequate for diagnosis, % were considered questionable, and only % were adequate. by comparison, in the second laboratory (with samples from experienced practitioners) % of slides were inadequate, % were questionable, and % were considered adequate for diagnosis. in the case of distinguishing between lymphocytic intestinal infiltrates (commonly known as inflammatory bowel disease) and lymphocytic neoplasia (small cell lymphoma), endoscopically obtained samples can give an incorrect diagnosis. many of these problems can be minimized with experienced operators and careful case selection from prior ultrasonography. radiography is most useful for identifying foreign bodies or signs of intestinal obstruction from other causes. the major findings are noted below in the section intestinal obstruction. contrast radiography can aid the diagnosis for both discrete and linear foreign bodies but should be used with caution, because intestinal perforation may be present. nonionic iodinated agents that are typically used for myelography (such as iopamidol or iohexol) should be used, since barium irritates the peritoneum and oral iodine compounds are hypertonic. hypertonic compounds may draw fluid into the stomach and intestines after oral administration, with the potential of creating further fluid and electrolyte imbalances in an already compromised patient. ultrasonography is a useful diagnostic adjunct and helps to detect and characterize localized thickening of the stomach or intestinal wall, lymphadenopathy, radiolucent foreign bodies, and changes in the size and echogenicity of the pancreas, liver, kidneys, or spleen. abdominal effusions can be assessed and sampled. ultrasound-guided fine-needle aspiration can be used to sample masses, bile, or peritoneal fluid. it should be recognized that in most cases of gastrointestinal disease, imaging will not give a definitive diagnosis and biopsy will be required, usually using either endoscopy or laparotomy. ultrasonography can be a considered as a means to "survey the field," assessing • the nature of the underlying disease, such as • thickened intestines with or without discrete layers • lymph node involvement • other organ involvement • the location of disease, for example, • diffuse or focal • proximal duodenum (reachable by endoscope) versus distal ileum these factors may be used to assess the appropriateness of endoscopy versus laparotomy to obtain diagnostic samples. most commonly used antiemetics all control vomiting by different mechanisms and include mirtazapine, metoclopramide, dolasetron/ondansetron, maropitant, and the phenothiazines (tables - and - ) . metoclopramide functions both as an antiemetic and prokinetic in cats, while cisapride functions solely as a prokinetic. mirtazapine, a piperazinoazepine, antagonizes the presynaptic alpha -adrenergic receptor, increasing noradrenergic and serotonergic neurotransmission; the primary mechanism targeted for its use is as an antidepressant in humans. mirtazapine is also a potent antagonist of the postsynaptic serotonergic receptors ( -ht and -ht ) and histamine h receptors. because of its antiserotonergic and antihistaminic effects, mirtazapine is used as an entiemetic and appetite stimulant in cats. anorexia is a common clinical problem in ill cats, and in some anorexic or partially anorexic cats the use of an appetite stimulant as adjunctive therapy to nutritional support (i.e. feeding tubes) may be of clinical benefit. prior to the development of mirtazapine, cyproheptadine was used as an appetite stimulant in cats, with variable clinical results. recently, the pharmacokinetics and pharmacodynamics of mirtazapine have been reported in cats. in a group of healthy cats, mirtazapine was found to be an effective appetite stimulant, with a shorter half-life than that reported in humans. the recommended oral dose is . mg/cat every hours. a in humans, age and kidney and liver dysfunction affect mirtazapine metabolism (hepatic cyp enzymes) and clearance (excreted in urine and feces), suggesting that dose adjustment may be necessary. a side effects reported in cats treated with mirtazapine include behavior changes (vocalization and interaction), tremors, muscle twitching, and hyperactivity. a, a metoclopramide is both an antiemetic and prokinetic drug that acts peripherally on the gastrointestinal tract and centrally within the central nervous system (cns). at low doses metoclopramide inhibits dopaminergic (d ) transmission, and at higher doses it inhibits serotonergic -ht receptors in the chemoreceptor trigger zone (crtz). , metoclopramide also acts peripherally as a prokinetic at the level of the gastrointestinal smooth muscle of the stomach and duodenum, triggering gastric emptying and duodenal contractions. multiple mechanisms mediate metoclopramide's prokinetic activity, including augmentation of acetylcholine release and increased smooth muscle sensitivity to cholinergic neurotransmission, which may in part be because of antagonism of dopamine, but more recently, serotonergic ht receptor activation has been suggested. , metoclopramide has been reported to increase the lower esophageal sphincter tone in humans, although in cats metoclopramide's affect on the lower esophageal sphincter is reported to be weak. adverse central nervous system, extrapyramidal signs occur secondary to dopamine (d ) antagonism, including excitement and behavior changes. extrapyramidal signs are most often seen at the higher doses needed to block -ht receptors. because of metoclopramide's prokinetic properties, an intestinal obstruction should be ruled out prior to its use. dopamine is a less important neurotransmitter in the chemoreceptor trigger zone of cats than alpha adre nergic and -ht -serotonergic receptors, suggesting that d -dopaminergic antagonist may be a less effective antiemetic in cats. clinically metoclopramide commonly controls vomiting in cats, although this clinical response may be secondary to -ht antagonism and/ or its prokinetic effects. , extrapolated from the short elimination half-life of metoclopramide in dogs ( minutes), frequent be clinically significant side effects. phenothiazines have the potential to lower the seizure threshold; their use is not recommended in patients with a known seizure history. other cns-associated side effects linked to d antagonism occur at higher doses and produce extrapyramidal signs, including rigidity, tremors, weakness, and restlessness. antagonism of the histaminergic receptors carries the risk of sedation. because of the need for frequent dosing ( . to . mg/ kg subcutaneously every hours) and the risk of hypotension and sedation, the clinical use of phenothiazine antiemetics is limited to hospitalized patients with refractory vomiting and should be avoided in patients who are dehydrated or hypotensive. cisapride is a serotonergic -ht agonist that increases propulsive gastrointestinal motility from the lower esophageal sphincter to the colon. cisapride binds serotonergic -ht receptors in the myenteric plexus, increasing the release of acetylcholine in gastrointestinal smooth muscle. in dogs cisapride has greater prokinetic activity in the stomach relative to metoclopramide. cisapride has no direct antiemetic effect, although it is indicated in a vomiting cat with colonic dysmotility secondary to megacolon. colonic distention can trigger the vomiting reflex in cats. cisapride induces colonic smooth muscle contractions in cats with megacolon that is dependent on the influx of extracellular calcium and is only partially cholinergic dependent. other potential indications include refractory generalized ileus or gastroesophageal reflux. dosage recommendations based on the pharmacokinetics in healthy cats is . mg/kg orally every hours. prior to the use of cisapride, an intestinal obstruction should be ruled out because of its strong prokinetic effects. side effects reported in humans are cramping and diarrhea. potentially life-threatening side effects include qt prolongation and ventricular arrhythmias, the primary concern in humans that led to cisapride's removal from the market in the united states. in cats qt prolongation associated with cisapride administration requires times the therapeutic dose. because of the risk of prolongation of the qt interval and ventricular arrhythmias, the concurrent use of cisapride and dolasetron is not recommended. other potential drug interactions associated with cisapride include concurrent therapy with azole antifungals (ketoconazole and itraconazole), because of their inhibition of hepatic cyp a isoenzyme system and the inhibition of cisapride metabolism. diet trials are commonly used in cats with idiopathic gastrointestinal signs or in cats with suspected or known intermittent dosing or delivery by a constant rate infusion (cri) is necessary. empirical dosing in cats is . to . mg/kg subcutaneously or orally every hours or to mg/kg/day as a cri. approximately % of metoclopramide is excreted in the urine, thus dose reduction is recommended in cats with underlying renal azotemia. dolasetron and ondansetron are selective serotonin antagonists that inhibit central and peripheral -ht receptors. their main antiemetic effect is through antagonism of the peripheral -ht receptors in the gastrointestinal tract. in cats -ht antagonism of the crzt is also likely important in the antiemetic effect of dolasetron and ondansetron. dolasetron and ondansetron were originally used for vomiting secondary to chemotherapy because of their superior clinical efficacy. the clinical use of dolasetron and ondansetron in cats has not been associated with reported side effects, and experimental studies report minimal toxicity in animals at doses times the antiemetic dose. side effects reported in humans include headaches, elevated liver enzymes, rare hypersensitivity reactions, prolongation of the qt interval, and arrhythmias. , dolasetron is commonly used for parenteral administration and ondansetron for oral administration, dictated primarily based on the tablet sizes available and cost. recommended dosing of dolasetron is . to mg/kg intravenously every hours and ondansetron . mg/ kg orally every hours. maropitant is a neurokinin- (nk- ) receptor antagonist, blocking the binding of substance p to the nk- receptors located in the emetic center, crtz, and the enteric plexus. in cats maropitant has been reported to be efficacious in treating xylazine-induced vomiting and motion sickness. recommended dosing in cats is mg/ kg intravenously, subcutaneously or orally every hours for up to days. maropitant is reported to be well tolerated in cats. prochlorperazine and chlorpromazine are considered broad-spectrum antiemetics by antagonism of d dopaminergic, histaminergic (h and h ), and cholinergic (muscarinic) receptors within the crtz and, at high doses, the alpha-adrenergic receptors (alpha and alpha ) within the vomiting center. in cats alpha -receptors play a key role in emesis (recall xylazine is the emetic of choice in cats), suggesting cats may be more sensitive to the antiemetic effects of the phenothiazines. prochlorperazine and chlorpromazine produce an antiemetic effect at relatively low doses, thus avoiding profound sedation; although, because of antagonism of the alpha-receptors, vasodilation and hypotension can hyperacidity alone is not considered a common cause for vomiting in cats, but famotidine is effective in treating vomiting in cats associated with gastric ulcers or gastritis. recommended dosage in cats is . mg/kg every to hours. ranitidine is also a competitive inhibitor of the h receptor associated with gastric parietal cells. in addition, ranitidine increases lower esophageal sphincter tone and functions as a prokinetic agent (increasing gastric emptying and stimulating intestinal motility, including colonic motility), because of its anticholinesterase food hypersensitivities. dietary strategies used to control vomiting in cats focus on either a highly digestible diet or an elimination (novel protein/carbohydrate or hydrolyzed protein) diet. the empirical use of elimination diets in cats is reported to be relatively successful, with approximately % of cats with idiopathic gastrointestinal signs responsive to a novel protein/carbohydrate diets within to days. interestingly, traditional diet trials are recommended for a minimum of to weeks, but in this group of diet-responsive cats with chronic gastrointestinal disease, clinical improvement was reported within days. thus if a cat is going to be diet responsive, clinical improvement to a diet trial should be noted relatively early. highly digestible diets enable more effective absorption and assimilation of nutrients in the face of a compromised digestive tract. these diets contain highly digestible proteins and carbohydrates, moderate to low fat, soluble fiber but low concentrations of insoluble fiber, and are supplemented with omega- fatty acids. these diets are recommended when food allergy or intolerance is suspected. these diets contain a single highly digestible novel carbohydrate source and novel protein source. alternatively, diets formulated with hydrolyzed proteins can be used as an alternative to novel protein/carbohydrate diets. see tables - and - for information on gastrointestinal ulcers. famotidine has no direct antiemetic effect but is a competitive inhibitor of the histamine (h ) receptors associated with the gastric parietal cells. the h -receptor is the dominant receptor involved in gastric acid secretion. h receptor antagonism is reported to result in a % to % reduction in acid production. famotidine is more effective at suppressing gastric acid secretion relative to ranitidine. famotidine is well tolerated, although, with chronic therapy, there is the potential for hypoacidity and gastric bacterial overgrowth. in humans dose reduction is recommended in association with renal dysfunction. famotidine is not an inhibitor of the hepatic microsomal cytochrome p- enzyme system, therefore significant drug interactions are not anticipated. activity. , significant drug interactions associated with hepatic microsomal cytochrome p- enzyme system inhibition are not a clinical concern with ranitidine. an adverse effect to be aware of in cats treated with ranitidine is transient hypotension associated with ranitidine administered as an iv bolus. in humans dose reduction is recommended in patients with renal azotemia. ranitidine is effective in decreasing gastric acid in cats. ranitidine would be a logical choice in a cat with gastrointestinal ulceration and/or atony. the reported dosage recommendation for ranitidine in cats is . mg/ kg orally every hours or . mg/kg intravenous every hours. omeprazole omeprazole is a proton pump inhibitor that targets the h + /k + atpase pump on the luminal surface of partial cells. omeprazole is effective at suppressing parietal cell acid secretion, and its effects persist for ≈ hours after drug withdrawal because of drug accumulation in the parietal cell (by ion trapping). indications for omeprazole therapy are for the treatment and prevention of nonsteroidal antiinflammatory drug (nsaid)-induced ulcers. omeprazole is enteric coated to prevent its degradation by gastric acid; therefore oral formulations should not be crushed. based on human studies, omeprazole is a hepatic microsomal cytochrome p- enzyme inhibitor with known drug interactions with diazepam. the extent of clinically significant drug interactions in cats has yet to be studied. omeprazole is reported to be effective in reducing gastric acid secretion in cats. the recommended empirical dosage in cats is . to mg/kg orally once daily. long-term use in humans and dogs is associated with gastric polyps and parietal cell hyperplasia, respectively, but the effect of long-term use in cats is currently unknown. sucralfate is a disaccharide complexed with aluminum that dissociates to sucrose octasulfate and aluminum hydroxide upon exposure to gastric acid. the sucrose octasulfate spontaneously polymerizes, producing a viscous material capable of binding ulcerative lesions in the gastric mucosa. once bound to the exposed mucosa, it prevents back diffusion of h + , inactivates pepsin, absorbs bile acids, and increases mucosal prostaglandin synthesis, collectively supporting ulcer healing. sucralfate is not systemically absorbed but does prevent the absorption of drugs capable of chelating with aluminum, including fluoroquinolones, tetracyclines, and digoxin. if sucralfate is indicated in a cat being treated concurrently with fluoroquinolones, tetracyclines, or digoxin, the recommendation is to administer the other drug hours prior to the administration of sucralfate to optimize drug absorption. clinical indications for the use of sucralfate in cats are for the treatment of gastric ulcers and esophagitis. dosage recommendation in cats is mg orally every hours. sucralfate can be crushed, suspended in water, and administered as slurry. diet trials are used in some cats with diarrhea if the underlying cause is from known or suspected food hypersensitivities. dietary management includes either a highly digestible diet, an elimination (novel protein/ carbohydrate or hydrolyzed protein) diet (see above for both), or a diet high in fiber. high-fiber diets contain a mixture of both soluble and insoluble fiber that can be beneficial in patients with signs of large bowel diarrhea. insoluble fiber, such as cellulose, functions to increase the bulk of the stool, bind fluid, and regulate intestinal motility. soluble fiber, including fruit and vegetable pectins and beet pulp, functions as a source of butyric acid that can be used by the colonic mucosa and decreases proinflammatory cytokines. , cobalamin cobalamin (vitamin b ) is an essential vitamin needed by a number of different enzymes, including key enzymes involved in methionine metabolism and the conversion of methylfolate to tetrahydrofolate needed for dna synthesis. cobalamin and folate are intimately linked, and hypocobalaminemia can lead to a functional deficiency of folate. ingested cobalamin requires intrinsic factor binding for enterocyte absorption at the level of the ileum. hypocobalaminemia is commonly associated with distal small intestine diseases in cats, including inflammatory bowel disease. in addition, low cobalamin has a negative impact on enterocyte function; therefore in many cats with intestinal disease and hypocobalaminemia, cobalamin supplementation is necessary for resolution of clinical signs. , quantification of serum cobalamin levels is recommended in cats with clinical signs of small bowel diarrhea, ones suspected to have an infiltrative disease of the small intestine (inflammatory bowel disease or gastrointestinal lymphoma), or ones with pancreatic dysfunction. when hypocobalaminemia is identified, supplementation is recommended mannanoligosaccharides, inulin, chicory, and lactosucrose. reports on the use of prebiotics in cats are limited to their use in healthy cats; healthy cats fed fructooligosaccharides were reported to have a trend toward an increase in fecal concentrations of lactobacilli and a decrease in concentration of c. perfringens and e. coli relative to the controls. to date no reports are available on the use of prebiotics in cats with gastrointestinal disease. probiotics and prebiotics potentially have a supportive role in the treatment of gastrointestinal disease in cats. the important clinical consideration in the use of probiotics as an adjunctive therapy is to ensure the use of live nonpathogenic microorganisms that have been documented to colonize the intestinal tract of cats. gastrointestinal flora co-evolve with their host. gastrointestinal microorganism colonization varies among species and within each individual animal. the distribution of fecal microflora for a given individual is considered unique but stable over time. antimicrobial and antiparasitic therapies for the treatment of feline diarrhea are indicated based on the specific diagnosis of infectious diarrhea, bacterial enteritis, or as adjunctive therapy for inflammatory bowel disease. infectious pathogens more commonly associated with feline diarrhea include bacterial enteropathies (clostridium, campylobacter), protozoal enteropathies (tritrichomonas foetus, giardia spp.), and helminthic enteropathies associated with ascarids, hookworms, whipworms, and tapeworms. only the more common anthelminthic, antimicrobial, and antiprotozoal therapies are discussed below (tables - and - ) . more information about antimicrobials and antiparasitics is found under specific infections in the discussions of infectious enteritis and gastrointestinal parasites. fenbendazole is an anthelmintic used to treat common helminth infections, including ascarids, hookworms, whipworms, and a single species of tapeworm, taenia pisiformis. giardia spp. are also considered susceptible to fenbendazole. fenbendazole binds beta-tubulin subunits of microtubules, interfering with their polymerization. side effects include vomiting and diarrhea, although both are considered rare. fenbendazole is not approved for use in cats in north america but is commonly used clinically, and an empirical dosage of mg/kg ( µg/cat every days) while the underlying cause of cat's malabsorption is being investigated and at initiation of targeted therapy. probiotics probiotics are ingested live microorganisms intended to benefit the host, specifically to support the microflora environment of the gastrointestinal tract as well as to provide an overall benefit to the body's immune function by immunomodulation. , , probiotics chemically modify ingesta and intestinal mucus, as well as affect immune cells, enterocytes, and goblet cells within the intestinal mucosa through direct receptor interactions and indirectly through the action of cytokines. the microorganisms commonly used are nonpathogenic bacteria and yeast that have a vital role in gastrointestinal health, including lactobacillus spp., enterococcus faecium, bifidobacterium spp., and saccharomyces spp. for example, lactobacilli synthesize b vitamins, digestive enzymes, and folate coenzymes. clinical indications for the use of probiotics are diverse, including primary gastrointestinal disease, chronic renal disease, and pancreatitis. the rational use of probiotics in the treatment of gastrointestinal diseases include their ability to modulate gastrointestinal flora, minimize colonization by pathogenic bacteria, and decrease the likelihood of bacterial translocation. in healthy cats, lactobacillus acidophilus is reported to reduce fecal clostridium counts. when lactobacillus acidophilus was used adjunctively with antimicrobial therapy, fecal shedding of campylobacter was reduced in cats with campylobacter-induced diarrhea relative to cats treated with antimicrobials alone. specifically, in cats with gastrointestinal disease, available research supports the probiotic enterococcus faecium as clinically beneficial in resolving diarrhea in kittens. relative to the control group, the kittens treated with probiotics had increased fecal bifidobacteria and blood iga concentrations and decreased fecal counts of clostridium perfringens. prebiotics are dietary supplements used to select for the more beneficial enteric flora, support gastrointestinal function, and prevent the overgrowth of pathogenic bacteria, including salmonella, escherichia coli, clostridium, or campylobacter. for a food additive to be considered a prebiotic, it must be nondigestible by the gastrointestinal tract (resistant to gastric acidity, gastrointestinal hydrolysis and absorption), yet fermentable by gastrointestinal microflora to short-chain fatty acids to stimulate the growth of "good" intestinal bacterial. prebiotics include nondigestible oligosaccharidescommonly, oligofructose, fructo-oligosaccharides, pyrantel pamoate is a nicotinic anthelmintic used primarily for the treatment of ascarids, but its spectrum of activity also includes hookworms and the stomach worm, physaloptera spp. pyrantel is toxic to susceptible parasites through its selective action on their nicotinic acetylcholine receptors, resulting in depolarization and spastic paralysis. pyrantel is not approved for use in cats but is considered safe in cats and is commonly used clinically. the dosage recommendation in cats is mg/ kg orally once, repeat in weeks, and finally repeated in months. metronidazole is a nitroimidazole antibiotic with an anaerobic antibacterial spectrum with antiprotozoal activity against giardia spp. in an anaerobic environment, metronidazole is converted to unstable intermediates (nitroso free radicals) that disrupt bacterial dna synthesis. immunomodulatory properties capable of inhibiting cell-mediated immunity have been described for metronidazole, although its immunomodulatory properties are reported at dosages well beyond what is recommended for clinical use, raising questions about the clinical use of metronidazole as an adjunctive therapy for treating inflammatory bowel disease. , resistance to metronidazole is considered rare. the most common adverse reaction is gastrointestinal upset, including inappetence, anorexia, nausea, and vomiting. profuse salivation can occur in cats after oral administration of metronidazole base (formulation used in standard tablets), which has lead to the use of metronidazole benzoate (a compounded formulation not approved by the food and drug administration) in some cats because of its better oral palatability. at high doses (> mg/ kg/day) benzoic acid is reported to be neurotoxic in cats, but with appropriate clinical dosing of metronidazole benzoate benzoic acid toxicity is unlikely. dose-related metronidazole toxicity in cats results in cerebellovestibular ataxia secondary to gamma-aminobutyric acid (gaba) inhibition at dosages greater than or equal to mg/kg/day , ; clinical signs include nystagmus, head tilt, ataxia, seizures, and obtundation. in cats with inflammatory bowel disease, the dosage recommendation for the metronidazole base is to mg/kg/day. metronidazole benzoate contains approximately % metronidazole base by weight, translating to an empirical dosage of mg/kg/day of metronidazole benzoate (equivalent to . mg/kg/day of metronidazole base). little is known about the safety of chronic metronidazole use in cats, but oral metronidazole has been reported to disrupt dna within feline peripheral mononuclear cells following days of therapy. this metronidazole-induced genotoxicity is reversible and is no longer detected days after antibiotic therapy is discontinued. ronidazole is a nitroimidazole antibiotic (similar to metronidazole) and available as a powder-on-feed antibiotic. ronidazole is not approved for use in cats but has immunosuppressive therapies used in cats with inflammatory bowel disease include glucocorticoids, cyclosporine, and chlorambucil (tables - and - ). more information on the treatment of inflammatory bowel disease is found elsewhere in this chapter. glucocorticoids are considered first-line therapy in the treatment of cats with inflammatory bowel disease. glucocorticoids bind their intracellular glucocorticoid receptors, modifying the expression of genes with glucocorticoid response elements. immunomodulation is achieved through inhibition of cytokine release and response, including decreasing leukocyte phagocytosis, chemotaxis, and antigen expression. the more common side effects in cats include gastrointestinal ulceration, opportunistic infections (e.g., urinary tract infections), pancreatitis, and diabetes mellitus. cats are less susceptible to iatrogenic hyperadrenocorticism than dogs. initial therapy is usually with oral prednisone or prednisolone. prednisone is a prodrug that is metabolized to its active form prednisolone. cats are reported to be less efficient in the conversion of prednisone to prednisolone ; therefore prednisolone may be preferred in cats, especially in cats refractory to prednisone therapy. been used off-label to effectively treat tritrichomoniasis in naturally and experimentally infected cats ( mg/kg orally every hours for days). t. foetus reduces nitroimidazoles to their nitroso free radicals. ronidazole has been reported to have better in vitro and -fold higher in vivo activity against t. foetus relative to metronidazole. , , ronidazole resistance is beginning to be reported in t. foetus isolates from cats with diarrhea. side effects include hepatoxicity and neurotoxicity. neurotoxicity is associated with high doses and has been reported in cats. the use of ronidazole is recommended only for confirmed cases of t. foetus, and dosing should not exceed mg/kg once daily in cats, especially in cats at risk for neurotoxicity. ronidazole is not registered for human or veterinary use in the united states; therefore its use in cats requires owner informed consent and client education of the potential human hazards. immunosuppressive therapies are considered the standard of care for cats with gastrointestinal biopsies consistent with inflammatory bowel disease (lymphoplasmacytic or eosinophilic inflammation). the common alternative forms of glucocorticoids can be considered in specific patient populations. in patients with severe malabsorption, injectable dexamethasone may provide improved bioavailability and clinical response. also dexamethasone maybe preferred in patients with a history of heart failure, fluid retention, or hypertension because of its lack of mineralocorticoid activity relative to prednisone/prednisolone. dexamethasone's potency is to times that of prednisolone; therefore a dose reduction is necessary when prescribing dexamethasone (the dexamethasone dose is one seventh that of prednisolone). , budesonide is an oral, locally active, highpotency glucocorticoid that is formulated to be released in the distal gastrointestinal tract (based on the ph differential between the proximal and distal small intestine), where it is absorbed and is locally immunomodulating at the level of the enterocyte. the amount of systemically absorbed budesonide is minimized, because % to % of the budesonide absorbed from the gastrointestinal tract undergoes first-pass metabolism in the liver. some systemic absorption does occur, as evidenced by a blunted adrenocorticotropic hormone (acth) stimulation test in dogs treated with budesonide at mg/m for days. , the use of budesonide in cats remains anecdotal, with a suggestive empirical dose of . to mg/cat/day. initial glucocorticoid therapy for cats with inflammatory bowel disease consists of antiinflammatory ( . to mg/kg/day) to immunosuppressive ( to mg/kg/ day) dosages, with dosages based on the potency of prednisone/prednisolone. the goal of therapy is to achieve clinical remission and slowly taper the dose of glucocorticoids to the lowest dose that will control the cat's clinical signs. some cats may be completely weaned off therapy, while others require long-term lowdose therapy. the tapering of therapy should be slow, with a % to % dose reduction every to weeks. cyclosporine is considered a second-tier immunosuppressive drug used to treat inflammatory bowel disease in cats. use of cyclosporine in the treatment of diarrhea associated with inflammatory bowel disease in cats is extrapolated from its use in dogs to treat glucocorticoid refractory inflammatory bowel diarrhea. cyclosporine suppresses t-lymphocyte-mediated inflammation in the gastrointestinal tract secondary to suppression of inflammatory cytokines. specifically, cyclosporine attenuates t-lymphocyte activation and proliferation through the inhibition of interleukin- (il- ) production. side effects of cyclosporine in cats include dose-dependent inappetence and vomiting, which may occur at the onset of therapy and are generally responsive to dose reduction. other less common side effects reported in cats are opportunistic infections, including toxoplasmosis and hepatoxicity. the microemulsion formulation of cyclosporine has higher oral bioavailability and less variable pharmacokinetics. a suggested initial dosage of cyclosporine is mg/kg every or hours. serum cyclosporine levels can be used to monitor for excessive trough plasma concentration (> ng/ml) as determined using a highperformance liquid chromotography (hplc) analytical method. chlorambucil is a slow-acting nitrogen mustard that alkylates and effectively cross links dna, leading to altered protein production. the immunosuppressive effects of chlorambucil are the result of its cytotoxic effect on lymphocytes, similar to other nitrogen mustards. bone marrow suppression is considered mild to moderate and is rapidly reversible. neurotoxicity and myoclonus has been reported in a cat accidently overdosed with chlorambucil. chlorambucil is used as a second-tier drug in cats to treat immune-mediated disorders, in part because of ease of administration and its low risk of myelosuppression. for the treatment of inflammatory bowel disease, the recommended dosing in cats is mg/cat every hours in cats greater than kg and mg/cat every hours in cats less than kg. chlorambucil is commonly used in combination with glucocorticoids in the treatment of immune-mediated diseases, including inflammatory bowel disease, overlooked. awareness about feline esophageal diseases is low, the clinical signs are often not specific, and imaging beyond survey radiographs may be required for diagnosis. the esophagus is composed of four layers (from inner to outer): mucosa, submucosa, muscularis, and adventitia (there is no serosal layer). in the dog, the muscle layer is entirely composed of skeletal muscle, but in cats, the distal third of the esophagus is composed of smooth muscle. the upper esophageal sphincter prevents reflux of esophageal contents into the pharynx and minimizes aerophagia. the lower esophageal sphincter prevents gastroesophageal reflux and relaxes during swallowing to allow food and fluid to enter the stomach. clinical signs of esophageal disease include drooling, dysphagia, pain on swallowing (odynophagia), and, most classically, regurgitation. weight loss may occur secondary to inadequate food intake when disease is severe or chronic. other clinical signs, such as anorexia, cough, dyspnea, and fever, may occur if complications such as aspiration pneumonia or esophageal perforation occur. regurgitation is passive expulsion of food or fluid from the esophagus. the food is undigested and often accompanied by mucus and saliva. mucosal erosions may produce frank blood in the regurgitated material. regurgitation must be differentiated from vomiting (table - esophageal disease is uncommon in the cat when compared with dogs, but it is also likely that problems such as esophagitis and esophageal strictures are often salivation, retching, and abdominal contractions. the vomitus consists of partially digested food from the stomach and/or intestines and may be mixed with bilestained fluid. some cats will have both vomiting and regurgitation. expectoration may also be confused with vomiting or regurgitation. expectoration is associated with coughing, but cats that cough excessively may also stimulate vomition so that a careful history is needed to characterize the clinical signs correctly. coughing may also occur in cats that have aspirated as a result of regurgitation. drooling, dysphagia, and odynophagia are most commonly seen with conditions of the oropharynx and/or proximal esophagus. odynophagia is most commonly associated with esophagitis and foreign bodies. dysphagia and regurgitation together most commonly indicate oral or pharyngeal dysfunction; if regurgitation is not accompanied by dysphagia, esophageal dysfunction is likely. regurgitation in cats with esophageal disease is caused by obstruction or muscular dysfunction. causes of obstruction include vascular ring anomaly, foreign object, stricture, and neoplasia. causes of muscular dysfunction include congenital disease, esophagitis, myopathies, neuropathies, and dysautonomia. regurgitation may occur immediately after eating if the lesion is in the proximal esophagus. however, a dilated esophagus provides a reservoir for food and fluid so that regurgitation may not be associated in time with eating. young cats with signs of esophageal disease should be suspected of congenital defects, such as vascular ring anomaly, or a foreign body. adult cats with esophageal disease may have a recent history of general anesthesia, administration of certain oral medications, or ingestion of irritant chemicals. acute onset of clinical signs may suggest a foreign body, while chronic, slowly worsening signs may indicate a stricture or tumor. all cats suspected of esophageal disease should have a minimum database as part of the diagnostic plan (complete blood cell count, serum chemistries, urinalysis, and other tests as indicated by age or concurrent diseases, such as serum total t and blood pressure measurement). an important part of diagnosis is observation of the cat while eating food, to localize the location of the dysfunction. if the cat is unwilling to eat while in the veterinary clinic, the owner can make a video of the cat eating at home for the clinician to view. the general diagnostic approach to regurgitation in cats is found in figure - . plain and contrast radiography and endoscopy are important diagnostic tools for esophageal disease. fluoroscopy is valuable for the diagnosis of motility disorders, but availability is limited to universities and referral centers because of the cost of equipment. ultrasonography is limited to evaluation of * references , , , , , . the cervical esophagus and a small segment of abdominal esophagus between the cardia of the stomach and the diaphragm. the entire esophagus should be evaluated with cervical and thoracic radiographs. thoracic radiographs may also show evidence of complications such as aspiration pneumonia or esophageal perforation. the normal esophagus is not visualized on plain radiographs, but may be seen if food or fluid are retained or a foreign body or mass is present. radiographic contrast agents useful for esophagrams in cats include liquid or paste barium. a water-soluble iodinated contrast agent (e.g., iohexol, gastrografin) is preferred if there is any risk the esophagus is perforated, because these agents are less irritating and more rapidly reabsorbed. esophagrams are most useful for diagnosis of luminal obstructions, extraluminal compression, mucosal irregularities, and possibly alterations in motility. dilute liquid barium can be administered with a syringe or it may be mixed with canned food, especially if a motility disorder or stricture is suspected. multiple lateral radiographs are taken rapidly, starting within seconds of swallowing the contrast agent. contrast is rapidly cleared from the normal esophagus by peristalsis. if the contrast in the esophagus terminates abruptly, an obstruction is likely. if the contrast is retained throughout the esophagus, muscular dysfunction is suspected. some conditions, such as esophagitis, are difficult to diagnose radiographically, because contrast agents may or may not adhere to ulcerated mucosa. flexible endoscopy is a noninvasive diagnostic tool for esophageal disorders and is often used if plain and contrast radiographs have failed to establish a diagnosis. it is most sensitive for diagnosis of masses, ulcers, perforations, and obstructions. in addition, it is often possible to retrieve foreign bodies using endoscopy as well as to assist with dilatation of strictures or placement of gastrostomy feeding tubes if required. biopsy of the esophageal mucosa is more difficult than biopsy of gastric or intestinal mucosa and is not commonly performed with the exception of mass lesions. esophagitis may result from various causes of inflammation, such as contact irritation from foreign bodies (including trichobezoars lodged in the esophagus), chemical irritants or caustic medications, gastroesophageal reflux, persistent vomiting, hiatal hernia, or general anesthesia. inflammation disrupts the esophageal mucosa and exposes the submucosa. an important part of the treatment plan is identification and treatment of the underlying cause. clinical signs include dysphagia, regurgitation, salivation, and repeated swallowing, although signs may be absent in cats with mild esophagitis. cats with odynophagia may repeatedly extend the head and neck while swallowing. if the esophagitis or underlying disease is severe, weight loss and dehydration may occur secondary to anorexia. if the submucosa and muscularis are damaged, strictures may form as a result of the production of fibrous connective tissue and compromise the esophageal lumen. neoplasia is an important cause of esophageal stricture in humans, but not in cats. most cases have single strictures, but multiple strictures are possible. in two studies, the mean stricture diameter was reported as mm. , most strictures are less than cm in length. clinical signs associated with strictures appear to days after the esophageal injury and may be present for weeks before definitive treatment is pursued. regurgitation typically occurs immediately after eating, although if the stricture is long standing, a pouch may form cranial to the lesion where food accumulates. survey radiographs may be normal in cats with esophagitis and strictures, but are useful to rule out other causes for the clinical signs, such as a foreign body, or to detect related problems, such as aspiration pneumonia. in some patients, dilation of the esophagus with fluid or air may be seen. a contrast esophagram may disclose irregularities of the mucosa in cats with severe esophagitis. segmental dilation may occur with severe inflammation. strictures may be diagnosed with an esophagram (figure - ) ; however, in some cases, it may be difficult to differentiate a stricture from intramural thickening (e.g., because of neoplasia). endoscopy is useful for diagnosis of esophagitis; findings include mucosal erythema, hemorrhage, and erosions or ulcerations. if gastroesophageal reflux is present, the lesions will be most severe in the distal esophagus, and the lower esophageal sphincter may be dilated. endoscopy is often used for definitive diagnosis of esophageal stricture as well as to visualize the lesion during treatment by bougienage or balloon catheter dilation. strictures appear as a ring of white fibrous tissue that narrows the esophageal lumen. if endoscopy is performed after a barium esophagram, hours should be allowed to elapse between the procedures or the barium will obscure visualization with the endoscope. general anesthesia is an important cause of esophagitis (sometimes leading to stricture formation) in cats, probably because gastroesophageal reflux appears to occur commonly in anesthetized cats.* for example, in a series of seven cats with benign esophageal stricture, recent anesthesia for ovariohysterectomy was the suspected cause in five cases. clinical signs appeared up to days after anesthesia. abnormal esophageal tissue was performed in two cases. the authors noted that the esophageal mucosa may appear grossly normal, but submucosal inflammation may be found on histopathologic examination of biopsies. a consequence of chronic severe gerd in humans is the development of metaplastic columnar epithelium (barrett esophagus) that replaces the normal squamous epithelium. one case series reported on barrett-like esophagus in three cats. two cases were associated with hiatal hernia and one with cardial incompetence. drug-induced esophageal damage and stricture formation is well known in humans and cats (see . in humans over drugs have been implicated, and most are antibacterials or nsaids. implicated drugs in the cat include tetracycline, doxycycline, and clindamycin in tablet or capsule form administered without a food or water bolus. , , , , clinical signs (dysphagia, regurgitation, salivation, anorexia) appear to days after drug treatment is started. strictures commonly form in the midcervical esophagus or over the heart base in the thoracic esophagus. doxycycline hyclate is most commonly associated with esophageal strictures in cats, and the principle reason for its irritating properties is an acidic ph. the monohydrate salt of doxycycline is less irritating and is marketed as tablets and a palatable paste licensed for use in dogs and cats in some countries. in humans esophageal ulceration after doxycycline therapy is more common than stricture formation. although the development of strictures in cats would appear to be uncommon, it seems possible the incidence of esophagitis is underestimated, because the clinical signs (e.g., odynophagia, chest pain) may go unrecognized. esophageal transit studies of normal cats have shown that the passage time of dry-swallowed tablets and capsules is often prolonged (longer than seconds). , complete entrapment (retention for more than minutes) in the midcervical region occurs commonly. however, a small bolus of food or water is sufficient to ensure immediate passage of the medication into the stomach. , the risk of esophageal retention can also be lessened by coating a tablet or capsule with butter or a gel dietary supplement (nutri-cal; vétoquinol, fort worth, tex.). one study determined that tablets or capsules administered using a one-step pill gun with flavored liquid (flavorx pill glide; flavorx, columbia, md.) or a pill delivery treat (greenies pill pockets; nutro products, franklin, tenn.) ensured an average transit time of seconds or less. delayed esophageal transit of medications allows tablets and capsules to disintegrate within the esophagus, exposing the mucosa to irritating chemicals. cats may be at risk of delayed esophageal transit, because they do not typically drink water with medication, and they do not have an upright posture. in addition, medications are often given to sick or dehydrated patients many preanesthetic drugs and induction agents reduce lower esophageal sphincter pressure. , other predisposing factors may be intraabdominal surgery and a head-down position on the surgery table. reflux fluid with a ph less than is likely to cause esophageal mucosal damage, as is prolonged contact time. esophageal defense mechanisms include clearance of the reflux fluid by peristalsis and neutralization of the acidic ph by the bicarbonate present in saliva. in a study of kittens less than weeks of age, risk of gastroesophageal reflux during anesthesia was evaluated with use of a laryngeal airway mask versus endotracheal intubation. gastroesophageal reflux was observed in % of kittens with use of the laryngeal airway mask but more importantly in % of kittens with endotracheal intubation. the reflux episodes occurred shortly after anesthesia induction. in a study of cats anesthetized with thiopentone or propofol, gastroesophageal reflux occurred in %. reflux also occurred shortly after anesthesia was induced and lasted for a mean of minutes. it is unknown why esophageal strictures form only in a small number of cats that experience gastroesophageal reflux during anesthesia. gastroesophageal reflux disease (gerd) is a commonly reported cause of esophagitis in humans, but it is rarely reported in cats when not associated with general anesthesia. , the true incidence is unknown, and diagnosis may be hampered by scant knowledge about the clinical presentation and diagnosis. clinical signs and diagnostic procedures are as for other causes of esophagitis. in one case series of three cats, diagnosis of gerd was based on clinical signs, contrast radiography, and endoscopic findings. biopsy and histopathology of obtained from a compounding pharmacy in most countries and can only be given orally. h -receptor antagonists are competitive inhibitors that block parietal h receptors and decrease the amount of gastric acid produced. proton pump inhibitors are noncompetitive inhibitors that act on the h + /k + atpase enzyme system at the secretory surface of gastric parietal cells. they are considered superior for decreasing gastric acid secretion and are therefore the first choice, despite their greater cost. a drawback of proton pump inhibitors is that they must be administered orally. sucralfate may be beneficial for reflux esophagitis, because it binds to mucosal erosions in an acid environment and provides a protective barrier. it is given as oral slurry, ideally separate from meals or other medications. antibiotics are not commonly recommended unless aspiration pneumonia is present or the eroded mucosa is at risk of bacterial infection in a patient with severe disease or a compromised immune system. corticosteroids are often recommended for cats with esophagitis to reduce esophageal inflammation and impair the formation of fibrous connective tissue. however, the benefit of corticosteroids in cats with esophagitis has not been investigated and administration must be weighed against potential adverse effects, especially in patients with aspiration pneumonia. treatment of esophageal stricture typically requires dilation with either bougienage or a balloon catheter; both are used with endoscopic visualization under general anesthesia. appropriate analgesia should be provided, because dilating the stricture is painful. it does not appear that placement of a gastrostomy feeding tube is specifically required to recover from dilation procedures, although a tube may be placed in some anorexic cats to ensure nutritional intake and administer oral medications. a bougie is a long, narrow, oblong, mechanical dilator available in various sizes (typically -to -mm sizes are used in cats) that is gently passed through the stricture, usually over a guide wire. established criteria for selection of bougie diameter and dilation end points are not available. in one study, the initial bougie chosen was approximately the same size as the estimated diameter of the stricture, or no more than mm larger. once the first bougie is passed, subsequent bougies of increasing diameter are employed. two to four bougies of increasing size may be passed in a single session, with the goal of dilating the stricture without causing esophageal tear or perforation. determining when dilation should be stopped is a matter of clinical judgment. the procedure may be repeated as needed to maintain improvement; the total number of procedures required is variable. in one retrospective case series of eight cats treated with bougienage, the median number of procedures was . , and a good outcome was achieved in % of the cases. in some cases, the endoscope tip itself has been used for that may be at greater risk of esophageal retention of medication. all oral medication given to cats in tablet or capsule form should be followed with food or a liquid. mild esophagitis will resolve on its own, especially if an underlying cause can be removed or treated. frequent meals of canned food should be provided. cats with moderate to severe esophagitis will require medical therapy, and those with difficulty eating or weight loss may also require gastrostomy tube feeding. esophagostomy or pharyngostomy feeding tubes should be avoided in these patients. treatment is provided to control inflammation and promote healing while reducing gastric acid secretion and increasing lower esophageal sphincter tone. the length of medical treatment will vary from about one week to several weeks, depending on the underlying cause and severity of disease. medications indicated for esophagitis include prokinetics, h -receptor antagonists, proton pump inhibitors, and sucralfate (table - ) . prokinetic drugs enhance gastric emptying and increase lower esophageal sphincter tone. metoclopramide also has antiemetic effects, which may be beneficial in patients with chronic vomiting. it can be administered by the subcutaneous (sc) route, an advantage in a vomiting or regurgitating patient. cisapride may be more effective at enhancing both gastric emptying and lower esophageal sphincter tone, but it must be stent placement has recently been described in cats with esophageal strictures with variable results. a -year-old cat presented with a -week history of dysphagia and regurgitation caused by a single cervical esophageal stricture after treatment with oral clindamycin. guided balloon dilation was performed times over a period of weeks, but stricture formation always recurred. a self-expanding metal stent was placed using endoscopy and fluoroscopy after another dilation procedure. the cat did well eating a canned diet from an elevated position for months, but by months, the cat was no longer able to eat even liquid food and was euthanized. on necropsy, the stent had migrated and was obstructed by swallowed hair. in another case, a biodegradable self-expanding stent was used to successfully treat an -year-old cat that presented with a stricture in the cervical esophagus after anesthesia for dentistry. balloon dilation was performed twice, but regurgitation recurred days after the last procedure. the stricture was dilated a third time with a balloon catheter, and a tubular selfexpanding polydioxanone stent was placed with fluoroscopic guidance. the life span of the stent was estimated to be to weeks, sufficient time to allow healing of the esophagus. foreign bodies are less commonly found in the esophagus of the cat than in other gastrointestinal locations. reported foreign bodies include string, needles, fish hooks, and bones. trichobezoars may cause obstruction when they become lodged in the esophagus during vomiting ( figure - ). recurrent esophageal trichobezoars have been infrequently reported in the literature. , it is not known if an esophageal motility disorder is the underlying cause for recurrent obstructions. in one case, an esophageal diverticulum developed in association with recurrent trichobezoars. treatment for recurrent trichobezoars includes prokinetic drug therapy (e.g., cisapride), moderate to high-fiber diets, and shaving of long-haired cats. common areas for foreign bodies to lodge include the thoracic inlet, the heart base, and the esophageal hiatus in the diaphragm. obstruction of the esophageal lumen may be complete or partial. clinical signs include acute onset of gagging, salivation, repeated swallowing, dysphagia, and regurgitation. however, chronic esophageal foreign bodies have been reported in cats with dysphagia, intermittent regurgitation, and weight loss over a period of weeks or months. bougienage when bougies or balloon catheters were not available. balloon catheter dilation has become a popular method in recent years. , , although some clinicians feel this is a safer procedure than bougienage, there is no data in the literature to support this assumption. the catheter can be placed through the endoscope biopsy channel, alongside the endoscope, or with the aid of a preplaced guide wire. as for bougienage, established criteria for selection of balloon diameter and dilation end points are not available, and the clinician's best judgment must be used. various balloon sizes are available; in one study, the size was selected so that the inflated diameter was mm larger than the stricture diameter. the balloon is passed into the stricture with endoscopic guidance. it is then inflated to a predetermined pressure for to minutes to stretch the stricture, usually with saline, but contrast agents may also be used if fluoroscopy is used. as for bougienage, some cases may require more than one dilation procedure (typically two to four). cuffed endotracheal tubes are not appropriate substitutes for balloon catheters. regardless of the method used, after the dilation procedure, the endoscope should be used to look for other strictures and should be passed into the stomach to look for potential causes, such as causes of chronic vomiting. after treatment, medical management to decrease ongoing gastroesophageal reflux, resolve inflammation, and prevent further stricture formation should be instituted (as described previously). most cats are able to eat the day following the dilation procedure. corticosteroid treatment after dilation is controversial, and no controlled studies in animals are available. antibiotics are not routinely recommended. the prognosis for cats undergoing esophageal dilation is generally good based on the ability to eat canned food with minimal episodes of regurgitation. however, published studies show % to % of cats died or were euthanized despite multiple episodes of dilation, and up to % could only be fed liquid diets. , , , even among cats with good outcomes, a return to a dry kibble diet may not be possible. the dilation technique employed may be dictated by the clinician's experience, the equipment available, and the cost. potential complications of both methods include esophageal tear or perforation, hemorrhage, infection, and aspiration. esophageal tears or perforations may lead to pneumothorax or pneumomediastinum. repeated stricture formation is also possible, leaving only less desirable treatment options, such as long-term percutaneous gastrostomy tube feeding or surgery. esophageal surgery is generally avoided whenever possible, because it is difficult and invasive (requiring a thoracotomy), with risk of serious complications, such as failure of anastomosis, necrosis, and stricture formation. closure of incisions in the esophagus is following uncomplicated foreign body removal, the esophagus should be carefully inspected for lesions and bleeding before the endoscope is withdrawn. food and water should be withheld for to hours. supportive care includes fluid therapy and analgesia; a gastrostomy feeding tube may be required in selected cases for nutritional support. broad-spectrum antibiotics are administered to control bacterial infection and therapy for esophagitis should be instituted as described previously. careful follow-up should include evaluation for stricture formation. if an esophageal perforation has occurred, conservative management may be sufficient if the defect is small. a broad-spectrum antibiotic should be administered along with other supportive care, such as fluid therapy and analgesia. feeding through a gastrostomy tube for several days is recommended as well as close monitoring for complications such as pleuritis. large perforations require thoracotomy for surgical repair. megaesophagus is a diffuse hypomotility disorder that may be classified as congenital versus acquired or idiopathic versus secondary to other diseases. it is uncommon in cats compared with dogs. at least two dog breeds have been identified with heritable congenital megaesophagus. a heritable form of megaesophagus has been suggested for cats, particularly for siamese cats, although no detailed studies have been performed. , it is often frustrating to determine the underlying cause of acquired megaesophagus. megaesophagus may be a manifestation of neuromuscular diseases, such as dysautonomia or myasthenia gravis (see chapter ) . megaesophagus may also develop secondary to esophagitis from chronic vomiting or gerd. , other uncommon causes of megaesophagus are found in the literature. one case report describes a young cat with megaesophagus secondary to a large nasopharyngeal polyp that extended into the cervical esophagus. megaesophagus resolved once the polyp was removed. in another report, a young cat with diaphragmatic hernia was diagnosed with megaesophagus and gastric dilation. megaesophagus resolved with medical treatment and surgical correction of the diaphragmatic defect. clinical signs are typically those of esophageal dysfunction; regurgitation is the most consistently found sign. regurgitation may not be closely related in time to eating if the esophagus is markedly distended and holds food. cats with long-standing disease may suffer from weight loss or secondary rhinitis. the appetite is typically normal or increased. additional signs may occur if systemic neuromuscular disease is present. aspiration pneumonia may cause fever, dyspnea, and cough. two case reports describe cats with idiopathic cough, mucopurulent nasal discharge, and fever may be found if aspiration has occurred. trauma to the esophagus may cause esophagitis and even esophageal stricture. perforation of the esophagus by the foreign body may lead to pneumothorax, pneumomediastinum, or pyothorax with signs of depression, anorexia, fever, and dyspnea. if the perforation occurs in the cervical esophagus, swelling, cellulitis, and drainage of serous or purulent material may be noted. many foreign bodies are readily diagnosed with survey radiographs, especially if they are radiopaque. other radiographic findings include an esophagus dilated with fluid or air. radiolucent objects may be detected with an esophagram. care must be taken when performing esophagrams on cats that may have an obstruction, because aspiration is a concern. if abnormalities that could be consistent with an esophageal perforation (e.g., periesophageal gas or fluid, pleural effusion) are detected on survey radiographs, an aqueous iodine contrast solution should be used. removal of esophageal foreign bodies should be performed as soon as possible to minimize esophageal trauma and pressure necrosis. endoscopy can be used to confirm the diagnosis and often to remove the object. both rigid and flexible endoscopes may be used along with accessories such as various forceps and foley catheters. care should be taken to remove the object as atraumatically as possible, especially if the object is sharp or pointed. if the object is in the caudal esophagus and it cannot be grasped and removed, an attempt should be made to gently push it into the stomach, where it can be retrieved using laparotomy and gastrotomy. if esophageal perforation has occurred, esophagotomy is recommended and is described elsewhere. , removal of fish hooks may require a combination of surgery and endoscopy. , a surgical approach to the esophagus is made, but the esophagus is not incised; rather, the portion of the hook protruding through the esophagus is cut and removed, and the endoscope is used to retrieve the remainder. clinical sign is regurgitation, and most patients are underweight. a distended cervical esophagus may be palpated, and secondary aspiration pneumonia may occur. a history of regurgitation since weaning is very suggestive of a vascular ring anomaly, but other causes of regurgitation must be ruled out. survey radiographs show a dilated esophagus cranial to the heart, while the caudal esophagus is usually normal. the bulge of the aortic arch normally seen on a ventrodorsal radiographic view is absent. an esophagram is used to confirm the location of the obstruction and the severity of disease. definitive treatment is surgical repair of the vascular defect (i.e., ligation and transection of the ligamentosum arteriosum). some patients will require nutritional support through gastrostomy tube feeding and treatment for aspiration pneumonia before surgery. early diagnosis and surgical intervention brings the best prognosis for return of normal esophageal function. some affected cats are left with residual esophageal hypomotility, which is managed as for idiopathic megaesophagus. esophageal neoplasia is rare in the cat as in the dog. although parasitic granulomas caused by spirocerca lupi are associated with esophageal neoplasia in dogs, this parasite does not infect cats. both primary and metastatic esophageal tumors can occur in the cat. squamous cell carcinoma is the most common primary esophageal tumor in cats and is often found in the caudal two thirds of the esophagus. , , , affected cats are middle aged or older. clinical signs are typically those associated with esophageal obstruction, such as regurgitation, dysphagia, odynophagia, and salivation. patients with advanced disease may suffer anorexia, depression, and weight loss. on physical examination, an esophageal mass may or may not be palpable. survey and contrast radiographs reveal esophageal dilation, a soft tissue mass, or periesophageal lesions that displace the esophagus. computed tomography is useful to identify periesophageal or intraluminal masses. definitive diagnosis is made with endoscopy and biopsy. mucosal biopsies are difficult to obtain, because the esophageal mucosa is tough; exfoliative cytology may also be helpful. treatment is rarely undertaken, because disease is often advanced at the time of diagnosis, and many patients have complications such as aspiration pneumonia. palliation may be attempted with chemotherapy or radiation, although data on efficacy is unavailable. in general, squamous cell carcinomas in other anatomic locations respond poorly to treatment. surgical resection may be attempted if anastomosis can be accomplished without excessive tension. megaesophagus and chronic vomiting associated with intermittent gastroesophageal intussusception. , survey and contrast radiographs may identify a dilated esophagus (figure - ), but contrast fluoroscopy is the diagnostic tool of choice when available, because it allows for assessment of peristalsis. care must be taken with contrast studies because of the risk of aspiration. treatment of megaesophagus is largely symptomatic and supportive unless an underlying disorder can be identified and treated. frequent small meals are offered with the cat feeding in an upright position. the upright position should be maintained for at least minutes after eating to allow for gravity-assisted passage of food into the stomach. this is best accomplished by having the owner hold the cat over their shoulder so that the esophagus is in a vertical position. different types of diets should be offered to determine which is best for the individual patient; calorically dense diets may be beneficial for patients with weight loss. prokinetic drugs, such as cisapride, stimulate smooth muscle, but since most of the esophagus is skeletal muscle, the efficacy of such drugs is questionable for treatment of megaesophagus. prokinetic drugs also increase lower esophageal sphincter tone and may increase esophageal transit time, neither of which is desirable in patients with megaesophagus. vascular ring anomalies are congenital malformations of the great vessels that entrap the thoracic esophagus and cause obstruction. the most commonly reported anomaly is persistent right aortic arch. the esophagus is entrapped by the aorta on the right, the ligamentum arteriosum and the pulmonary trunk on the left, and the heart base ventrally. other vascular anomalies are rarely described in cats, such as a double aortic arch described in a siamese cat. onset of clinical signs occurs around the time of weaning to solid food so that most affected cats are presented at less than months of age. the most common surgery. surgery is the treatment of choice for large defects, especially in young cats with congenital disease or cats that have failed medical management. various reconstructive surgical techniques have been described. disorders of the hiatus are rare in cats. hiatal hernia is protrusion of the distal esophagus and stomach through the esophageal hiatus of the diaphragm into the thoracic cavity; the protrusion may be intermittent ("sliding") or persistent. other organs are occasionally involved, such as the omentum. this is distinct from a gastroesophageal intussusception where the stomach is prolapsed into the lumen of the distal esophagus. , both congenital and traumatic hiatal hernias have been described in cats. , , , , congenital hernias appear to be more common than acquired hernias, and affected cats typically present with clinical signs before year of age. it is suspected that increased inspiratory effort associated with upper airway obstruction, such as a nasopharyngeal polyp, may also lead to development of hiatal hernia. hiatal herniation reduces lower esophageal sphincter pressure. clinical signs associated with hiatal hernia, such as intermittent vomiting and regurgitation, may be because of reflux esophagitis, hypomotility, or obstruction. large hernias and secondary aspiration pneumonia may be associated with respiratory distress. survey radiographs may reveal a gas-filled soft tissue density in the caudal dorsal mediastinum. an esophagram will show the gastroesophageal junction and gastric rugae cranial to the diaphragm (figure - ) . both fluoroscopy and endoscopy may be useful for diagnosis but are not typically necessary. the prognosis for cats with hiatal hernia is considered to be good. a trial of medical management (as for reflux esophagitis) for month has been recommended before the stomach is a frequent site for gastrointestinal problems in cats, and the most common gastric problems are described in this chapter. some conditions such as gastric dilatation-volvulus are often reported in dogs but rarely reported in cats. in one report of three feline cases, all were associated with diaphragmatic hernia. gastric parasites, the diagnostic approach to the vomiting cat, the gastric emptying time of normal cats is shorter than that of other mammals. in one study, the gastric emptying half-time for solid food in normal cats was . to . hours. this implies prolonged fasting (longer than hours) in preparation for anesthesia and surgery is unnecessary. the main clinical sign of gastric disease is vomiting, but it is important to note that vomiting is also associated with many nongastric problems, including concurrent intestinal disease, such as enteritis or colitis. vomiting patients therefore require a thorough physical examination and diagnostic plan to determine the cause. vomiting must be distinguished from regurgitation, which is primarily associated with esophageal disease (see table - ). vomitus often contains food, hair, refluxed bile, and therapeutics for vomiting are covered elsewhere in this chapter. the anatomy of the feline stomach is similar to that of other mammals having a simple glandular stomach. most of the stomach is situated on the left side of the abdominal cavity. it has five regions, starting from the lower esophageal sphincter: cardia, fundus, body, antrum, and pylorus ( figure - ) . the pylorus of the cat is unique compared with other species in that it is narrow and has high resistance in order to maintain a tight seal ( figure involve a wide variety of objects, including linear objects (e.g., dental floss, thread with or without a needle, tinsel, string). the owner may or may not be aware of the ingestion. ingestion of multiple foreign bodies may be seen in cats with pica ( figure - ). in one case report, a young domestic shorthair cat required gastrotomy for removal of copper pennies. some patients require multiple surgeries, because of repeated foreign body ingestion. in such cases, a behavioral diagnosis should be sought and treatment instituted (see chapter ) . trichobezoars (large masses of hair) also represent a type of foreign object. both long-and shorthaired cats may be affected. hair is normally ingested during grooming and is eliminated in vomitus and feces. cats lack the strong peristaltic contractions ("housekeeper" contractions) that clear the stomach of undigested contents normally found in other species. this may explain why cats seem to be susceptible to gastric trichobezoars. gastric motility dysfunction is suspected to cause repeated gastric trichobezoars in some cats. intestinal , and esophageal , obstruction with trichobezoars has also been documented. traditional treatments for cats with recurrent trichobezoars include regular grooming, shaving the hair coat of long-haired cats, flea control, or blood. fresh blood may appear as large or small clots. older blood clots have a brown "coffee ground" appearance. gastric bleeding may also cause melena. other clinical signs may be associated with gastric disease, such as anorexia, weight loss, pain, lethargy, bloating, and nausea. gastritis may be acute or chronic in nature, and this distinction may be useful in assessing the potential cause. for example, cats with acute gastritis may be suspected of foreign body or plant ingestion, drug or toxin exposure (see chapter ), or dietary indiscretion. cats with chronic gastritis may be suspected of parasitism, helicobacter spp. infection, or dietary intolerance or hypersensitivity (see chapter ) . chronic lymphocytic plasmacytic gastritis of unknown etiology is also a common cause of chronic vomiting. whenever possible, a specific underlying cause should be sought and treated. patients with sudden onset of vomiting may have an obvious cause in the history (e.g., dietary indiscretion), but in many cases, the cause is not apparent. abdominal radiographs should be taken if foreign body ingestion is possible, especially in a young cat. if the patient is systemically well, further diagnostic testing may be postponed pending response to therapy. treatment for uncomplicated acute gastritis is symptomatic and supportive. clinical signs are expected to resolve in to hours; if signs persist, re-evaluation and further investigation is warranted. subcutaneous fluid therapy using an isotonic balanced electrolyte solution may be used to correct mild fluid deficits (< %). oral intake of fluids and food should be discontinued for up to hours. a highly digestible diet, either commercial or homemade, is introduced with a gradual transition back to the normal diet over the next several days. antiemetic therapy may be indicated for acute uncomplicated gastritis if the vomiting is frequent or the cat has signs of nausea (see table - ). protectants, such as kaolin and pectin, are difficult to administer to cats and are without proven efficacy. bismuth subsalicylate is controversial; it is considered contraindicated by some experts, because of the cat's sensitivity to salicylates, yet is commonly used in clinical practice. cats ingest foreign bodies less commonly than dogs. in one study of cases of gastrointestinal foreign body ingestion, only % were in cats. foreign body ingestion is most likely to be seen in young cats and may a b taken just before surgery to ensure the object has not moved further down the gastrointestinal tract. postoperative management after gastrotomy includes maintenance of hydration and electrolyte balance. hypokalemia is common with anorexia and vomiting and should be treated by supplementation of iv fluids with to meq/l potassium chloride (not to exceed . meq/kg/hour). refractory vomiting should be treated with an antiemetic. a highly digestible diet can be introduced the day after surgery. in general, the prognosis for recovery is good. in one study, % of cats with gastrointestinal foreign bodies survived to discharge. those cats that did not survive had linear foreign bodies of long-standing duration with subsequent peritonitis. helicobacter are spiral or curved gram-negative bacteria that inhabit the glands, parietal cells, and mucus of the gastric antrum and fundus. helicobacter contain large amounts of urease, which alters the ph in the vicinity of the bacteria and allows for colonization of the acidic environment of the stomach. in the early s, the discovery of the association of helicobacter pylori with gastric disease (gastritis, peptic ulcers, and neoplasia) in humans revolutionized treatment of those diseases. since then, helicobacter spp. have been associated with gastric disease in various veterinary species, including cats and dogs. several helicobacter spp. (e.g., h. heilmannii, h. bizzozeronii, h. felis) have been identified in cats, some of which have the potential to infect humans, although transmission is thought to be rare. , the prevalence of helicobacter infection in cats varies geographically and may be very high (> %) in some locations. , , , , the importance of helicobacter as a cause of gastric disease is cats is unclear; the bacteria may be found in the stomach of both clinically normal cats and cats with gastritis. the prevalence of helicobacter infection is not higher in cats with gastritis compared with normal cats. determination of the role of helicobacter is also hampered by the paucity of controlled clinical trials that evaluate eradication of gastritis and clinical signs in infected cats. an immune response to infection characterized by gastric lymphoid hyperplasia is common, although the local immune response in cats is generally less severe than the response in humans infected with h. pylori. to date gastrointestinal ulcers have not been associated with helicobacter infection in cats. recent studies have suggested a possible association between helicobacter infection and gastric lymphoma in cats, although more research is needed to confirm the association and understand the pathogenesis. , helicobacter spp. may be commensal in most cats, and perhaps loss of tolerance explains the development of gastritis in some individuals. another possibility is that the inflammatory response is normally well managed and disease may treatment of underlying dermatologic disorders, and administration of semisolid petroleum laxatives. more recently, commercial diets have been formulated for control of trichobezoars. cats with recurrent trichobezoars causing illness and suspected motility disorders may benefit from treatment with prokinetic drugs such as cisapride. clinical signs of gastric foreign bodies are variable but typically involve intermittent or persistent vomiting because of gastric outflow obstruction, distention, and mucosal irritation. gastric obstruction may be partial or total. patients with complete obstruction will present with more dramatic signs, including anorexia and depression. the base of the tongue should always be examined, because linear foreign bodies are sometimes anchored either in this location, or they may be lodged in the pylorus, causing intestinal plication. gastric foreign bodies may also be asymptomatic and found incidentally. physical examination may be unremarkable or may reveal dehydration or abdominal pain. if the stomach is markedly distended, the foreign body may be palpable in some patients. survey radiographs are always indicated when foreign body ingestion is suspected. radiopaque foreign bodies may be readily diagnosed, although some, along with radiolucent objects, will require a contrast study for diagnosis ( figure - ) . barium is commonly used as a contrast agent, although if gastric perforation is suspected, an aqueous iodinated agent is preferred. ultrasonography is also useful for detection of gastrointestinal foreign bodies. removal of some foreign bodies can be attempted endoscopically, particularly if the object does not have sharp edges and is not too large. successful removal of fish hooks, particularly single-barb hooks, using endoscopy has been described. otherwise, foreign objects are best removed using gastrotomy through a ventral midline laparotomy. a radiograph should always be to know when treatment should be attempted. one expert recommends treating only patients with clinical signs of gastritis that have biopsy-confirmed helicobacter infection with a treatment regimen of amoxicillin ( mg/kg, every hours, po), clarithromycin ( . mg/ kg, every hours, po) and metronidazole ( mg/kg, every hours, po) for days. a common dilemma would be determining the treatment of choice for patients with lymphoplasmacytic inflammation of the stomach and small intestine and confirmed helicobacter infection. are such patients best treated for inflammatory bowel disease, helicobacter infection, or both? currently, guidelines for determining the best treatment approach are lacking. also, few studies on the efficacy of combination therapy have been conducted in cats. long-term eradication of infection may be difficult, and histopathologic resolution of gastritis may not be possible, which raises the question of whether helicobacter is the true underlying cause. , in one study, two cats with clinical gastritis and helicobacter infection were treated with oral metronidazole, amoxicillin, and bismuth subsalicylate for weeks and were also fed a commercial elimination diet. posttreatment gastric biopsies were obtained a mean of weeks after the cessation of treatment. resolution of clinical signs occurred rapidly, and clearance of helicobacter spp. was achieved at that time point, but gastric inflammation persisted in post-treatment biopsies. in another study, cats with asymptomatic helicobacter infection were treated with oral omeprazole, amoxicillin, metronidazole, and clarithromycin for days. treatment failed to eradicate infection in of the cats based on molecular analysis of post-treatment gastric biopsies. it is unclear if treatment failure is because of recrudescence or reinfection. the reader is referred to excellent reviews of helicobacter in cats for more information. , , chronic gastritis chronic gastritis is common in cats with chronic intermittent vomiting. ollulanus tricuspis is a worm that infects the stomach of cats, causing chronic gastritis, and it is difficult to diagnose (see below, gastrointestinal parasites). the worm is occasionally found on histologic examination of gastric biopsy samples. it is reasonable to treat empirically (fenbendazole mg/kg, once daily, po × days) for this parasite when the cause of gastritis is not apparent. the frequency of vomiting in cats with chronic gastritis is highly variable, ranging from once or twice per week (and not necessarily every week) to more than once daily. most patients are otherwise well, although other clinical signs (inappetence, anorexia, depression, or weight loss) are possible depending on disease severity. results of routine laboratory testing are typically normal but may show neutrophilic leukocytosis, result when there is an abnormality of the immunoregulatory system. the most commonly used methods for diagnosis of helicobacter infection in cats are based on gastric specimens obtained during endoscopy (or laparotomy): exfoliative cytology, histopathologic examination of biopsy specimens, and rapid urease testing of biopsy specimens. however, it is important to note that even when helicobacter organisms are identified, the infection may not be the cause of the patient's clinical signs, and other causes of vomiting should always be evaluated. exfoliative cytology is the least expensive and most easily performed diagnostic test. in one study, it was also the most sensitive diagnostic method when compared with urease testing and histologic examination. brush cytology samples gathered during endoscopy are airdried on microscope slides and stained with wright's stain. the slide is examined at × magnification under oil immersion. spiral bacteria are readily seen if present. at least oil-immersion fields on two slides should be examined before determining a specimen is negative for helicobacter-like organisms. since helicobacter produce abundant urease, a rapid urease test (e.g., clotest, ballard medical products, draper, utah) may be used for diagnosis. the kit consists of an agar gel impregnated with urea and a ph indicator. a gastric biopsy sample is applied to the gel, and if urease is present, ammonia will form and change the ph (and thus the color) of the gel. the gel may change color rapidly (within minutes), but hours must elapse before the test can be considered negative. the more rapidly the color changes, the higher the bacterial load. both false-positive and false-negative results are possible with rapid urease testing for various reasons, giving the test a sensitivity of % to %. , histopathologic examination of gastric biopsy samples using hemotoxylin and eosin (h&e) or silver stains is highly sensitive and specific in human studies for detection of helicobacter-like organisms. the organisms are not equally distributed; so, examination of biopsy specimens from multiple sites will increase sensitivity. the bacteria may be seen in mucus on the surface epithelium as well as in the gastric pits, glandular lumen, and parietal cells. organisms may also be seen submucosally within gastric lymphoid follicles. histopathologic examination of biopsy samples also allows for assessment of other abnormalities. mild to severe lymphocytic-plasmacytic or lymphocytic gastritis may be present. in humans combination therapy with antibiotics and antisecretory drugs is recommended to reduce the risk of gastric ulcers and cancer from h. pylori infection. treatment is highly successful at eradicating both clinical signs and histologic changes in the gastric mucosa. since helicobacter infection is common in cats, yet no clear pathogenic role has been established, it is difficult cases. depending on the underlying cause and severity of disease, abdominal pain, anorexia, lethargy, pale mucous membranes, and drooling may also be seen. cats with neoplastic disease may have prolonged clinical signs and are more likely to present with anorexia and weight loss. cats with perforated ulcers may or may not present with signs of shock. diagnosis may be problematic because the clinical signs and physical examination findings are often not specific, even in cats with perforated ulcerations. the causes of gastric ulceration in cats are not well characterized. in dogs the most common cause is the administration of ulcerogenic drugs, particularly nsaids, either alone or in combination with corticosteroids. several cases of nsaid-induced gastroduodenal ulceration or perforation have been reported in cats. , , additional cases may be reported in the future, because long-term administration of these drugs is gaining in popularity for treatment of chronic diseases such as osteoarthritis. nsaids cause direct mucosal damage and interfere with prostaglandin synthesis. although inhibition of the cox- enzyme is thought to be the cause of adverse effects, such as gastric ulceration, even cox- -selective drugs have been associated with adverse effects, and safety in sick cats is not well evaluated. recently, guidelines for the long-term use of nsaids in cats were published by the international society of feline medicine and the american association of feline practitioners. the recommendations include administering nsaids either with or shortly after food, withholding therapy if inappetence or anorexia develops, determining dose based on lean body weight, and titrating to the lowest effective dose. neoplastic causes of gastric ulceration include systemic mastocytosis, mast cell tumor, lymphosarcoma, adenocarcinoma, and gastrinoma (zollinger-ellison syndrome). cats with chronic renal disease may suffer mucosal damage from uremic toxins and increased gastric acid production secondary to hypergastrinemia (because of decreased renal metabolism of gastrin). hepatic disease is a cause of gastric ulceration in dogs but is uncommonly reported in cats. recent anesthesia and surgery have been implicated as a cause of gastric ulceration and perforation, perhaps through hypovolemia, hypoperfusion, or stress. , other non-neoplastic causes reported for gastric or gastroduodenal ulceration in cats include parasites (e.g., ollulanus tricuspis, toxocara cati, aonchotheca putorii, gnathostoma spp.), bacterial infections, toxins, inflammatory bowel disease, and foreign bodies. one case report describes a cat with severe gastric ulceration caused by intoxication with dieffenbachia leaves. in some case reports, the cause for the gastric ulcerations could not be determined. a minimum database should be collected for cats suspected of gastric ulceration, to identify underlying diseases. anemia, usually regenerative, may be present. eosinophilia, or hypoproteinemia. survey and contrast radiographs are often normal. the most common finding on histopathologic examination of biopsy samples is lymphocytic plasmacytic (lp) gastritis ( figure - ) . some patients will also have concurrent evidence of lp inflammation in the small intestine, pancreas, and/or liver. such patients will be treated for their concurrent problem; treatment of inflammatory bowel disease, pancreatitis, and cholangiohepatitis is covered elsewhere in this chapter. some cats with chronic lp gastritis respond to treatment for dietary intolerance or hypersensitivity with a limited antigen diet (see chapter ) . patients with moderate to severe lp gastritis may be best treated with a limited antigen diet and immunosuppressive therapy (prednisolone to mg/kg/day, po tapering to every other day at the lowest dose that controls clinical signs). patients that fail this initial treatment approach may require additional immunosuppressive therapy, such as chlorambucil (see table - ) . occasionally, cats with chronic gastritis are diagnosed with eosinophilic inflammation on histopathologic examination of biopsy specimens. treatment is similar to that for lp gastritis, although such patients should be evaluated for evidence of hypereosinophilic syndrome and eosinophilic enteritis. eosinophilic fibrosing gastritis was suspected to be caused by toxoplasmosis in one case report. gastric or gastroduodenal ulcerations are uncommon in the cat compared with the dog and may be caused by a variety of disorders, both gastric and nongastric. classical clinical signs include vomiting, hematemesis, and melena. however, in one review of eight cats, hematemesis and melena were present in less than one third of suturing of the ulcer site as well as collection of biopsy samples for histopathologic examination. the prognosis for recovery was excellent in two studies, particularly for cats with non-neoplastic causes of gastric or gastroduodenal ulceration. , in one study of seven cats with perforated gastric or duodenal ulcers, the survival rate was low ( %). disorders of gastric motility are better characterized in dogs than in cats. the most common clinical sign is vomiting of undigested food hours or more after a meal. if outflow obstruction is present, vomiting may be projectile. there may also be a history of recurrent trichobezoars. various disorders are associated with impaired gastric motility, such as chronic gastritis, drug therapy (e.g., anticholinergic and narcotic drugs), dysautonomia, gastric neoplasia, metabolic disorders (e.g., hypokalemia), and temporary postsurgical gastroparesis. in some cases of chronic motility dysfunction, no cause can be identified. outflow obstruction may be caused by neoplasia, foreign bodies, and extragastric masses. pyloric stenosis is infrequently documented in young cats, often siamese cats. , , since the range of underlying disorders is diverse, the diagnostic approach should allow for detection of both gastric and nongastric disorders. a minimum database (cbc, serum chemistries, urinalysis, feline leukemia virus [felv] and feline immunodeficiency virus [fiv] serology) is used to establish overall health status. radiographs are used to confirm presence of food in the stomach for longer than hours. ultrasonography may detect gastric lesions, such as masses. endoscopy is used to identify outflow obstruction as well as other lesions, such as ulcers, and evidence of gastritis. assessment of gastric emptying using nuclear scintigraphy is the most accurate method but is limited to referral centers. gastric emptying times for liquids, canned food, and dry diets have been established using nuclear scintigraphy. , , however, emptying times are variable, depending on the amount and type of diet fed as well as the amount of water ingested. even the shape of kibble affects emptying time. radiographic contrast series are widely used, but gastric emptying times are variable for barium in either liquid form or mixed with canned food. contrast radiography using liquid barium ( to ml/kg) is performed in a fasted patient. radiographs are taken immediately after administration of the barium and again at and minutes, in some cases, also at and hours. liquid barium is expected to enter the duodenum no more than minutes after administration, and the stomach should be completely empty of barium within hours. the clinician should be aware that some cats with gastric motility disorders will have other findings will be dependent on the presence of underlying diseases; for example, azotemia and isosthenuria may indicate renal disease. electrolyte and acidbase abnormalities may be because of chronic vomiting and anorexia. survey and contrast radiographs and ultrasonography are primarily useful to rule out other causes for the clinical signs, such as foreign bodies. cats with perforated ulcers may have evidence of pneumoperitoneum (sometimes severe) on plain radiographs or ultrasonographs, and this is an indication for surgical exploration. , , , , evidence of peritonitis on imaging studies should be followed with peritoneal fluid analysis. a definitive diagnosis may be made using endoscopy, which allows direct visualization of lesions and collection of biopsy samples. however, some cats with gastric ulceration present in poor condition, which may preclude the use of endoscopy because of anesthetic risk and risk of ulcer perforation. the location of ulcers is typically pyloroantral or fundic in cats with nonneoplastic disease. , areas of erosion may appear pale or hemorrhagic; the mucosa is often friable and bleeds easily. fresh or clotted blood may be seen in the stomach lumen. in some cases, mucosal ulceration must be distinguished from ulcerated tumors. nsaid-induced ulcers are typically found in the antrum and do not have marked mucosal thickening; ulcerated tumors frequently have thickened edges and surrounding mucosa. biopsy samples should be taken at the periphery of the ulcer to avoid perforation. treatment should be directed at any underlying disorder. treatment for nsaid toxicity is described in chapter . general supportive measures include fluid therapy and electrolyte replacement; blood transfusion may also be required (see chapter ) . gastric acid production can be decreased with the use of h -receptor blockers or proton pump inhibitors, and sucralfate is used as a mucosal protectant (see table - ). sucralfate may inhibit absorption of other oral medications and should be given hours apart from other drugs. if vomiting is severe or persistent, antiemetic therapy is warranted (see table - ). analgesia should be provided for painful patients; a good choice is the opioid buprenorphine (see table - ). broad-spectrum antibiotic therapy is indicated for patients with significant mucosal barrier dysfunction, perforation, leukopenia and/or neutrophilia, fever, and melena. surgical intervention is warranted for patients with life-threatening hemorrhage, failure to respond to medical management, or evidence of perforation. the entire abdominal cavity and gastrointestinal tract should be thoroughly explored to locate extragastrointestinal lesions, non-perforated ulcers, and multiple ulcers. in one case series, nonperforated ulcers were detected at laparotomy by association with adhesions or a gastric mass. surgical management includes débridement and months. physical examination findings are nonspecific, although occasionally a gastric mass or gastric thickening may be palpated if the stomach is markedly enlarged. results of routine diagnostic testing are generally nonspecific; anemia may be associated with ulceration. survey or contrast radiography may reveal a mass ( figure - , a); other findings include delayed gastric emptying, impaired motility, and mucosal ulceration. ultrasonography is also useful for diagnosis and can be used to guide needle aspirates of masses ( figure - , b). endoscopy allows for visualization of lesions as well as the ability to obtain partial thickness biopsy samples. problems with interpretation of endoscopic biopsy samples include detection of necrosis, inflammation, and ulceration rather than the primary lesion. in dogs some neoplastic lesions are submucosal, making it very difficult to obtain diagnostic samples by endoscopy. therefore several biopsies should be taken and masses should be biopsied multiple times in the same place to sample deeper tissues. the center of ulcerated lesions should not be biopsied. surgical biopsies are more reliable for diagnosis. a normal gastric emptying time with liquid barium. barium can also be mixed with canned food and fed as a meal; retention of barium-containing food in the stomach for more than to hours is abnormal. gastric emptying time may also be established with the use of barium impregnated polyspheres (bips; med i.d. systems, grand rapids, mich.) and radiography. gastric emptying times for bips have been established in healthy fasted and fed cats as well as in sedated cats, , but the values do not correlate well with scintigraphic studies. a mixture of small ( . mm) and large ( mm) spheres are administered with food, and two to four radiographs are taken over the next hours. the small spheres are intended to mimic liquid transit time and the large spheres solid transit time. however, studies assessing the clinical relevance of this method are lacking. one review concluded that bips are probably sufficiently sensitive to detect grossly delayed gastric emptying. treatment of gastric emptying disorders is directed at identifiable causes. treatment for gastric ulcers, chronic gastritis, and foreign bodies is described elsewhere in this chapter. pyloric stenosis is managed surgically. if no outflow obstruction exists, treatment with prokinetic agents, such as metoclopramide or cisapride, may be beneficial (see table - ). gastric tumors account for less than % of malignancies in dogs and cats. benign gastric tumors are even less common than gastric malignancies. gastric smooth muscle hamartoma has been reported in one -year-old cat. although adenocarcinoma is the most common gastric cancer of the dog, lymphoma is the most common gastric cancer in the cat. feline gastrointestinal lymphoma occurs as two major types: small cell (lymphocytic) and the more aggressive large cell (lymphoblastic) form. small cell lymphomas are more frequently enteric. in one study of cats with gastric lymphoma, diffuse large b-lymphocyte tumors of immunoblastic nuclear type predominated. gastric lymphoma is not associated with felv, and the role of helicobacter in the development of gastric lymphoma in cats requires investigation. adenocarcinoma, , , plasmacytoma, and gastric carcinoid have also been described. the siamese cat may be predisposed to adenocarcinoma. , as would be expected, most cats with gastric neoplasia are older cats. as for most gastric diseases, vomiting is the most common clinical sign of neoplasia. the vomitus may contain blood and melena may be present. other clinical signs include anorexia, weight loss, bloating, and depression. perforation of the tumor may occur, leading to pneumoperitoneum or septic peritonitis. clinical signs present gradually and are often present for weeks to surgical resection is the most common treatment for gastric neoplasia other than lymphoma ( figure - ). the prognosis for most patients is poor, typically because of debilitation, concurrent diseases, and recurrent or metastatic disease. the success of chemotherapy for lymphoma depends on cell type, with small cell tumors carrying a better prognosis than large cell tumors. in diarrhea can be defined as increased volume and/or increased frequency of defecation of stools with increased water content. approaches to diarrhea, as for any clinical sign, need to take into account the individual animal. for example, neoplasia is much less likely to occur in a kitten than in a geriatric cat. in many cases, the precise diagnosis of gastrointestinal disease cannot be reached without biopsy samples. the decision to obtain biopsy samples should follow a logical pathway that is appropriate to the cat's condition. these are summarized in figure - . for example, many cases of acute diarrhea in a well cat can resolve with limited or no intervention, and so do not require a precise diagnosis. the diagnostic steps are . signalment and clinical history . physical examination . fecal assessment . blood and urine testing . imaging (radiography, ultrasonography) . biopsy samples these steps do not include treatment/diet trials or other empiric therapies that are appropriate in many cases. steps and are often undertaken at the same time, and there is no definite order for these steps. they are divided here for reasons of clarity. in a younger cat, where infectious causes are more likely, thorough fecal testing is more important; in an older cat, extragastrointestinal diseases, such as hyperthyroidism, are more likely; so, blood and urine testing is more important, but fecal assessment should not be neglected. the decision to proceed to step (and each subsequent step) should take into account several considerations. the main considerations in assessing and managing a cat with diarrhea are • is there an acute onset or a chronic time course? • are there any dietary changes or indiscretion? consider treatment trials: antibiotics (e.g., amoxicillin/ clavulanate ؉ metronidazole). food trials with novel proteins (e.g., rabbit, kangaroo, venison). • is the cat well or unwell? • is there primary or secondary gastrointestinal disease? • is there small or large bowel diarrhea? the components of the clinical history for cats with diarrhea are detailed in table - . after establishing the cat's age, breed, vaccination, and deworming history, it is important to establish the duration and nature of the diarrhea. chronic diarrhea is usually defined as greater than weeks in duration and mostly warrants at least some degree of a diagnostic workup, whereas acute diarrhea is often self-limiting in a well cat. a description of the feces helps determine whether the diarrhea is small or large bowel in origin (table - ); this will affect how any investigations might proceed. important questions to ask concern frequency of defecation (and how this compares with the normal state), tenesmus (straining usually indicates large bowel diarrhea, since an irritated colon leads to urgency), volume of feces (smaller volumes are typical of large bowel diarrhea; larger volumes are more typical of small onset and duration of diarrhea acute versus chronic? acute diarrheas are abrupt in onset and of short duration, and generally they are self-limiting. chronic diarrheas persist usually longer than weeks and fail to respond to symptomatic therapy. appearance of diarrhea quantity and quality of the stool (color, consistency, character, presence of blood or mucus)? loose to watery feces that contain fat droplets, undigested food, melena, and variable colors suggests small intestinal disease. the volume is always increased with small intestinal disease. loose to semisolid feces containing excess mucus and fresh blood (hematochezia) indicates large intestinal disease. the volume may be normal to slightly decreased with large intestinal disease. description of defecation process tenesmus (straining) and dyschezia (painful defecation)? these are hallmarks of large intestinal disease (e.g., inflammatory or obstructive lesions of the colon, rectum, or anus). frequency is normal to slightly increased with small bowel disease, but greatly increased with large bowel disease. associated physical signs vomiting, anorexia, weight loss, and dyschezia may help localize the disorder to a specific part of the gastrointestinal tract. clinical signs relating to problems in other organs or body systems should be noted and may suggest a more generalized disease. vomiting may occur as a consequence of small intestinal inflammation in some cats with diarrhea. weight loss may result from decreased caloric intake (anorexia), decreased nutrient assimilation (maldigestion/malabsorption), or excessive caloric loss (protein-losing enteropathy or nephropathy). weight loss is observed uncommonly with large bowel disease. in many cases, the answers to these questions are obvious. for example, a cat may seem well but has had access to lilies (the author has seen diarrhea as a primary presenting sign for this!) or has a palpable abdominal mass. substantial weight loss is an indicator that further investigations are warranted sooner rather than later. if the decision is made for empiric management and outpatient care, it is vital to follow up either by scheduling a recheck visit or calling the client, because simple acute problems can turn into complicated chronic problems. if the diarrhea has been present for less than a week and the cat has no weight loss, dehydration, fever, or palpable abdominal abnormalities, it is appropriate to manage the cat as an outpatient. even in the absence of fecal testing, it is appropriate to deworm the cat (see the section gastrointestinal parasites). the cat should be fasted for hours ( hours, if less than months old) and then fed a bland diet (such as plain, cooked, skinless chicken, or low-residue prescription diets designed for cats with gastrointestinal problems). it is appropriate to maintain the cat on the low-residue diet for at least to days and then slowly reintroduce the regular diet. fecal assessment is mostly used to assess infectious agents, such as parasite-associated diarrhea, but the importance of assessing feces, even when parasitic or bacterial infections are not suspected, should not be underestimated. gross examination of feces can determine if melena or fresh blood or mucus are present to help distinguish large from small bowel disease when the owner's observations may be misleading. occult fecal blood can be an indicator of gastrointestinal inflammation in cases of subtle disease, and undigested starches and fats can indicate maldigestion or malabsorption. for assessment of feces for parasites, the fecal sample should ideally be fresh (< hour old). refrigeration (for no longer than one week) can preserve ova, oocysts, and cysts but not protozoal trophozoites. feces should be assessed by a. to assess for trophozoites bowel), how formed the stool is (from soft stool to cow-pat consistency to liquid tea; usually more watery stool relates to small intestinal disease), color (darker indicates digested blood), and presence of any mucus or blood (presence relates to large bowel). most household toxins, such as plants, cause signs additional to diarrhea such vomiting or neurological signs, but it is important to ascertain if the cat has had access to anything unusual. likewise, it is important to find out if the cat has had any possible exposure to dietary indiscretions; this can include if the cat has been seen with or is known to hunt prey including insects. cockroaches carry pathogenic bacteria , and other prey such as birds and rats can carry salmonella; salmonellosis in cats has been dubbed songbird fever. simple causes of self-limiting diarrhea include dietary change (either a new flavor or a new style of food, such as dry food for the first time); so, the owner must also be quizzed if anything new has been offered, either new cat food or treats (such as greasy fish or chicken). although the physical examination will usually determine how unwell a cat is, the owner's impressions are also important, because cats can hide signs from strangers, particularly in a practice setting. lethargy and inappetence are important signs, as ill cats typically do not eat well. the cat's general demeanor can be an indicator of how unwell a cat is and therefore dictate the extent of diagnostic testing required. this can be noted by assessing how interested the cat is in its surroundings or any behavior changes from previous visits, such as if a normally difficult-to-handle cat is placid. body weight should be assessed and, if possible, compared with that of previous visits (even those noted on a clinical record from another veterinarian). the body condition score (bcs) should also be assessed and can be very important when there is no prior weight information. dehydration is usually a sign that a cat needs more involved management. abdominal palpation should be performed to assess pain (where?), any masses (foreign bodies, lymph nodes, or even focally thickened intestines, such as with neoplasia), or turgid intestines. fever often indicates infection but can also reflect neoplasia or other inflammatory changes. a thorough examination of all body systems should always be performed, no matter what a cat presents for. in the case of diarrhea, extragastrointestinal signs can be of vital importance, such as a palpable thyroid and tachycardia suggesting hyperthyroidism. after the clinical history has been taken and the physical examination performed, the veterinarian must make the important decisions of whether any interventions are required and whether the patient should be factors affecting interpretation include whether the growth is a heavy and pure growth of a known pathogen, such as salmonella, campylobacter, yersinia, or clostridium difficile. further information about the relevance of culture and pcr results is contained below in the section infectious enteritis. investigations begin by assessing if the diarrhea is the result of primary gastrointestinal disease or secondary to another process, by performing routine serum/plasma biochemistries, hematology, urinalysis, and total t (for older cats). in most cases of secondary gastrointestinal disease, diarrhea is not usually the primary presenting complaint, but since the approach to investigations and management diverge so much, this is an important step to take. biochemistry and urine tests may also show the consequences of diarrhea, such as dehydration and electrolyte abnormalities. a. can aid in the visualization of internal structures of some protozoa . fecal flotation (preferably with centrifugation) a. to find cysts, oocysts, and ova fecal culture should be undertaken with the understanding that bacteria will be cultured; so, interpretation is based on the relevance of the positive culture result. used to evaluate the smear for the presence of trophozoites, such as giardia spp. and tritrichomonas foetus. . place peppercorn size amount of feces on a warm slide and mix with a drop of . % saline (smear must not be too thick, because trophozoites will be easily missed). . apply coverslip. . evaluate systematically for motile organisms using the × magnification. . confirmation at × magnification. adding iodine to a wet mount through the edge of the coverslip can aid in the visualization of internal structures of some protozoa. the direct wet preparation must be examined without any stain for motility first, because staining the preparation kills the organism. methylene blue is useful for identifying trophozoites, particularly those of entamoeba histolytica. this method has little to no diagnostic value for the diagnosis of bacterial-associated diarrhea. used to find cysts, oocysts, and ova in feces. standing (gravitational) flotation methods are easier and quicker but have much poorer sensitivity than centrifugation methods. solutions used in centrifugation flotation methods include zinc sulfate and sheather sugar. . weigh out to g of feces. . mix feces with approximately ml of flotation solution. . pour mixture through a tea strainer into a beaker or fecal cup. . pour strained solution into a -ml centrifuge tube. . fill tube with flotation solution so that a slight positive meniscus forms, being sure not to overfill the tube. . place a coverslip on the tube, and put the tube in the centrifuge. . make sure the centrifuge is balanced. . centrifuge at rpm ( × g) for minutes. . remove the tube and let stand minutes. . remove the coverslip, and place it on a glass slide. systematically examine the entire area under the coverslip at × magnification (i.e., × objective). you may wish to use the × objective lens to confirm your diagnosis and make measurements; however, with practice, most parasites can be identified using the × objective ( × magnification). (fpli) are useful markers of intestinal and pancreatic disease, [ ] [ ] [ ] [ ] but it is important to note that they typically do not give a precise diagnosis. cobalamin and folate are water-soluble vitamins and are readily found in commercial cat foods so that dietary insufficiency is rare, and decreased levels are almost always because of gi disease. these vitamins are taken up by specific receptors in different areas of the small intestine. chronic inflammatory gastrointestinal disease may damage the receptors and lead to decreased serum concentrations of one or both vitamins, provided the disease process is severe and long standing enough to deplete body stores. serum cobalamin and folate concentrations may also be decreased in cats with exocrine pancreatic insufficiency (epi). trypsin-like immunoreactivity is a pancreasspecific marker, and assessment of serum tli is used for diagnosis of epi and pancreatitis in the cat, although the sensitivity of the assay for pancreatitis is low. pli is a marker for pancreatic inflammation and is more sensitive than tli for the diagnosis of pancreatitis. since inflammation of the small intestine may be seen concurrently with pancreatitis, serum tli and pli are useful adjunctive tests in the diagnosis of diarrhea. tli, pli, and cobalamin are stable in serum at room temperature for several days, but folate is unstable so that samples for cobalamin/folate analysis should be frozen (table - ) . samples submitted for folate concentration should not be hemolyzed, because red blood cells contain high levels of folate. in addition, folate is light-sensitive, and samples should be wrapped to exclude light. severe lipemia may interfere with common assays for tli and pli. the main utility of these tests are to indicate that further investigation of gastrointestinal disease is warranted. when a cat presents for weight loss with no overt signs of gi disease, decreased cobalamin or folate can indicate that further investigations with imaging and, ultimately, biopsy sampling are warranted. many clients are more willing to proceed with hematology can be normal in some cats, with changes expected, and so should not be used to rule out any condition. it can be useful, for example, if there is a left shift neutrophilia, indicating acute infection, or eosinophilia, reflecting parasitism. monocytosis can suggest chronic disease that was not suggested by the clinical history. in the case of acute onset diarrhea, the cat may be unwell as a consequence of the diarrhea (e.g., from dehydration) and not because of the cause of the diarrhea. if rehydration is required (with intravenous or subcutaneous fluids, depending on severity of illness), then it is important that biochemistry tests are performed before fluid administration so that any diagnostic clues are not lost by alteration of the profile from the fluid therapy. fever and neutrophilia may indicate the need for antibiotic therapy. if infection is suspected, fecal sampling (see step ) should occur before starting antibiotics. if a cat is unwell from dehydration, then further testing may not be warranted. the clinician should be alert that linear foreign bodies can result in diarrhea (see the section intestinal obstruction). diarrhea of chronic duration (greater than weeks) does require a more thorough investigation at the outset. however, if clinically well, the cat can be managed as an outpatient in the first instance, at least while waiting for results of diagnostic testing. a diet trial with a novel protein is appropriate for a well cat with stable weight. as with any patient managed as an outpatient, follow-up is vital and, in this scenario, includes scheduling revisits. cobalamin, folate, feline trypsin-like immunoreactivity (ftli), and feline pancreatic lipase immunoreactivity invasive diagnostics when a specific marker of the disease in the organ involved has been recognized. caution should be exercised, because either cobalamin or folate may not be reduced with gi disease. in one study of small cell lymphoma, only % of cats were hypocobalaminemic, meaning that if this was the only instigating factor to investigate, nearly one fourth of cats would not have been investigated further. also, cobalamin may be reduced in nonalimentary illness. . to detect hypocobalaminemia that may indicate the need for supplementation for clinical improvement. to recognize pancreatic pathology when fpli is increased. it is important to note that an elevated value gives no indication of the nature of the pancreatic pathology. . to make a diagnosis of epi when the ftli is low. it should be noted that epi can result from other pathology that may require further investigations. veterinarians if the patient is new to the practice) are usually helpful. body condition scoring (using a -point or -point scale) for every cat seen is helpful in recognizing those that are underweight. weight loss often occurs with loss of muscle mass in cats, and muscle mass can be assessed over the ribs and pelvis as well as scapulae and nuchal crest. thickened intestines are also a subjective finding; it is the author's opinion that thickened intestines are actually intestines with increased turgidity, since differences between normal intestines and those with inflammatory infiltrates can be as little as . mm. perhaps more important during the history taking and physical examination are those signs that can point to extragastrointestinal disease. when confronted with a cat showing weight loss or vomiting or diarrhea (or a combination of signs), the clinician should start with trying to distinguish the signs as being either primary gastrointestinal or secondary signs. examples of clues pointing to extragastrointestinal diseases include tachycardia and palpable thyroid nodule, indicating hyperthyroidism, or polydipsia/polyuria, which has a variety of causes but is not typical of primary intestinal disease. inflammatory bowel disease has traditionally been considered an immune-mediated disease. the local immune system of the intestinal mucosa no doubt plays an important role, but recent work has also shown the importance of the normal bacterial population in perpetuating and, perhaps, even initiating pathology. it is known for certain that ibds are an expression of an overanxious immune response, with a recent study indicating increases in inflammatory (il- ), type- immunity (il- p ), and immunomodulatory (transforming growth factor [tgf]-beta, il- ) cytokines. other researchers have found an association with bacterial counts (enterobacteriaceae, e. coli, and clostridium spp.) and abnormalities in mucosal architecture, indicating that mucosal bacteria are involved in the etiopathogenesis. we can summarize these theories by saying that ibds are likely to be a consequence of hypersensitivity reactions to antigens from the intestinal lumen (e.g., bacterial, parasitic, or dietary antigens). this hypersensitivity may occur because of failed immunoregulation (suppressive function) of the gut-associated lymphoid tissue (galt). it is known that granulomatous colitis in boxer dogs is associated with infection, and pathogens may well be found in at least some cases of ibd in cats that cause the immune response and subsequent inflammatory infiltrate of the lamina propria typically seen. although not described specifically in cats, chronic intestinal inflammatory change can impair motility. inflammatory bowel disease (ibd) refers to intestinal inflammatory infiltrates of the small or large intestine (or both) of unknown etiology. the term ibd should strictly be applied to mean idiopathic ibd, thus excluding inflammatory enteritis because of food sensitivities, although common usage has led to ibd referring to intestinal inflammatory infiltrates of both known and unknown causes. ibd is not a diagnostic end point but a description of a series of intestinal diseases that have similar histopathology. recent efforts by the world small animal veterinary association (wsava) gastrointestinal standardization group have led to both diagnostic and classification guidelines , , that encompass chronicity, nonresponse to symptomatic treatment, no specific cause found, as well as histologic confirmation of non-neoplastic intestinal inflammatory changes. there are no obvious breed or gender predispositions, and although cats of any age can be affected, inflammatory intestinal diseases are more likely to occur in middle-aged to older cats ( to years of age or older) than in younger cats. presenting clinical signs include vomiting, diarrhea, and weight loss with increased or decreased appetite. these signs can occur in isolation or together. weight loss without vomiting or diarrhea deserves special mention because not only have several studies , shown this to be the most common presenting sign for ibd, but many veterinarians do not consider primary intestinal disease without the presence of vomiting or diarrhea. weight loss despite normal to increased caloric intake can represent poor absorption of food because of small intestinal disease, although it can also represent maldigestion associated with exocrine pancreatic insufficiency or increased metabolism associated with hyperthyroidism, or even lack of energy utilization associated with diabetes mellitus. conversely, appetite may be reduced, most likely because of nausea. if the large bowel is affected, signs are typically discomfort when defecating, resulting in frequent small volumes of diarrhea, often with mucus and blood; if the large bowel alone is affected, there may be no weight loss. physical examination findings are often nonspecific, but the most consistent findings for small intestinal disease are weight loss (or being underweight in a cat not seen previously) and palpably "thickened" intestines. noting a cat as underweight can be subjective, and prior recorded weights (even from previous paper has suggested that ultrasonographic thickening of the muscularis layer is more likely in cats with intestinal small cell lymphoma than those with ibd, but this change was also seen in % of cats with normal small intestine. inflammatory bowel diseases require histologic findings obtained from biopsy samples for diagnosis, but diagnosis should not be made solely on these findings. the wsava international gastrointestinal standardization group has proposed "an all encompassing definition of inflammatory bowel disease" that comprises clinical criteria, imaging criteria, as well as pathophysiologic criteria. the clinical criteria for the diagnosis of ibd include there are no typical laboratory findings in ibd, and many cats may have entirely normal results from routine biochemical and hematologic investigations. moderate liver enzyme elevations may be seen , , , even in the absence of recognizable hepatic pathology, and this may reflect subclinical secondary hepatic disease, secondary cholestasis, or showering of the liver with inflammatory cells from the small intestine through the portal circulation. other changes can reflect consequences of the intestinal disease, such as azotemia or hemoconcentration reflecting dehydration, or hypokalemia reflecting inappetance. the chronic inflammation may be reflected by neutrophilia, monocytosis, , , , or hyperglobulinaemia. hypocobalaminemia can reflect ileal inflammation, and low serum folate can reflect proximal small intestinal inflammation. typical ultrasonographic findings consistent with ibd are focal or diffuse intestinal wall thickening ( figure - ) ; normal wall thickness is less than or equal to . mm for the duodenum and less than or equal to . mm for the ileum, and large mesenteric lymph nodes with hypoechoic changes may be seen. one study found that ultrasonographic findings correlated with histologic grade of ibd. there is no clear distinction between ultrasonographic changes from ibd and those from small cell lymphoma. one recent therapeutic trial, follow-up visits are vitally important. many cats with small intestinal disease may show initial improvement simply because of the diet having lower residue, since there is decreased substrate for intestinal bacteria to digest and lower osmotic potential. the corollary of this is that failure of one novel protein diet does not mean that all novel protein diets will fail. when food sensitivities are responsible for gastrointestinal clinical signs in cats, the responsible food ingredient is usually a dietary staple. commonly incriminated ingredients are beef, fish, wheat, and corn gluten. a careful dietary history is therefore important. large bowel inflammation typically improves with higherfiber diets, , and attempting a trial with such a diet is certainly appropriate. immune suppressive therapy is the mainstay of ibd treatment, and glucocorticoids, such as prednisolone, are most commonly used. sulfasalazine use for large bowel signs has not been critically evaluated but seems safe and effective. in cats with substantial weight loss or severe clinical signs, such as chronic diarrhea, the author prefers to start with corticosteroid therapy, even if dietary causes have not yet been ruled out. the diet should also be changed to one containing a novel protein, and, if and when clinical signs resolve, an attempt is made to wean the cat from corticosteroid therapy, hopefully to the point of being discontinued. a diet challenge can then be used to confirm the diagnosis of food sensitivity. there are no universal guidelines for doses of corticosteroids. the author prefers the use of orally administered prednisolone to reduce the chance of side effects and will choose the starting dose based on the severity of disease. the starting dose is usually mg/kg, once daily, po ( mg/cat/day for most cats) starting days after biopsies have been obtained to allow time for the mucosa to heal. if there is an improvement noted after a recheck at weeks, the higher dose is maintained for a further to weeks, at which point, many cats are back to their normal weight and are not exhibiting clinical signs. if this is the case, the corticosteroid dose can be weaned down to mg/kg, po (often mg/cat/day) for several months, with continued rechecks scheduled to assess weight, clinical signs, and diet. the goal is to wean down to the lowest effective dose. if hypocobalaminemia is present, cobalamin supplementation may be required. cobalamin is administered parenterally at µg/cat subcutaneously weekly for weeks, then every second week for weeks, then monthly. owners can be shown how to inject their cats (as practitioners routinely do with diabetics). clinicians often consider the assessment of histologic samples to be out of their hands; however, it is important to work with the pathologist by providing good quality samples and a good clinical history, as well as having an open dialogue if the findings are not within expectations. for example, with lymphocytic/plasmacytic infiltrations, the pathologist has the difficult task of distinguishing diseased from normal tissue in a site that is laden with lymphocytes in the healthy state. once deciding the tissue has pathology, the pathologist's next task is distinguishing inflammatory infiltrate from neoplastic infiltrate with normal, mature lymphocytes (as seen in small cell lymphoma). inflammatory change also results in changes to normal tissue architecture, with thickened villi, edema, or erosion of the epithelium being typical changes. clinicians should expect morphologic descriptions as well as assessments of degree and type of inflammation. these difficulties are further compounded with the recognition that histologic grading of mild, moderate, or severe does not necessarily correlate with severity of clinical signs. this means that a cat with severe clinical signs of weight loss and vomiting or diarrhea may have only mild histologic changes (and vice versa). concurrent inflammation of the pancreas and liver with intestinal inflammation was first described in the mid- s, and despite constant reference to this phenomenon at conferences and veterinary websites, there has been little description since then, though one study found % of ibd cases had liver inflammation and % had pancreatic inflammation. the term "triaditis" has frequently been used, but the author prefers to spell this "tri-iditis" to distinguish it from inflammation of the hepatic portal triads. there has been no assessment of prognosis when the pancreas and/or liver are involved, but the author has found no difference in prognosis. many cases diagnosed with intestinal inflammatory infiltrates have these changes because of dietary sensitivity. in one study, % of cats with histologic gastrointestinal changes improved with dietary elimination therapy alone. interestingly, improvement was noted within days compared with the longer duration of weeks often recommended for improvement of dermatologic manifestations of food sensitivities. this careful study made note of the cat's prior diets and likely dietary causes of sensitivities. another study found dietary therapy to be unsuccessful in of cats but no specifics of diets tried are noted. as with any national cancer institute working formulation (nci wf) system. for most veterinarians in practice, the most important distinction is the histologic grade, because low-grade (lymphocytic or small cell) lymphoma has a much better prognosis (and requires different treatment) compared with high-grade (often lymphoblastic) or intermediategrade lymphoma. for the purposes of simplicity and practicality, only small cell lymphoma and high-grade lymphoma will be addressed here. the prognosis and treatment for intermediate-grade intestinal lymphoma should be considered as for high-grade lymphoma. small cell lymphoma was first described in human pathology in . earlier, small lymphocytes were considered end-stage cells without the ability to divide. in cats small cell lymphoma is most commonly associated with the gastrointestinal tract or skin. small cell neoplasia can be a confusing concept, since our traditional ideas of malignant neoplasia focus on rapidly dividing cells. the confusion is compounded by various terms used in the literature, such as lymphocytic lymphoma, low-grade lymphoma, well-differentiated lymphoma, or diffuse lymphoma; another term, epitheliotropic malignant lymphoma predominantly applies to small cell lymphoma, and other papers fail to distinguish these lymphomas from lymphoblastic lymphosarcoma (the traditional, aggressive form). "small cell lymphoma" seems to be most widely used term, though the author prefers "lymphocytic lymphosarcoma," since it is more descriptive. intestinal small cell lymphoma can be considered as a severe lymphocytic intestinal infiltrate, the most common form of which is commonly called ibd. not only is lymphocytic ibd hard to distinguish histologically from lymphocytic lymphosarcoma, but the approaches and treatments are similar. several reports have suggested a relationship between the two conditions in that inflammatory infiltrates may become neoplastic over time. , , prevalence the true prevalence of intestinal small cell lymphoma is unknown, but several recent studies have indicated similar rates to inflammatory bowel diseases, with kleinschmidt et al noting small cell lymphoma cats compared with with intestinal lymphocytic infiltrates, evans et al reporting cases compared with with ibds , and baral et al diagnosing cases compared with with ibds. traditionally, % of feline lymphosarcoma is regarded as intermediate or high grade, but this may not be the case within the gastrointestinal tract. fondacaro et al found % of gastrointestinal lymphoma to be lymphocytic ; a more recent paper found some cats seem resistant to conventional therapy. if this is the case, the diagnostic findings should be re-assessed to ensure no steps were missed or findings disregarded; the cat should be reexamined to look for emergence of other signs; and the pathologist who reads the histology should be contacted to recheck the findings. some cases of apparently resistant ibd are actually food sensitive, but it can be difficult to find the incriminating diet source, and commercial diets are not always effective. if underlying infectious causes have been entirely ruled out and the practitioner is certain of the diagnosis of idiopathic disease, immune suppressive therapy can be increased by either increasing the dose of prednisolone or using other agents, such as chlorambucil, typically at mg/cat, po, every second day. it has been suggested that cats with eosinophilic inflammation may be more likely to be refractory to standard therapy. side effects of immunosuppressive therapy are rare but include inducing diabetes mellitus, immune suppression, delayed healing, and gastrointestinal ulceration. reported doses of sulfasalazine to manage large bowel ibd are to mg/kg, po, once daily for to days. because this drug is usually only available as mg tablets, one eighth of a tablet, providing a dose of . mg, is usually appropriate for most cats. in some countries, it is possible to have a compounding pharmacist formulate the drug into more convenient tablet sizes or as an oral suspension. cats are generally regarded as susceptible to salicylates, and possible side effects include vomiting or diarrhea, or anemia. the exact pharmacodynamics of this drug are not known; so, caution for extended use should be exercised and the drug withdrawn if any possible adverse signs are noted, but there are anecdotal reports of extended use of this drug without adverse consequences. a survey of the online veterinary cancer registry (http://www.vetcancerregistry.com) identified % of all submitted feline tumors to be intestinal tumors. approximately % of reported feline small intestinal tumors were lymphomas. adenocarcinomas accounted for %, and other tumor types reported included mast cell tumors and leiomyosarcomas. "lymphoma in veterinary medicine: no longer a oneword diagnosis" was the title of an editorial in a recent issue of the veterinary clinical pathology journal, and this is nowhere truer than in the feline gastrointestinal tract! a recent study classified cases of feline gastrointestinal lymphoma both histologically and immunophenotypically, and it found eight different categories according to the revised european and american lymphoma/world health organization (real/who) classification system and six categories according to the approximately equal numbers of high-grade and lowgrade gastrointestinal lymphoma. older cats are more at risk of small cell lymphoma, with mean or median ages reported from to years. younger cats with the disease have, however, been recognized. , , , no breed or gender predispositions have been definitively recognized. two larger studies have suggested a skew to males with males compared with females in one report, and males compared with females in the other ; most other studies looking at gender and breed did not clearly distinguish between lymphoblastic and lymphocytic neoplasia. clinically, it is impossible to distinguish cats with ibds from cats with small cell lymphoma. this is hardly surprising when even histologic distinction can be difficult! therefore cats will present with weight loss or vomiting or diarrhea at a similar frequency to those with ibd. weight loss has been recognized as a presenting sign in % to % of cases, diarrhea in % to % of cases, and vomiting in % to % of cases, with various combinations of these signs also possible. other variable signs are lethargy and inappetence or, conversely, polyphagia. , , , these findings can be summarized by stating that cats with gastrointestinal small cell lymphoma can present with any combination of signs relating to the gastrointestinal tract. intestinal small cell lymphoma is typically a diffuse disease, and therefore multiple areas of the alimentary tract are usually affected. in studies where different locations of the small intestine were assessed, the jejunum was most commonly affected ( %), with the ileum frequently affected ( % to %), and duodenal pathology slightly less prevalent ( % to %). , although the numbers of cats assessed in these studies are small, the important fact that the duodenum is not always affected needs to be recognized, which has important implications for how biopsy samples are obtained, because lesions beyond the duodenum are likely to be beyond the reach of an endoscope. further difficulties in precise diagnosis may arise, since non-neoplastic lymphocytic infiltrates (e.g., ibd) are often found in other locations along the intestinal tract. , , the stomach is also affected in % to % of small cell lymphoma cases. , , although not fully assessed, involvement of the colon appears rare. local lymph node involvement is common, being noted in up to % of cases. this percentage may be even higher, because many studies assessed lymph node cytology from ultrasound-guided fine-needle aspirates, which may miss spread to the lymph node, because the population of neoplastic lymphocytic cells is indistinguishable from the normal population of lymph node cells. histology is required to assess changes in lymph node architecture. liver involvement is not uncommon but not thoroughly assessed. one study noted liver lymphocytic neoplasia in of cats with small intestinal lymphocytic neoplasia, another found of affected cats in which the liver was biopsied, another noted of cats had liver involvement, and a further study detected neoplasia "in the lymph nodes, liver, or both" in all cats with intestinal small cell lymphoma. the pancreas may also be involved. , this may be akin to the noted association of lymphocytic inflammation of intestine, pancreas, and liver that has been dubbed tri-iditis. ultrasound findings may not suggest extragastrointestinal involvement. in the case of liver pathology, ultrasonography may show no changes in as many as % to % of cases. , focal nodular changes and he patomegaly have been recognized as ultrasonographic signs of hepatic small cell lymphoma. both lymphocytic ibd and lymphocytic neoplasia are often recognized simultaneously in the same cat, , and numerous authors have suggested that lymphocytic ibd may be a precursor to intestinal lymphoid neoplasia. , if this is the case, then antigenic factors, such as bacterial population changes or food sensitivities, could be considered primary initiating factors for small cell lymphoma since they are potential underlying etiologies of ibds. however, neoplasia also requires genetic mutations to occur (often affecting regulation of cell death and cell survival), and these may be initiated by the inciting antigenic factors or the ongoing inflammatory changes. as opposed to other feline lymphoid neoplasia, no association has been made with felv infection. , , , intestinal lymphocytic lymphosarcoma begins in the superficial mucosa and progresses to involve the entire mucosa and submucosa; then advancing in a perivascular pattern into the tunica muscularis, eventually infiltrating all four intestinal tunics. lymph node and other organ (such as liver or pancreas) involvement likely represent metastasis through lymphatics and perhaps hematogenously. more distant metastasis is not reported. serum or plasma biochemistry and hematologic findings are typically nonspecific. however, this testing is important as part of the diagnostic workup to rule out extra-gi disease, such as hyperthyroidism or diabetes mellitus. common biochemistry findings are mild to moderate increase of liver enzymes, such as alanine aminotransferase (alt), aspartate aminotransferase (ast), and/or alkaline phosphatase (alp). , , , as with ibds, these liver enzyme changes may or may not represent overt hepatic disease. albumin may be reduced but is normal in most cases , , ; azotemia may be present and may be of prerenal origin or represent concurrent renal disease. in one study, of cats were hypocobalaminemic; of cats had low folate, but of had elevated folate; and of cats had increased ftli. hematologically, a mature neutrophilia with or without monocytosis is sometimes present, representing the inflammatory response; lymphopenia may be present as a stress response. anemia may be present and may occur as a result of chronic slow gi blood loss, and in some cases, ulceration, or it may be because of chronic disease; hemoconcentration is also possible, reflecting dehydration. , , , palpable or ultrasonographically visible thickened intestines ( % to % of cases) , , , or mesenteric lymph nodes ( % to % of cases) , , , are no more or less likely to be present in comparison with ibds. there are no defined ultrasound guidelines for cats with intestinal small cell lymphoma, because most prior papers do not distinguish between small cell and lymphoblastic neoplasia. , a more recent paper found of cats undergoing ultrasound examination had diffuse small intestinal wall thickening, with a mean of . mm (range, . to . mm; median, . mm), and focal mural thickening of mm was noted in one cat. in many cases, against expectations, intestinal wall layering was preserved. these findings also mean that of cats had ultrasonographically normal intestinal wall thickness (≤ . mm for the duodenum and ≤ . mm for the ileum). if affected, jejunal lymph nodes may appear as hypoechoic and enlarged; in the same study, of cats had lymph node changes with a mean diameter of . mm (range, . to mm; median, mm) compared with the normal diameter of less than or equal to . mm. none of these findings can definitively distinguish small cell lymphoma from ibds; although one recent paper has suggested that ultrasonographic thickening of the muscularis layer is more likely in cats with intestinal small cell lymphoma (figure - ) than those with ibd, this change was also seen in % of cats with a normal small intestine. however, thickening of the muscularis layer together with lymphadenopathy was recognized in % of those cats with small cell lymphoma compared with % of those with ibd and % of cats with no small intestinal pathology. biopsy samples and histopathology are required for definitive diagnosis. an example of jejunal and mesenteric lymph node appearance at laparotomy is shown in figure it is difficult to distinguish between lymphocytic inflammation and small cell lymphocytic neoplasia in any location; some histopathologic features that might help in differentiating the ends of the spectrum may include therefore become known as the fondacaro protocol. this consists of a combination of prednisolone and chlorambucil given orally by the client at home (table - ). the rationale is that a slow alkylating agent, such as chlorambucil, is more appropriate to use for the slowly dividing, well-differentiated lymphocytes that cause disease. this can be contrasted to the aggressive chemotherapeutic agents required for the rapidly proliferating cells in lymphoblastic neoplasia that is typically associated with lymphosarcoma. reported response rates to this protocol are excellent, with % to % of cats achieving complete clinical remission, reported median survival times ranging from to months for those cats responding to therapy, and reports of individual cats surviving as long as months. , , the original reported protocol comprised prednisolone ( mg/cat, po or mg/kg, po) given daily with chlorambucil pulsed by administration of mg/m for days every weeks. a more recent study dosed prednisolone similarly, but chlorambucil was given as continuous therapy of mg/cat, po every second or third day. no mucosal congestion, edema, or fibrosis in lymphocytic neoplasia, compared with ibd . epitheliotropism, or homing of neoplastic t lymphocytes to the mucosal epithelium in lymphocytic neoplasia these features can be seen in figure - . each of these criteria may be useful but are unlikely to be definitive. further studies that may not be routinely available but which may be helpful are immunophenotyping; most reports have found purely t lymphocytes in most cases of intestinal small cell lymphoma , , , (figure - ). . clonality; the detection of a clonal population of cells, as recently described for intestinal lymphocytic lymphosarcoma, would be closest to providing the basis for definitive diagnosis. effective treatment of feline intestinal small cell lymphoma was brought to light by fondacaro et al and has cat to be weaned off corticosteroids, with chlorambucil continued as monotherapy (as is often the case with humans). iatrogenic diabetes mellitus usually needs to be managed with insulin therapy, at least initially (see chapter ) . high-grade lymphoma or lymphosarcoma is the traditional style of aggressive, rapidly dividing lymphoid neoplasia that carries a much poorer prognosis than small cell lymphoma. most early studies do not distinguish grade of neoplasia; so, the prevalence of low-grade and high-grade alimentary lymphoma are difficult to assess. several recent studies found a similar prevalence of each, , but the seminal paper describing small cell lymphoma found only cases of lymphoblastic lymphoma compared with cases of small cell lymphoma. this ratio of approximately one high-grade gi lymphoma case for every three low-grade cases more closely approximates the rate found in the author's practice. the reported median ages of affected cats range from to years, but cats as young as year old have been diagnosed. most papers note that males are overrepresented, and siamese cats may also be overrepresented although most affected cats are domestic shorthairs. , , , , precise signalment is difficult to determine from the literature, because many papers assess all anatomic locations of lymphoma without necessarily breaking down epidemiologic data for each anatomic site. also, there are few comparisons to a reference population. the association of lymphoma with felv infection is well established and documented and is covered in chapter ; fiv has also been shown to be lymphomagenic. , since the control of felv through vaccination began in the s, nonretroviral-associated lymphoid neoplasia has become more common, and the rates of intestinal lymphoma have, in fact, increased since felv infection rates have decreased. the underlying causes for this increase are not known. the association with inflammation from ibds was noted for small cell lymphomas, and perhaps there is a spectrum from lymphocytic ibd to small cell lymphoma to high-grade lymphoma. that some cats are more likely to have inflammatory changes become neoplastic is suggested by a paper noting higher lymphoma rates in cats with vaccine-associated sarcomas (a neoplastic condition where the role of chronic inflammation is well noted). similar protocols are used in humans with both lowgrade (i.e., lymphocytic) lymphosarcoma and chronic lymphocytic leukemia. , some studies with humans have indicated that continuous therapy with chlorambucil results in prolonged survival, although metaanalyses have not been able to determine optimum dosing and scheduling of administration of chlorambucil or other alkylating agents in these conditions in humans. , although we do not have enough data to critically compare pulsed therapy to continuous dosing, the study assessing continuous dosing appeared to have a lower number of cats completely responding, although those cats that did respond had a longer median survival than those in the studies assessing pulsed chlorambucil dosing. , the differences may also relate to the definitions used for complete response. the chlorambucil dose of mg/cat, po every second day (or third day) is often chosen because of the ready availability of mg coated tablets, the breaking of which can expose the owner to these cytotoxic medications. chlorambucil can be compounded into smaller doses, thus allowing daily dosing of mg capsules. the author has used this dose to apparent good effect, but there has been no critical assessment. it is unknown whether involvement of lymph nodes or other organs, such as the liver, affects prognosis. the only study of substantial size to include extra-gi locations found anatomic location was not prognostic for response or survival time. in another study, of the five cats with liver involvement, two cats did not survive more than months, yet the other three lived longer than years, with two surviving longer than years. a study of hepatic small cell lymphoma suggests the density of neoplastic lymphocytes may influence survival, and density may relate to the stage of the disease when diagnosis occurs. adverse effects of chlorambucil are rare, but gastrointestinal signs, myelosuppression, and myoclonus have all been reported. gastrointestinal signs, such as vomiting, diarrhea, or inappetence, can be difficult to distinguish from continuation of the gastrointestinal disease diagnosed. these signs are usually self limiting. myelosuppression is also possible with thrombocytopenia reported. , monoclonus has been reported on one occasion. it is ideal to check hematologic parameters every months for cats receiving chlorambucil. continuous therapy using lower doses of chlorambucil may be less likely to lead to these adverse effects. high doses of corticosteroids can induce diabetes mellitus, and thus blood glucose should be checked regularly. if diabetes occurs, the author has found that budesonide ( mg budesonide is generally considered to be equivalent of mg prednisolone) can be substituted for prednisolone, since it has reputed lower systemic effects (though no assessments of this drug's effectiveness in cats have been made). an alternative is for the of distinction of intestinal layering as shown in figure - . the area of lymphomatous infiltration is hypoechoic, because it contains a uniform cell population without much reactionary fibrous tissue. mesenteric lymphadenomegaly is common (figure - ) , as are changes in other organs, such as kidney, liver, or pancreas. ascites may also be seen. , although ultrasonographic distinctions predominate, there is considerable overlap between ultrasonographic findings with small cell lymphoma and high-grade lymphoma. the clinician must not lose sight of the fact that microscopic distinctions are required to diagnose either condition. cytologic diagnosis of high-grade lymphoma from fine-needle aspirates (fna) is much more likely than with small cell lymphoma. this is because there is usually a focal lesion, and the neoplastic cells are a monomorphic population of large, immature cells (i.e., that are not normally seen in tissue). sometimes, mixed lymphoid whether the underlying cause is retroviral or chronic inflammation or anything else, the pathogenesis of highgrade intestinal lymphoma, as with small cell lymphoma and other neoplasia, depends on chromosomal changes that affect regulation of cell growth and death, resulting in malignant transformation and clonal expansion of immature lymphocytes. metastasis can occur in one third to two thirds of cases, , with involvement of mesenteric lymph nodes most commonly noted, but spread to liver, spleen, kidneys, and thorax is also possible. a recent survey of gastrointestinal lymphoma found that most cases ( of ) involved the small intestine (including that also involved the stomach and that also involved the large intestine), and of cases involved the large intestine only. cats with high-grade alimentary lymphoma often present similarly to those with other gastrointestinal diseases. typical clinical signs are weight loss, anorexia, lethargy, vomiting, diarrhea, or a combination of these signs. repeated studies have found cats with no vomiting or diarrhea; in one study, of cats had only anorexia or weight loss on presentation. cats with large bowel pathology usually present, as with other causes of colitis, with increased urgency, and small, frequent amounts of diarrhea, often with blood or mucus. cats with large bowel neoplasia of any form can present for constipation caused by intestinal obstruction. palpation of an abdominal mass has been recognized in % to % of cases, , but the corollary of this is that % to % of cases did not have a palpable mass. it is also important to note that up to % of cats with intestinal small cell lymphoma, and a number with ibds, have palpable mesenteric lymph nodes; so, a palpable abdominal mass is not a specific indication of high-grade neoplasia. many cats have palpably thickened bowel loops. hematology and plasma or serum biochemistry findings are also nonspecific. increased liver enzymes may or may not indicate liver involvement. anemia may be recognized and can be non-regenerative, reflecting chronic disease or slow blood loss, or regenerative if there is more substantial blood loss associated with mucosal ulceration. hypoalbuminemia can be because of blood loss or intestinal protein loss. hypercalcemia of malignancy is a possibility but not commonly reported. despite nonspecific signs, laboratory testing is important to rule out extra-gi diseases and help manage consequences of enteric disease, as with small cell lymphoma and ibds. ultrasonography commonly shows a focal intestinal thickening (of to mm) with partial or complete loss although noted as the next most common intestinal neoplasia, after the various forms of lymphoma, adenocarcinoma is seen relatively infrequently in practice. most cats are more than years old, , , males may be overrepresented, and several studies have recognized a distinct overrepresentation of siamese cats. , three distinct forms have been described : cats typically present with nonspecific signs of gastrointestinal disease but can present with obstructive signs. cats with large bowel neoplasia can present for tenesmus or hematochezia and even constipation, if the lesion is obstructive (or partially obstructive). on physical examination, an abdominal mass is palpable in approximately % of cases, but other findings are usually nonspecific. anemia can be found if mucosal ulceration has occurred, but there are no distinctive laboratory findings. lesions can occur anywhere along the intestinal tract. one study of cases found % of feline intestinal adenocarcinoma lesions were present at the ileum or ileocolic junction. twenty-five to percent of cases have metastasis at the time of the diagnosis, and this is a poor prognostic indicator. , , radiology may show a mass lesion or intestinal obstruction, and ultrasonography can localize lesions to an intestinal origin. the ultrasonographic appearance of the proliferative, circumferential, outwardly expansile populations (of immature lymphoblasts and mature lymphocytes) are seen if a germinal lymphoid follicle is aspirated, and precise diagnosis may be difficult if there are a large number of lymphoblasts. fna samples are best obtained with ultrasound guidance. the cytologic sample quality is greatly improved by not aspirating when the needle is visualized in the mass but merely "pecked" into the mass so that the needle is merely acting to finely "core" the mass. on removing the syringe and needle, the hub of the needle is removed before drawing air into the syringe, the hub is replaced, and the sample within the needle is expressed onto a slide. usually, the decision to diagnose by cytology from fna is based on the ultrasonographic appearance of a mass. since there is substantial crossover of ultrasonographic appearance of intestinal masses, laparotomy for excision is often performed with the affected bowel submitted for histology. except when intestinal obstruction has resulted, there is no therapeutic benefit of excising a gastrointestinal lymphoma (which requires excision and anastomosis), but there is minimal room for doubt when a histologic diagnosis is achieved. the response to therapy for high-grade intestinal lymphoma is significantly worse than that for small cell lymphoma. , further, response to therapy for highgrade intestinal lymphoma appears to be worse than for lymphoma in other anatomic locations. precise remission rates and survival times are difficult to quantify, because many studies assess lymphoma from multiple locations and do not necessarily differentiate response of gastrointestinal lymphoma or report the grade of lymphoma. with remission rates reported from % to % , , and a median survival time of up to weeks (range, to weeks), it can be said with some certainty that some cats respond to therapy for reasonable durations. multiple authors have noted that the best prognostic indicator is response to an initial treatment cycle, , , which should prompt clinicians to encourage owners to start therapy and decide whether to continue based on the cat's response. there are several published chemotherapeutic protocols, , , , , but all follow the same principles of using medications to target specific phases of the cell division cycle (such as l-asparaginase and vincristine) with other medications that interrupt multiple phases of the cell cycle (cyclophosphamide and doxorubicin). targeting the cancer cell in different ways enables more cells to be killed, reduces the toxicity of the individual drug used, and reduces the likelihood of resistance to a specific drug. several authors have noted increased success with the addition of l-asparaginase and doxorubicin to protocols. , , chemotherapy for lymphoma is covered in more detail in chapter , oncology. look promising, but only two cats assessed had gastrointestinal mast cell neoplasia. lomustine was used unsuccessfully in one cat with sclerosing mct; another cat with sclerosing mct received eight treatments of vinblastine and had a survival time of greater than years. adenomatous polyps have been reported in the duodenum and ileum and can result in intussusception. cats of asian ancestry, predominantly siamese, are greatly overrepresented, and most reported cases have been males. cats usually present for vomiting or hematemesis that, surprisingly, can be very acute in onset; complete intestinal obstruction may result. , , resection is curative, with survival times of more than years reported. eosinophilic sclerosing fibroplasia has recently been described in a series of cases and is not strictly neoplasia. the ulcerating mass lesions that can occur anywhere from the stomach to the colon are often grossly and histologically mistaken for neoplasia. there appears to be no breed predisposition or age predisposition (with ages ranging from weeks to years), but of cases ( %) were castrated males cats compared with of ( %) female spayed cats. eighty-four percent of cats presented for vomiting, % presented for weight loss, and of ( %) cats had peripheral eosinophilia. all cases had a palpable abdominal mass. the pyloric sphincter was the most common site, and lesions in this location were mostly considered unresectable. fourteen of cats ( %) had bacterial colonies within microabscesses and necrotic foci within the lesion. the bacteria recognized were predominantly gram-negative rods, but antibiotics did not seem to be clinically effective. the bacteria are suspected to initiate the lesions, having been embedded after foreign body penetration. there are no specific treatment recommendations, but excision, where possible, would be prudent; corticosteroids appear to be helpful adjunctive therapy. survival times are difficult to estimate since many cats were euthanized because neoplasia was suspected and follow-up times were short (up to months) for the remaining cats. there are very few reports of intestinal leiomyosarcomas in cats, , which have been reclassified as gastrointestinal stromal tumors. these tumors may be more likely to arise from the ileocecocolic junction. resection, if possible, is usually recommended, with survival times of to months reported before recurrence. the author owns a cat with this tumor where resection was not form is better described than the annular, constrictingband form with minimal outward enlargement. in these cases, sonographically, a solitary segmental intestinal mural mass is present and characterized by circumferential bowel wall thickening with transmural loss of normal sonographic wall layers. the thickening can vary in echogenicity but may be hypoechoic and may be symmetric or asymmetric. there is no definitive distinction, however, from lymphosarcoma, mast cell tumor, smooth muscle origin tumors, or even segmental benign inflammatory bowel disease. surgical resection is the treatment of choice. there seem to be two distinct groups in terms of survival time postresection: . short-duration survival (euthanasia or death within weeks of surgery) . long-duration survival (mean survival time of months, with a number of cats surviving greater than years) , because clean margins improve prognosis, for large bowel adenocarcinoma, subtotal colectomy may be required for complete excision. because of the potential for success after resection, it is recommended to excise unidentified masses at the time of surgery. other forms of intestinal neoplasia are recognized infrequently, and include intestinal mast cell tumors, adenomatous polyps, eosinophilic sclerosing fibroplasia, gastrointestinal stromal tumors (leiomyosarcoma), and hemangiosarcoma. mast cell tumors (mct) are often cited as the third most common form of feline gastrointestinal tumor, but the intestines are a far less common site than cutaneous, splenic, or hepatic mast cell neoplasia. , masses are usually segmental nodular thickenings that occur in older cats. the masses are indistinguishable ultrasonographically from other tumors, such as lymphoblastic lymphosarcoma. a recent series of cases described a variant of feline intestinal mast cell tumor, dubbed sclerosing mast cell tumor, for which neoplastic cells form a trabecular pattern with dense stromal collagen. additionally, eosinophilic infiltrates were moderate to marked in most cases. these cases can be confused histologically with eosinophilic enteritis, gastrointestinal stromal tumor, or fibrosarcoma. surgical resection is recommended, but lesions are commonly infiltrative or metastasize widely, and there are few reports of successful treatment. lomustine (dosed to mg/m , po every to weeks) has recently been assessed as adjunctive chemotherapy for mast cell neoplasia in various locations, and results recognized in that many intestinal bacteria can be found in healthy animals. further antibiotic administration can result in increase of other bacteria. fungal causes of diarrhea are usually recognized from histology of biopsy samples. it remains to be seen whether the recent ready availability of pcr panels looking at a number of infectious causes of diarrhea will be beneficial for recognizing pathogens that had previously been misdiagnosed or a hindrance for readily recognizing commensal organisms not necessarily causative of the clinical signs being investigated. the most common viral, bacterial, and mycotic causes of diarrhea in cats are described below. parasitic gastrointestinal diseases are covered later in this chapter. viral causes of diarrhea are not usually specifically diagnosed, since, with the exception of the canine fecal elisa possible, and the cat appears healthy months past diagnosis (figure - ). cats with intestinal hemangiosarcoma often present with anemia, and the disease appears to be highly metastatic. the intestines appear grossly thickened by dark red tissue. the small and large intestines seem to be affected with similar frequency. removal of macroscopic disease is recommended, but often the full extent of the severity is only recognized at surgery. the prognosis is poor. suspicions of infectious causes of diarrhea should be aroused in younger cats, cats from shelters, or cats with immune suppression. when considering infectious causes of diarrhea, clinicians should assess whether the diarrhea is large bowel or small bowel in origin and correlate this with specific pathogens that are likely to cause clinical signs as shown in table - . to increase the diagnostic yield of fecal examination for parasitic causes of diarrhea, wet smears and appropriate fecal flotations should be performed on fresh fecal samples (< hour old). it is appropriate to administer broad-spectrum anthelminthics, even if fecal tests are negative. bacterial and viral causes of diarrhea should be considered when the cat is systemically unwell with fever. fecal culture should be performed in these circumstances, but the limitations of this testing need to be yersinia enterocolitica current theory of fip pathogenesis involves initial infection with fecv and then mutation to fipcv in small numbers of susceptible individuals. , routine serologic testing for fecv in cats with diarrhea would neither prove correlation with the clinical signs nor affect how the disease is managed and so is not recommended. cats with fecv diarrhea should be managed with symptomatic therapy of fasting, then reintroducing a bland diet and supportive care with fluid therapy if necessary. other viruses, such as astrovirus, reovirus, rotavirus, and torovirus-like agent, have been recognized to cause diarrhea in cats, but their roles as pathogens are unclear. they are not routinely recognized in practice, since electron microscopy of fecal samples is necessary for diagnosis and is not routinely performed. management is supportive care with appropriate fasting, then reintroduction of bland diets and fluid and electrolyte replacement if necessary. successful identification of a known bacterial pathogen from a fecal sample does not necessarily mean that the agent found is the cause of disease in the cat. although a number of bacterial pathogens have been demonstrated to cause diseases when specific pathogen-free (spf) cats are experimentally infected, these same organisms can be found in healthy cats. the differences between healthy and diarrheic cats that have bacteria found in their feces may relate to virulence factors of the organism, or host factors (local or systemic immunity) of the cat. there is no definitive answer for this quandary. the author's opinion is that • if a diarrheic cat is systemically unwell and has a fever, then feces should be cultured. • if an organism is isolated that is known to cause signs consistent with those the cat is showing, the cat should be treated appropriately. campylobacter diarrhea is usually caused by c. jejuni. clinical signs of infection are poorly documented, but most cats are asymptomatic. younger cats are more likely to have clinical signs and hemorrhagic, mucoid diarrhea has been reported. diagnosis can be from culture of feces or swabs, and the organism is quite hardy; so, it usually survives transport to the laboratory. in individual cases, the organism has not been cultured after antibiotic treatment, , but it is not definitively proven that antibiotic therapy affects the natural course of the disease. antibiotics that can be used are amoxicillin-clavulanate ( mg/kg, every hours, po) for parvovirus, routine definitive tests are not available. clinical signs of panleukopenia (feline parvovirus infection) are more likely to occur in kittens, with the highest morbidity and mortality occurring between and months of age. subclinical cases in older (susceptible) cats probably go unrecognized. the organism is very stable in most environments, and infections mostly occur from environmental contact. peracute cases can result in death within hours, with little or no warning signs. acute cases often have fever, depression, and anorexia, with signs beginning approximately to days before presentation. vomiting is usually bile tinged and unrelated to eating. diarrhea does not always occur, and when it does, it is usually later in the course of the illness. leukopenia is not pathognomonic, because this can also occur with acute bacterial infection (e.g., salmonellosis can present identically). commercially available elisa tests for canine parvovirus antigen in feces can detect feline parvovirus; however, shedding may have ceased by the time clinical signs occur, and vaccination can result in positive test results for up to weeks. aggressive fluid therapy, usually at twice maintenance rates, is usually required. broad-spectrum antibiotic coverage is used to prevent or treat secondary bacterial infection from viral injury of intestinal mucosa. parenteral antibiotics are preferred to prevent the possibility of further gastrointestinal irritation. the author recommends calculating iv doses and introducing appropriate amounts of antibiotics to the fluids bag to create a constant rate infusion (cri); cefazolin can be used in this way at mg/kg/ hours, and betalactam cris are commonly used in human medicine. aminoglycosides or fluoroquinolones can be used concurrently at routine doses if fever persists after hours or the cat is moribund on presentation, but care must be used with these agents. aminoglycosides are potentially nephrotoxic, and fluoroquinolones have been reported to result in cartilage damage in growing animals, although this has not been demonstrated clinically in cats. fluoroquinolone retinal toxicity has been seen in all animals. cats that survive the first week usually recover, and prior infection imparts lifelong immunity. vaccinations are highly effective for disease prevention. feline enteric coronavirus (fecv) mostly causes mild, self-limiting diarrhea and must be distinguished from feline infectious peritonitis coronavirus (fipcv), which is essentially always fatal and for which diarrhea is not a typical sign (but is possible). the most widely accepted or fluoroquinolones, such as enrofloxacin ( mg/kg, once daily, po) for durations of to days. macrolides, such as erythromycin ( to mg/kg, every hours, po), are regarded as the drug of choice for humans but can cause gastrointestinal side effects. clostridium difficile has been recognized in up to % of diarrheic cats. clinical signs are typically acute onset watery diarrhea and anorexia. diagnosis has been made with detection of toxin a or toxin b in fecal samples using elisa. although these tests have not yet been validated for cats, they may prove to be a useful aid to diagnosis and are available for testing of equine feces at some commercial laboratories. nontoxigenic strains exist; so, positive culture alone does not ensure diagnosis. metronidazole ( mg/kg, every hours, po) for approximately days is the treatment of choice. clostridium perfringens typically results in large bowel diarrhea with tenesmus, mucus, and hematochezia, but small bowel signs can also be seen. pcr testing for enterotoxin a is commercially available and may prove to be a useful adjunct in diagnosis. antibiotics that can be used include metronidazole ( mg/kg, every hours, po), tylosin ( to mg/kg, twice daily, po), or amoxicillin-clavulanate ( mg/kg, every hours, po) for days. escherichia coli is a ubiquitous organism within the feline intestinal tract, and it would be unusual not to successfully culture e. coli from the feces of both healthy and unwell cats. when e. coli is associated with clinical signs of gastrointestinal disease, it is mostly as an opportunistic pathogen, with overgrowth resulting from changed environmental conditions, such as inflammation from other pathology or another pathogen. there are also specific strains of e. coli that are true pathogens because of virulence factors not present in commensal e. coli; these include enteropathogenic e. coli and enterotoxigenic e. coli, which both induce a watery diarrhea, and enterohemorrhagic e. coli, which produces a diarrheal syndrome with copious bloody discharge and no fever. pcr testing is commercially available to identify pathogenic strains of e. coli , ; although not offered at routine veterinary laboratories, this testing is available to veterinarians, and laboratories offering these services can readily be found online. diagnosis should also document histologic lesions corresponding to the strain of e. coli identified. there is emerging resistance to e. coli worldwide in all species of animals, including humans. this includes the typical therapies for gram-negative bacteria of beta-lactamenhanced penicillins and fluoroquinolones. a major risk factor is prior antibiotic usage, because commensal organisms are exposed to antibiotics. pcr testing does not enable antibiotic sensitivity testing, and fecal culture may not be able to distinguish pathogenic from nonpathogenic strains; so, sensitivities may not be an accurate reflection of the pathogenic organism. pcr testing for genes that impart resistance to e. coli have recently been described but are not yet commercially available. in some circumstances, supportive care with fluid and electrolyte replacement may be all that is required while the cat's immune system combats the infection. empiric therapy could include beta-lactam-enhanced penicillins (such as amoxicillin-clavulanate at mg/kg, every hours, po), fluoroquinolones (such as enrofloxacin at mg/kg, once daily, po), or cefovecin ( mg/kg, every weeks, sc), but the clinician must be aware of possible drug resistance. salmonella typhimurium infection is possible from ingestion of infected prey, infected food sources, or from a contaminated environment, including the veterinary hospital. the resulting clinical signs depend on the number of infecting organisms, the immune status of the cat, and the presence of concurrent diseases. infection rates in cats (and humans) have been correlated with seasonal bird migrations, and the illness has been dubbed songbird fever, but there is no distinction between this and other salmonella infections. clinical signs usually begin to days after exposure, starting with fever (often > ° c [ ° f]), malaise and anorexia, and progressing to diarrhea, vomiting, and abdominal pain. hematology can show leukopenia with a left shift and nonregenerative anemia, and biochemistry results are usually nonspecific. diagnosis is based on isolation of the organism by culture or identification with pcr, but care should be taken to correlate pathogen identification with clinical signs since, as with most gi pathogens, the organism can be isolated from healthy animals. as with e. coli, antibiotic resistance is widespread, with one united kingdom survey finding the multiple drugresistant strain dt to be the most frequent bacteriophage type identified. treatment should be reserved only for those cats showing systemic signs, because routine antibiotic use in treating salmonellosis induces drug-resistant strains and prolongs the convalescent excretion period. antibiotic choice should be based solely on sensitivity findings, since resistances are so widespread and unpredictable. this means that if the organism has been identified by pcr, then culture of feces must also be undertaken. the duration of treatment must be long enough to eliminate fecal excretion of the organism, prevent the chance of relapse, and reduce the chance of resistance developing; up to days has been advocated. , these cautions are particularly important because of the zoonotic potential of salmonellosis. linear foreign bodies have traditionally been considered more common than discrete foreign bodies in cats, , , but a study from a primary care facility indicated only % of foreign body cases were because of linear foreign bodies. the larger case load of linear foreign bodies at referral institutions noted in earlier studies may indicate the abilities of primary care practitioners to recognize and effectively deal with discrete foreign body obstructions. most studies have found that cats with intestinal foreign bodies are generally younger (mean, . to . years), with a notable exception being obstruction from trichobezoars where three of five cats in one study were years or older; the greatest risk factor appears to be length of hair coat. no specific breed predispositions have been described but siamese and siamese-related cats have been noted to have oral fixations and so may be expected to be overrepresented with intestinal foreign bodies. clinical signs will vary depending on the type of foreign body (linear or discrete), the position of obstruction, and the time since obstruction. most cats present for anorexia or vomiting. partial obstruction can result in diarrhea (which can be bloody). foreign body obstruction is typically considered an acute condition, with duration of obstruction because of a linear foreign body, measured from the onset of clinical signs to diagnosis, reported to range from to days. , , however, one paper demonstrated chronic, intermittent, gastrointestinal disease from a linear foreign body of a -month duration demonstrating that partial obstruction can result in a chronic course. physical examination may or may not reveal abdominal pain, palpable abdominal mass (or plication), dehydration, or fever. all cats presenting for anorexia or vomiting should have the underside of the tongue evaluated for the presence of a linear foreign body. applying gentle pressure with a thumb in the intermandibular space to elevate the tongue is an effective way to visualize lesions or foreign bodies in the sublingual area (see figure - ) . life-threatening consequences can result from the interactions of local and systemic factors that arise from intestinal obstruction. locally, damage to the mucosa from traction and pressure of the foreign object can cause hemorrhage, ischemia, and necrosis. systemically, hypovolemia, toxemia, and acid-base and electrolyte imbalances can ensue. complete intestinal obstruction by discrete masses results in gas and fluid distention of the lumen proximal other bacterial causes of diarrhea have been reported in cats, such as yersinia enterocolitica, yersinia pseudotuberculosis, clostridium piliforme (tyzzer's disease), and anaerobiospirillum sp. specific diagnosis of these (and other bacterial infections) may be found in the course of investigation. management follows the principles of supportive care and appropriate antibiosis based on sensitivity testing. small intestinal bacterial overgrowth (sibo) has not been specifically described in cats. the criteria defined for dogs is a fasting bacterial count in duodenal juice of greater than organisms/ml and is often recognized with other chronic gastrointestinal diseases. healthy cats appear to have at least this number of upper intestinal bacterial with a range of to /ml recognized. bacterial overgrowth could potentially occur with ileus or intestinal inflammation of any underlying cause. foul-smelling small bowel diarrhea with no specific pathogen recognized may be an indicator of this condition, as could an increase in bacterial metabolites, such as folate. if suspected, it is appropriate to manage with broad-spectrum antibiotics, such as metronidazole ( to mg/kg, every hours, po) or amoxicillin ( mg/ kg, every hours, po) for an extended duration such as to days. alterations in intestinal flora have been recognized after such treatment ; however, any advice for this "condition" is entirely empirical. all efforts should be directed at identifying a precise underlying cause. mycotic and other infectious agents are only rarely recognized as intestinal pathogens in cats. diagnosis is made by histologic and microbial analysis of samples obtained at biopsy. possible agents include histoplasma capsulatum, aspergillus spp., candida albicans, and pythium insidiosum. intestinal obstructions arise most commonly as a result of neoplasia in older cats and foreign body ingestion predominantly in younger cats. , , less common causes include intussusception and granulomatous inflammation (e.g., from fip) ; tapeworm infection, with greater than worms acting as a linear foreign body, has also been reported. other listed causes are volvulus, intestinal torsion, incarceration of bowel in a hernia, adhesions or stricture, intramural abscess or hematoma, and congenital malformations. may not occur if the obstruction is partial or intermittent, or if vomiting results in less fluid present. since most foreign body obstructions in cats are proximal, identifiable dilatation may not be recognized for this reason. linear foreign bodies present further challenges for radiographic recognition; the following typical radiographic signs , may or may not be present: to the obstruction. most gas accumulation is a result of swallowed air, which is predominantly nitrogen that cannot be absorbed by the intestinal mucosa. gas also arises from bacterial fermentation. fluid accumulates as a result of increased secretions (saliva, bile, and secretions of gastric, pancreatic, and small intestinal origin) and retention of fluid already ingested, and it can be augmented by local hemorrhage. since most intestinal obstructions in cats do not reach the midjejunum, reabsorption of fluids that normally occurs at the jejunum and ileum is impaired. linear foreign bodies, such as string, dental floss, or elastic toys, require proximal anchoring, usually under the tongue or in the pylorus (for example, by part of a toy attached to elastic). peristalsis moves the free end of the "string" through the intestinal tract, resulting in pleats of intestines around the foreign body. as the foreign body is forced against the intestinal mucosa, the mucosa becomes edematous, and even partial penetration affects mucosal integrity, allowing systemic entry of bacteria. intraluminal bacterial populations increase for both discrete and linear foreign bodies as a result of stasis. mucosal permeability can be affected by prolonged luminal distention, allowing entry of bacteria and toxins systemically or into the peritoneal cavity. direct entry of bacteria to the peritoneal cavity, causing septic peritonitis, can result from perforation of the intestinal wall from linear foreign bodies or sharp discrete foreign bodies, such as toothpicks or plastic toys. definitive diagnosis requires identification of the foreign body retrieved at surgery or in some cases, by endoscopy. this may be aided greatly prior to surgery by diagnostic imaging. however, imaging findings, particularly in the case of partial obstructions, may be subtle enough that obstruction of no identifiable cause is recognized or no overt signs are apparent. laboratory findings are not helpful in the precise diagnosis but are important to assess fluid and electrolyte balances that must be corrected. cats rarely help practitioners by ingesting radiopaque objects, but on the rare occasions that they do, these can be observed easily on plain radiographs. nonopaque foreign bodies depend on dilatation of the intestine from gas and fluid accumulation proximal to the obstruction for radiographic recognition (figure - ) . one study has suggested that if the jejunal diameter is greater than . times the length of the cranial end plate of the second lumbar vertebra, then intestinal obstruction is the most likely abnormality. care must be taken that the jejunal and not duodenal diameter is measured and that the radiographs must be positioned strictly lateral, because an oblique view can alter the measurement of the lumbar vertebra. however, dilatation of an obstructed intestine successful treatment of foreign body obstruction requires evacuation or removal of the foreign body as well as correction of any bacteremia or endotoxemia, acid-base or fluid imbalances. discrete foreign body obstruction requires surgery or endoscopy to remove the object. in some specific circumstances, linear foreign body obstruction may be managed conservatively by cutting the anchor point below the tongue and allowing the cat to pass the foreign body by peristalsis. however, the decision to manage a cat conservatively must be done with the cat hospitalized, with fluid therapy and antibiotic coverage and a clear recognition on behalf of the practitioner and the owner that surgery may subsequently be required. cutting a sublingual linear foreign body may be achieved in a conscious cat by applying pressure with the thumb of one hand in the intermandibular space to elevate the tongue and gently grasping it using gauze with the other hand while a second person cuts the line with a suture cutter. there is a chance of a small nick on the sublingual surface. if the cat will not tolerate the procedure, sedation is appropriate. when cutting the line, the nature of the linear foreign body should be assessed (i.e., is it more or less likely to cut mucosa). in one study, cats with linear foreign bodies were managed conservatively with cats subsequently requiring surgery. the authors of that paper created guidelines that will be adapted here. conservative management should be attempted if the cat • is presented acutely (within days) after known ingestion of a linear foreign body • has a sublingually fixed linear foreign body that can be cut • has no overt signs of peritonitis • altered gas pattern with luminal gas collecting in small bubbles instead of normal curved tubular columns. this can be subtle when there is only minimal involvement of the intestine but overt when involving the entire small intestine. commashaped gas patterns are more likely to occur with linear foreign bodies. contrast radiography can aid diagnosis for both discrete and linear foreign bodies but should be used with caution because intestinal perforation may be present. nonionic iodinated agents that are typically used for myelography (such as iopamidol or iohexol) should be used, since barium is irritating to the peritoneum and oral iodine compounds are hypertonic. hypertonic compounds may draw fluid into the stomach and intestines after oral administration, with the potential of creating further fluid and electrolyte imbalances in an already compromised patient. ultrasonography is a very useful diagnostic tool, particularly for discrete foreign bodies, where, in most cases, there is overt distention of the small intestines with intraluminal fluid apparent (figure - ) . this modality has not been extensively assessed as an adjunct to diagnosis of foreign body intestinal obstruction in cats specifically, although there are several papers assessing dogs and small numbers of cats that agree with its utility. , , linear foreign bodies are more difficult to assess ultrasonographically, but plicated bowel can be recognized, sometimes with the foreign body seen as a hyperechoic line centrally. si a technique has been described for removal of linear foreign bodies by making a single enterotomy incision proximally and passing a red rubber catheter over the linear foreign body aborally, milking the foreign body within the catheter through the colon for retrieval from the cat's anus by an assistant. this technique is not always effective, because it can be hampered if the foreign body is knotted or does not run smoothly through the red rubber catheter. if the affected bowel segment demonstrates evidence of necrosis or perforation on the mesenteric border of the intestine, resection and anastomosis should be performed. necrosis is indicated by dark discoloration, thin intestinal wall, poor arterial pulsation, poor capillary bleeding, or lack of peristalsis. end-to-end anastomosis can be accomplished using a simple interrupted appositional pattern or a modified simple continuous appositional pattern with the same type of suture material used for enterotomy closure. , intraabdominal masses causing intestinal obstruction are often presumed to be neoplastic but can also be of infectious origin. resection, where possible, is always recommended, because resection of neoplasia (if no metastasis) can offer a good prognosis, , , , and infectious causes may be managed with adjunctive therapy after definitive diagnosis. intestinal obstruction in older cats is more likely to be secondary to neoplasia. any neoplasia can cause obstruction, but adenocarcinoma , and adenomatous polyps , are reported to cause obstruction more often surgical intervention is mandatory if • clinical signs (e.g., vomiting or anorexia) persist or deterioration occurs with conservative management • the cat has overt signs of peritonitis • the linear foreign body is fixed at the pylorus some authors disagree with attempting conservative management, since a perforated intestine from a linear foreign body reportedly carries a % mortality rate, , and early surgical intervention is never an incorrect decision. this should be balanced with the observation that cats can carry a linear intestinal foreign body, such as an elastic cord for a -month duration without intestinal perforation. however, fishing line, for example, would not be so forgiving! surgery to remove an intestinal foreign body (figures - and - ) should be considered an exploratory laparotomy. that is, the aim of the surgery is not only to remove the foreign body but to assess the entire intestinal tract and abdomen for other foreign bodies or pathology. enterotomy to remove discrete foreign bodies should always be distal to the obstruction, because the intestine is likely to be compromised proximal to as well as overlying the obstruction, thus delaying healing and creating the potential for surgical dehiscence. linear foreign bodies require multiple enterotomy incisions, since pulling the object out through a single incision could create iatrogenic intestinal perforation. the anchor point (either sublingual or pylorus by gastrotomy) must be released in the first instance. enterotomy incisions are closed with / synthetic, monofilament, absorbable suture material, such as polydioxyanone (pds) or equivalent, in either a simple interrupted or simple continuous pattern. , removal of a discrete foreign body (a piece of leather) at laparotomy. enterotomy to remove discrete foreign bodies should always be distal to the obstruction, because the intestine is likely to be compromised proximal to as well as overlying the obstruction. this is the same cat as in the radiology image in figure - . affected bowel is required, with anastomosis of the healthy tissue. there appears to be no benefit to enteroplication, which can result in significant ileus. there is no benefit to performing resection-anastomosis if the intussusception does reduce manually. , the prognosis depends on the underlying disease process and the chronicity of the intussusception, and therefore how debilitated the cat is at presentation, however, prognosis is mostly good, with survival reported in up to % of cases, though recurrence can occur in some cats with idiopathic disease, often at different locations of the intestinal tract. constipation is defined as infrequent or difficult defecation associated with retention of feces within the colon and rectum. prolonged constipation results in harder than other types of neoplasia. please refer to the sections on intestinal neoplasia earlier in this chapter for more details. granulomatous inflammation causing a single focal intestinal lesion can lead to obstruction in the same way that neoplastic change can. feline infectious peritonitis (fip) can present as focal lesions, often in the colon or ileocecocolic junction. in the case of fip, the focal lesion is usually an indicator of multisystemic disease; so, resection does not help prognosis. the fungus-like organism, pythium insidiosum has also been reported to cause granulomatous lesions, resulting in intestinal obstruction from large extraluminal masses that are approximately fist sized. resection with adjunctive itraconazole ( mg/kg) for months after surgery was a successful treatment. intussusception refers to invagination or prolapse of one portion of the intestine into the part of the tract that either precedes or follows it. there is a bimodal age distribution with intussusceptions in older cats, most likely associated with neoplasia (or ibd in some cases) ; underlying causes for younger cats are ill defined and may be idiopathic in many cases, , but associations with parasitism and, in one case, a linear foreign body, have been made. siamese and burmese cats seem to be overrepresented. the most common locations are the ileocolic region and the jejunum. , , , affected cats present with nonspecific signs of gastrointestinal disease, such as anorexia and lethargy. vomiting is not necessarily a presenting sign; diarrhea may occur. abdominal palpation reveals a mass in most cases. plain and contrast radiography only show evidence of obstruction and usually do not help define that the bowel has intussuscepted. , , , ultrasonography is very useful for diagnosis, because a distinctive pattern of alternating hypoechoic and hyperechoic concentric rings (figure - ) is present in transverse sections. , sometimes, the target lesion seen can be hard to distinguish from the pathology of other intraabdominal masses, such as lymph nodes, and in these cases, the size of the lesions can help, because the width will always be greater than mm with an intussusception (because the sum of at least four intestinal wall widths cannot be less) and is often greater than mm. surgical correction is always required, and manual reduction is typically not possible because there is usually significant venous infarction, edema, and congestion (figure - ) as well as adhesions from fibrin and effusions from the affected bowel. , if the intussusception does not reduce manually, resection of the and drier feces that become impacted, and this is known as obstipation. chronic, recurrent constipation and obstipation can result in megacolon, which refers to persistent increased bowel diameter that is not responsive to therapy. megacolon is not a specific disease entity; it may be considered the most advanced stage in the spectrum of chronic constipation. in most cases, constipation can be managed quite simply if the underlying cause is determined and dealt with. a comprehensive list of causes of constipation is noted in table of course, multiple factors can interact. for example, an older cat may have renal disease and so will be dehydrated to some degree and have arthritic hips and so be reticent to squat. the presenting signs of constipation are usually evident to owners and include straining in the litter box and producing hard dry feces, if at all. sometimes, however, owners can misinterpret signs. cats can strain because of lower urinary tract problems, and, if no urine is produced, some owners assume the problem is because of constipation. some constipated cats can intermittently have diarrhea because of direct colonic irritation from hard dry feces and so may present for diarrhea and not constipation. cats can also present for less specific signs, such as anorexia, lethargy, weight loss, and even vomiting. , vomiting can occur because of colonic receptors stimulating vagal afferent endings, which, in turn, can stimulate the chemoreceptor trigger zone. sometimes owners are concerned that their cat is defecating less, but the cat has just changed its diet to a much lower-residue diet and so is producing less feces. a full dietary history is an important aspect of the initial assessment. physical examination should confirm presence of feces in the colon and assess the degree of impaction. the presence of feces can usually be confirmed by abdominal palpation. in constipated cats, the colon is often palpated as a long firm tube extending cranially; sometimes, feces can be palpated to and around the colic flexure. alternatively, the feces may be palpated as large, discrete fecal concretions (that can sometimes be hard to distinguish from intraabdominal masses such as lymph nodes). if there is any doubt of the presence or degree of fecal impaction, survey abdominal radiographs should be taken. a lateral view taken in a conscious cat should be adequate to confirm the diagnosis. the physical examination should also assess for contributing causes, including musculoskeletal conditions. any recent trauma should be taken into account. the hips and lumbosacral region should be assessed for pain. the degree of flexion and extension of the hips should be gently assessed. the lumbosacral spine can be assessed by running two fingers on either side of the spinous processes. the cat will flinch in painful areas. any arthritic change is magnified in an underweight cat, since there may be less muscle mass and the joints may bear a heavier load. any suspicions of underlying musculoskeletal abnormalities can be confirmed with radiographs. neurologic assessment should also be performed. subtle changes just affecting colonic innervation will not be apparent on physical examination alone. however, an assessment of proprioception, placing reflexes, and gait should at least be performed to assess for lumbosacral spinal cord disease. anorectal abnormalities or lesions should be evaluated. impacted or infected anal sacs can lead to reticence to defecate; and therefore anal sacs should be assessed and expressed. because this is painful for most cats, the cat should be held by an experienced assistant. the author prefers expressing one anal sac at a time with well-lubricated gloves and the index finger within the rectum and the thumb positioned externally. a rectal exam can be performed with a well-lubricated (gloved!) middle finger, feeling over the pelvic rim for masses as well as assessing if the colon closes over (squeezes) the finger. if the colon feels open around the finger, this can be an indicator of impaired colonic innervation but does not imply that this is a permanent change. if there are impacted feces continuing to the anus, rectal examination is not possible until this has been cleared. if a cat finds anal gland expression or rectal examination too painful to tolerate (based on the clinician's judgment), these procedures should be done under sedation. hydration and electrolyte status are also important factors in the constipated cat. chronic renal disease is defined by azotemia (in conjunction with inadequately concentrated urine), which means the cat must be dehydrated to some degree. plasma or serum biochemistry and urinalysis can be used to diagnose renal disease, assess degree of renal disease, or recognize prerenal dehydration. electrolyte changes including hypokalemia and hypocalcemia may also contribute to reduced colonic smooth muscle function. in young to middleaged cats of apparent good health and hydration, blood difficulty defecating and pass hard, dry stools but do not have fecal impaction at the time of examination. after the obstructing feces have been removed, steps must be taken to ensure colonic motility and smooth passing of feces. medical management of constipation traditionally involves laxatives and prokinetic agents. these may not be required in straightforward cases. as long as there is no obstructive lesion, cisapride at . mg/ cat, every hours, po is very safe and can be instituted with a view to reducing the dose to once daily after to days and discontinuing if signs remain abated. doses of up to . mg/cat, every hours, po have been reported. cisapride is only available from compounding agencies in most countries. an osmotic or lubricant laxative (table - ) may be used concurrently at reduced doses as necessary. reducing the fecal bulk produced is an important part of long-term management. traditional dietary recommendations are to increase the amount of fiber. , , , increased dietary fiber results in production of shortchain fatty acids, which have been demonstrated to stimulate feline colonic smooth muscle contractions. however, dietary fiber is also classified as a bulk laxative and so, by definition, will increase fecal bulk. in humans dietary fiber has been considered a mainstay of therapy for constipation, but a recent review concluded that many patients with more severe constipation have worsening symptoms when increasing dietary fiber intake. because megacolon is believed to be the end result of chronic dilatation, , it is the author's firm belief that initial dietary efforts should be directed to reducing fecal bulk and thus introducing a low-residue diet. reduced dry matter intake reduces stool volume, and the author has found that recurrence rates of constipation reduce greatly when cats are transitioned to entirely wet food diets. wet food diets also help ensure adequate water and urine assessments are usually not required at an initial presentation for constipation. in all cases, the same principles of management apply: . ensure removal of obstructing feces . ensure colonic motility and smooth passage of feces . reduce fecal bulk . ensure adequate hydration . manage underlying problems the first step is to ensure obstructing feces are removed. in simple cases, the cat will evacuate feces after use of a glycerin or sorbitol pediatric rectal suppository. another option is administration of a microenema, such as microlax (mcneil consumer healthcare, fort washington, pa.), which contains ml of sodium lauryl sulfoacetate. these products act to lubricate the colon wall and therefore facilitate the passage of feces. the author prefers to use one or two of these within the consult room to observe the cat defecating (the cat must be provided with a litter tray!). the outside tube should be lubricated with the suppository contents before carefully inserting and then expressing the rest of the contents. there are also stimulant laxatives (containing bisacodyl) and emollient laxatives (containing sodium docusate) that have reportedly been used. if a rectal suppository vial cannot easily be inserted because hardened fecal content obstructs its entry, a more substantive enema will be required (sometimes requiring sedation or anesthesia), and this is covered in the next section on management. some cats present for the abdominal wall with the other hand, but great care must be taken with this maneuver, because the devitalized colon can be perforated more easily. , enemas as described are painful for the cat, and opioid analgesia is recommended at the time of anesthesia. opioids can reduce peristalsis in humans, but having evacuated the bowel, the pain relief is more important than this transient effect. an alternative to enemas is administration of an oral polyethylene glycol (peg ) solution (e.g., colyte, golytely). a nasoesophageal tube is placed and the solution is given as a slow trickle ( to ml/kg/hour) over to hours. defecation usually results in to hours. in a retrospective study of cats, median time to defecation was hours and the median total dose of peg was ml/kg. a no adverse effects were noted. a cat that has been obstipated needs supportive therapy when discharged. there are no controlled comparisons of the various therapies noted in table - ; the author prefers cisapride . mg, every hours to every hours, po (first thing in the morning, when the owner returns from work, when the owner goes to bed), and lactulose syrup ml/cat, every hours, po. a cat that has been so severely obstipated that an enema under anesthesia is required can be expected to continue these medications lifelong. to reduce fecal bulk and decrease the opportunity for recurrence, low-residue canned foods (or sachets) are preferred for cats that have become obstipated. some cats may benefit from high-fiber diets. as with simple initial episodes, canned food helps maintain adequate hydration, and at home subcutaneous fluids may be used additionally in cats with chronic kidney disease. with repeat episodes or severe obstipation, investigations for an underlying cause should be thorough and include evaluation for colonic mass obstructions. a review of published cases indicated that % of cases of megacolon are accounted for by idiopathic megacolon ( %), pelvic canal stenosis ( %), nerve injury ( %), or manx sacral spinal cord deformity ( %). although most cases are idiopathic, an attempt should be made to identify and treat any specific underlying causes. megacolon is not specifically defined in cats. it has been described as "generalized colonic dysfunction manifesting as severe colonic dilation and fecal impaction," or a "severely and irreversibly dilated and hypomotile" colon and "a subjective evaluation of the diameter of the colon, usually based on radiographic assessment." there are specific radiographic guidelines for humans with megacolon, in that a colonic diameter of more than . cm at the level of the pelvic brim is considered diagnostic. intake and therefore help maintain hydration. however, increased dietary fiber is beneficial for some cats, and trial and error may be required to determine whether a high-fiber or low-residue diet will be of benefit to each individual cat. in one report, cats with recurrent constipation refractory to traditional medical and dietary management were successfully treated with a psylliumenriched dry extruded diet. a after month on the diet, cats had no clinical signs of constipation. the remaining cat was clinically normal after months on the diet. improvement was noted in of cats after only days of dietary therapy. measures should be taken to ensure adequate hydration. maintaining adequate hydration is particularly relevant for cats with chronic kidney disease that have impaired ability to conserve water. changing to wet food diets helps increase water intake. some cats with chronic kidney disease may need additional fluid support, such as subcutaneous fluids administered by the owner at home on a regular basis. underlying problems may be minor and simple to manage, such as an anal gland abscess, or more involved, such as reduced pelvic outflow, as a result of prior trauma. arthritis is a common underlying factor in many older cats and may be managed with prudent use of nonsteroidal agents (see chapter ) . in cases of obstipation, the cat is more likely to be debilitated to some degree; so, laboratory investigations to assess plasma or serum biochemistry parameters as well as hematology and urinalysis are ideal. any hydration deficit or electrolyte abnormalities should be corrected before the anesthesia that is often required to remove the obstructing feces. rectal suppositories and microenemas are usually ineffective in obstipated cats. enemas are often required to remove impacted feces in such circumstances. the enema solution must be warmed and introduced slowly to avoid vomiting. the typical volume required is to ml/kg (so, up to approximately ml/cat). the enema solution can be an isotonic electrolyte solution or tap water, and mild soap can be added (but any soap used must not contain hexachlorophene, which is neurotoxic if absorbed); mineral oil can be used ( to ml/cat) as a lubricant or docusate as an emollient ( to ml/cat), but the two agents must not be used together since docusate promotes mucosal absorption. sodium phosphate-containing enemas must not be used, because they can induce severe hypernatremia, hyperphosphatemia, and hypocalcemia in cats. often, the enema solution alone is insufficient to reduce the fecal mass, and manual manipulation of the feces by abdominal palpation is required. sometimes the feces must be broken down by a gloved finger perrectum while the colon is massaged manually through radiographically, in the lateral view, the normal colon should be approximately the same diameter as the length of the body of the second lumbar vertebra. in cats, however, "there are no published guidelines for determining megacolon, so, diagnosis of abnormal colonic dilatation is subjective." however, one author has suggested that "as a rule of thumb, the diameter of the colon should be less than the length of the body of the seventh lumbar vertebra (l )." this author continues, "enlargement of the diameter of the colon beyond times the length of the body of l is indicative of chronic large bowel dysfunction and an explanation must be sought." a recent paper found that of cats with no gastrointestinal disease had a colon diameter greater than the length of l ; however, no assessment of constipated cats was made. in practice, many cats with megacolon have a colonic diameter far exceeding this guideline ( figure - ). one study of cats with megacolon found the mean diameter of the colon was . times greater than the length of the seventh lumbar vertebra (median, . ; range, . to . ), but in general, objective descriptions of this condition are lacking in the veterinary literature. the definition of megacolon in cats should include functional as well as radiographic guidelines. in the absence of broadly recognized radiographic recommendations, the author proposes that the o'brien rule-ofthumb guidelines (as noted above) be introduced until a more comprehensive study can establish other radiographic diagnostic criteria (or confirm these). the author therefore proposes to define megacolon as dilatation of the colon, to more than . times the length of the seventh lumbar vertebra, which is refractory to medical and dietary management. practitioners can expect the radiographic assessment of colonic dilatation to exceed this guideline in cats with megacolon and, conversely, there are likely to be cats having colonic distention greater than this amount that will respond to medical and dietary management and can therefore not be defined as having megacolon. by the definition used above, megacolon is refractory to medical and dietary therapy; so, to be defined as having megacolon, a cat may have had several episodes of obstipation managed by enema as well as dietary trials (with both low-residue and high-fiber diets) and medical therapy with cisapride and an osmotic or emollient laxative; yet the cat will still obstruct with feces. in these circumstances, the only possible therapy is subtotal colectomy. subtotal colectomy refers to surgical excision of % to % of the colon, whether it is grossly diseased or not with preservation of the ileocolic junction (icj). this approach has resulted in a more favorable clinical response than when the icj is also excised. , when preserving the icj, it has been noted that, in some rare cases, it can be difficult to join the proximal segment of colon to the distal piece of descending colon because of the tethering effect of the ileocecocolic blood vessels. in these cases, sacrificing these vessels and removing the icj (i.e., total colectomy) is recommended to facilitate approximation of the ileum to the distal colonic segment. a recently described technique using a biofragmentable anastomosis ring, compared with sutured anastomoses, showed no discernible effect on prognosis. prognosis following subtotal colectomy is generally good. a review of multiple papers, totaling over cats that had undergone subtotal colectomy, found the most commonly reported perioperative complication was diarrhea or loose stools immediately after surgery. in the majority of individuals, stool consistency improves without further treatment so that within to weeks of the surgery soft, formed stools are developed. diarrhea can persist in a small number of cases. in the longer term, in some cats, constipation can eventually return, but this can usually be managed by dietary and medical therapies. pathology of the rectum or anus is relatively rare in cats and therefore poorly described in the veterinary literature. consequently, published information is often not referenced, suggesting it expresses the authors' opinions. readers are directed to surgical texts for details and approaches about surgical corrections. the anal sacs are paired cutaneous evaginations situated between the internal and external sphincter muscles. these sacs store secretions from alveolar and sebaceous glands that reside within the sacs. each anal gland has an associated duct that opens to the skin surface just lateral to the anus. , normal anal gland secretions have only very recently been described and vary markedly; the color can be white, brown, orange, yellow, tan or gray, and consistency can range from watery to thick and creamy, with two thirds of cats having solid portions within the secretion. on microscopic examination, epithelial cells are commonly seen, with most cats having some neutrophils present. bacteria are commonly recognized as are, on some occasions, yeasts. bacteria seen in this study were mainly gram-positive cocci ( %) or gram-negative cocci ( %). gram-negative or grampositive rods were also seen but were rarely the dominant bacterial population. with such a wide range of normal secretions, it is difficult to diagnose any pathology from the nature of the secretion alone. however, blood is infrequently recognized, and neutrophils are typically present in only small numbers in normal secretions. anal sac diseases described in cats include impaction, inflammation (sacculitis), infection, abscessation, and neoplasia (essentially the same as in dogs). , it has been contended in dogs that sacculitis and abscessation are an extension of impaction. it is not known in dogs or cats what the predisposing causes are, but suggested underlying reasons are loose stools (that are less effective at expressing the sac during defecation), local swelling or edema occluding the duct, and obesity. the author's observations have also indicated that constipation can result in anal sac impaction because of less frequent expulsion of the sac contents; the resultant pain of the anal sac impaction can lead to further constipation, thus establishing a cycle. the retention of secretions may predispose to sacculitis, but impacted anal sacs do not always result in inflammation. abscessation is a likely sequel to sacculitis. cats usually present for licking, scratching, or biting at the perineal area and can present for scooting (or dragging their anus) as dogs do. other presenting signs can be inability to sit or settle, a lump seen by the owner, or a generally unwell state. expression is the only management required for impacted (and not infected) anal sacs. the author prefers expressing one anal sac at a time with well-lubricated gloves and the index finger within the rectum and the thumb externally. this is painful for most cats; so, the cat should be held by an experienced assistant, and it is sometimes not possible without some degree of sedation. with frequent episodes, underlying causes should be investigated. sometimes, trial-and-error diet change to manipulate the nature of the feces to either more (highfiber diets) or less (low-residue diets) bulk help reduce the frequency of episodes. obesity should be managed by reduced caloric intake, but dietary management for this should also take into account the nature of the feces. overt infection may be recognized by pus secretion from the anal sacs, which will have a high numbers of neutrophils. this can be managed by broad-spectrum antibiotics, such as amoxicillin/clavulanate or cephalosporins. a single treatment with a nonsteroidal antiinflammatory drug, such as meloxicam, can be given in animals with appropriate hydration and without other illness. anal sac abscesses often present already open and draining. many heal well by secondary intention with antibiotic treatment until they are closed over; so rechecks are required before the completion of an antibiotic course. large abscesses may require surgical drainage with the insertion of a penrose drain and management as for a cat fight abscess. it must be remembered that wounds in this area are easily re-infected by fecal contamination. recurrent impaction, sacculitis, or infection may require anal sacculectomy (as in dogs). this procedure should be delayed until infection is cleared. the procedure is similar to that performed in dogs. reports of anal sac/gland neoplasia were confined to sporadic case reports, , until a large case series was recently published. in this study, cases of anal gland carcinoma were recognized at a private diagnostic laboratory during a -year period, with submissions from practices. this indicates that, for most practices, this condition will be seen, at most, once every years. affected cats ranged in age from to years (median and mean, years); female (mostly spayed) cats were overrepresented ( % of cases), and siamese cats may have been over-represented ( . % of cases). the number of siamese cats with anal sac neoplasia was times greater than the number of siamese cats in the laboratory reference population. affected cats presented for dyschezia, recurrent constipation, change in the nature or volume of feces, b). a low-residue wet diet is recommended to reduce fecal bulk during the healing period. rectal prolapse occurs as a result of a disease process that causes chronic straining, such as intestinal conditions that result in diarrhea and tenesmus . conditions that result in constipation or other intestinal obstruction . lower urinary tract diseases . dystocia and/or perineal swelling or ulceration, sometimes with purulent or hemorrhagic discharge. most tumors were originally interpreted as and initially managed as anal sac abscess. presumptive metastasis in liver, lung, or abdominal lymph nodes was recognized by physical examination or radiography in six cats; one cat was hypercalcemic. excision appeared to be curative (with a -to -year follow-up period) in of cats undergoing surgery for resection or debulking (others had only incisional biopsy performed). for the remaining cats with known postsurgical outcome, median survival was only months, with a % -year survival rate (with none of these cats surviving to years). atresia ani is a developmental defect of the anal opening or terminal rectum (see figure - ). kittens usually present within days or weeks of birth with abdominal distention, discomfort, tenesmus, restlessness, vomiting, and/or loss of appetite. there are several anatomic variations : • type i: a membrane over the anal opening remains, with the rectum ending as a blind pouch just cranial to the closed anus. • type ii: the anus is closed as in type i, but the rectal pouch is located somewhat cranial to the membrane overlying the anus. • type iii: the rectum ends as a blind pouch cranially within the pelvic canal (rectal atresia), whereas the terminal rectum and anus are normal. • type iv: occurs in females and atresia ani exists with a persistent communication between the rectum and the vagina (rectovaginal fistula). this fistula can occur with a normal anal opening as well. most reported cases have been type iv, , , , , and this has also been recognized with concurrent sacrococcygeal agenesis. surgical correction has been described for type ii and type iv , atresia ani in cats. the reader should consult these references for surgical advice; possible complications include megacolon after prolonged obstruction, postsurgical anal stricture, and fecal incontinence because of sphincter dysfunction. foreign bodies in cats rarely obstruct the gastrointestinal tract distal to the jejunum ; however, large fecal balls resulting from constipation can, additional to constipation or obstipation, cause distention of the anus. this distention can result in inflammation of the anal sphincter with loss of tone ( figure - , a) , which, in the author's experience, is temporary with correction of the underlying cause of constipation. it can take some weeks for the dilated anus to return to normal (figure - , with antibiotics, such as cephalosporins, and regular cleansing. prolapses are usually classified in three ways. first degree: prolapse of only mucous membrane . second degree: prolapse of full rectal wall thickness . third degree: prolapse is sufficient to bring mesorectum outside the anus the prolapsed rectum is obvious but must be differentiated from ileocolic intussusceptions, which have been described with neoplasia. this distinction can be made by inserting a thermometer through the anus alongside the prolapsed mass. insertion will not be possible for an intussusception but will be for an anorectal prolapse. the prolapsed tissue must be assessed for viability, and management must include determining and managing the underlying cause as well as management of the prolapse. in simple cases where the mucosa is viable, the prolapse can be reduced with lubrication and gentle pressure. a temporary purse-string suture may be required to prevent recurrence. perineal dermatitis is often confused with gastrointestinal or urogenital disease, because there are often copious sebaceous secretions that can mimic fecal or urinary secretions. perineal dermatitis can result from flea or other allergies but also fecal or urine scalding associated with diarrhea or urinary incontinence, respectively. skin fold dermatitis can also occur in obese cats ( figure - ). episioplasty has been described to correct this, but the author has found that stringent dieting can result in improvement while managing the skin fold dermatitis these populations. the reported prevalence for each parasite varies greatly with the population studied, the geographic location of the population, and the sensitivity of the diagnostic test used to study that population. the presence or absence of diarrhea is not a reliable predictor of whether a particular cat is infected with or shedding a parasite. in fact, most cats with diarrhea do not harbor enteric protozoa. on the other hand, most cats with diarrhea because of enteric pathogens will shed those organisms, often intermittently. it is important to remember that infection with most gastrointestinal parasites may not cause clinical signs. therefore detection of a pathogenic parasite in a cat with diarrhea does not necessarily prove causation. a search should always be undertaken to identify other causes of diarrhea prior to convicting a cat of having diarrhea because of a particular parasite. in addition, co-infections or the presence of other noninfectious causes of diarrhea can result in more severe diarrhea that is often refractory to treatment for the parasite. treatment will be more rewarding if all potential causes of diarrhea are identified in the patient. enteric parasites with zoonotic potential occur commonly enough that cats, particularly those with diarrhea and who are owned by immunocompromised persons, should be evaluated for those pathogens. , the following is a discussion of the most common enteric parasites found in cats. for more on parasite prevention and control, see chapter , and for more on zoonotic enteric parasites, see chapter . ollulanus tricuspis is an almost microscopic nematode worm infecting the stomach of domestic and wild cats. the worm measures less than mm long. the larvae of o. tricuspis develop and hatch within the uterus of the female worm. they develop to maturity in the stomach of the cat where it is capable of re-infecting the host. the worm is transmitted to other cats that ingest the vomitus of an infected cat. clinical signs shown by infected cats include vomiting, anorexia, and weight loss. , histologic findings in infected cats include lymphocytic-plasmacytic gastritis, lymphoid hyperplasia, and mucosal fibrosis. gross lesions may be absent, or the cat may develop nodular gastritis. one report suggested the parasite may have been a contributing factor in the carcinogenesis of a gastric adenocarcinoma in an infected cat. . a common theme when discussing the prevalence of most gastrointestinal parasites in cats is that they occur more commonly in younger cats and in cats housed in crowded conditions, such as catteries and shelters. it is likely an increased chance for transmission exists in lungs. after further development in the lungs, the parasite migrates up the trachea and is swallowed. adult s. felis and s. planiceps burrow into the wall of the small intestine, while adult s. tumefaciens lives in the colonic mucosa. ova may be shed in the feces or hatch in the intestinal tract. autoinfection occurs if larvae become infective and penetrate the intestinal wall before being shed. ova and larvae that are shed develop into freeliving adult worms. the prepatent period is between and days. , clinical signs and diagnosis signs of a strongyloides spp. infection are usually absent. , lung migration may cause cough or respiratory distress. the presence of the parasite in the intestinal tract may result in diarrhea and weight loss. strongyloides tumefaciens is associated with the formation of small, worm-filled nodules in the colon. identification of strongyloides spp. larvae using the baermann fecal concentration technique is required to diagnose most infections. unless the infection is heavy, examination of a fresh fecal smear is insensitive for identification of these larvae. the nodules formed by s. tumefaciens infection can be visualized during colonoscopy. histopathology of the biopsied nodules should reveal many adult worms. infection with strongyloides spp. can be treated with fenbendazole, pyrantel pamoate, thiabendazole, , or ivermectin. to evaluate efficacy, repeat a fecal examination to days after the treatment ends. because of the presence of free-living adult worms in the environment and the ability of larvae to cause infection by penetrating intact skin, prevention is difficult. keeping cats indoors in warm, humid climates may be an owner's only means of preventing infection with strongyloides spp. parasites. infections with trichuris vulpis rarely occur in cats and are considered to be clinically unimportant. , the two species of roundworms commonly infecting cats are toxocara cati (figure - ) and toxascaris leonina (figure - ) . the latter also has the ability to infect dogs. cats are infected with t. cati in several ways. most commonly, infection is by ingestion of contaminated food, water, or infected paratenic hosts such as rodents. transuterine transmission has not been reported. the diagnosis of infection with o. tricuspis is difficult, because ova are not shed in the feces; rather, the vomitus must be examined for worms or larvae. the worms may also appear in gastric mucosal biopsy samples. a report of cats undergoing endoscopic examinations found the parasite in gastric biopsy samples from cats. fenbendazole may be effective in treating infections with o. tricuspis. preparations with febantel may also be expected to successfully treat these infections. transmission can be prevented by appropriately treating infected cats. other cats should not be allowed to ingest infected vomit. this parasite is of no zoonotic concern. physaloptera another parasite rarely inhabiting the stomach in cats is in the genus physaloptera. larger than ollulanus tricuspis, this blood-sucking worm infects cats that have ingested intermediate hosts, such as cockroaches, crickets, or flour beetles. preying on transport hosts, such as mice that have eaten an intermediate host, is another way cats become infected with this parasite. clinical signs of infection with physaloptera spp. include vomiting, anorexia, and melena. a diagnosis of physaloptera infection can be made after identifying the ova in the patient's feces or adult worms in the vomitus. occasionally, the worms may be seen during gastroscopy. the adult worms must be differentiated from ascarids. infection can be treated with ivermectin, pyrantel pamoate, or fenbendazole. because there is no migratory phase of the life cycle, the treatment does not need to be repeated. three species of strongyloides infect cats. strongyloides felis infects cats in india and tropical australia, , s. tumefaciens is a rare parasite of cats in the southeastern united states, and s. planiceps is found in cats in malaya and japan. strongyloides stercoralis, found in dogs and humans, produces experimental infections in cats, but natural infection with this species has not been observed. feline infection with strongyloides spp. is considered by most to be rare. however, one report from australia identified s. felis in of necropsied cats. infection with strongyloides spp. occurs after ingestion of infective larvae. infection can also take place after the larvae penetrate the skin of the cat. ingested larvae penetrate the intestinal wall and migrate through the diaphragm into the lungs. after cutaneous penetration, the larvae enter the venous circulation and enter the clinical illness because of roundworm infection is uncommon. illness, when it does happen, most often occurs in kittens signs may be mild and can include vomiting, diarrhea, weight loss, poor growth, and a "pot belly." a heavy infection with t. cati can result in catarrhal enteritis. severe infections can lead to intestinal obstruction and, possibly, perforation. much less dramatic changes arise after infection with t. leonina, although enteritis may occur. roundworms are frequently diagnosed with a fecal floatation. the centrifugal floatation technique is more sensitive than the simple fecal floatation technique many hospitals use. occasionally, adult worms will be passed with the feces. the goals of treating roundworms include disease prevention in an individual cat or kitten, prevention of environmental contamination by cats defecating outside, and the prevention of zoonotic infections. many effective and safe anthelmintics are available (table - ) . benzimidazoles, such as fenbendazole, act on the parasite's microtubular structure, leading to disintegration of the worm's intestines, muscular layer, and hypodermis. pyrantel in the pamoate formulation is poorly absorbed and causes paralytic parasite death. macrocyclic lactones, such as milbemycin, also lead to paralytic parasite death. these compounds act on the parasite's gamma-aminobutyric acid (gaba)-and glutamate-controlled ion channels. these channels are lacking in tapeworms, accounting for the lack of efficacy against these parasites. lastly, emodepside (a cyclic octadepsipeptide) has been combined with praziquantel in the product profender (bayer animal health). this topical parasiticide has been shown to be both safe and effective. these drugs appear to be so safe that overdosing is almost impossible. kittens can be dewormed starting at two weeks of age and again at , , , , and weeks. older kittens and adults can be dewormed every month to months. because of the safety of these drugs, the possibility of false-negative tests and, more importantly, the zoonotic potential of these infections, perhaps all kittens should be dewormed, not just those testing positive. roundworm ova are very hardy and can remain infective for years. they survive sewage treatment and composting, and there is no practical means of decreasing the ova population once the environment is contaminated. thus it is best to attempt to prevent contamination in the first place. when practical, keeping cats indoors allows appropriate control of potentially transmammary infection occurs, but only if the queen is acutely infected late in pregnancy. chronically infected queens do not pass t. cati ova in their milk. after ingestion, t. cati larvae migrate through the small intestinal wall, into the liver, and then to the lungs where they are coughed up and swallowed. these larvae then infect the small intestine. some of the migrating larvae become encysted in the cat's muscle tissue. larvae from ova ingested through the milk tend not to undergo migration and mature directly in the small intestine. the prepatent period is approximately weeks. infection with t. leonina occurs after ingestion of infective ova or an infected paratenic host. unlike t. cati, very few t. leonina larvae migrate through the cat's tissues. most develop in the wall of the small intestine. the prepatent period is to weeks. toxascaris leonina ova can become infective within days of being passed in the feces when the ambient temperature is ° c but normally require to weeks. lungs, and kidneys. ocular larval migrans results in granulomatous retinitis that is often misdiagnosed as retinoblastoma in older children. this can lead to unnecessary enucleation. toxocara cati appears, however, to be less important than t. canis as an infection in humans. the species of hookworms that infect cats are ancylostoma tubaeforme and ancylostoma braziliense (see figure - ). they are reported to be an uncommon infection in cats. , ancylostoma braziliense can also infect dogs. hookworm infections occur after ingesting food or water contaminated with hookworm larvae or eating contaminated fecal material. if the pet cat is allowed outdoors, attempts at preventing hunting may reduce the possibility of infection. keep children's play areas, such as sand boxes, inaccessible to cats when children are not at play. feeding only well-cooked food can prevent infection by contaminated food. finally, empirical, preventative deworming for cats that go outdoors should be performed to times yearly. any less frequently does not lead to an appreciable decrease in the prevalence of the parasite. roundworms easily infect humans who ingest the ova, particularly children. visceral larval migrans occurs after infection with toxocara canis in humans. infection can lead to the formation of nodules in the brain, liver, tapeworm infections are well tolerated by the cat. usually there are no signs of infection other than finding segments on the feces or attached to perianal hair. because both d. latum and spirometra tapeworms absorb vitamin b across the cuticle, megaloblastic anemia is possible, but unlikely. tapeworm infections are diagnosed by identifying the typical appearance of the segments or the egg packets within the segments. the segments of t. taeniaeformis are flat, while those of d. caninum have been described as appearing like a grain of rice. the segments should be handled carefully, because they are friable and rupture may result in exposure of the handler. the operculated ova of d. latum and spirometra spp. must be differentiated from trematode ova. even though tapeworm infections are well tolerated, cats should be treated for reasons of owner discomfort and public health concerns (see table - ) . these infections are easily treated, because drug treatment is highly effective. re-infection must be controlled using preventative measures, especially flea control to prevent re-infection with d. caninum. praziquantel and infected paratenic hosts. the larvae can survive for months in the tissues of paratenic hosts. infection also occurs after larval migration through the skin. in either case, the worm matures in the small intestine. unlike dogs, transmammary infection has not been reported in cats. , the prepatent period is between and days, depending on the route of infection. the time to patency after transcutaneous infection is longer than for direct colonization. infective l larva develop to days after the ova are passed. developing larvae attach to the mucosa of the small intestine where they ingest copious amounts of blood. because the worms can remove a significant volume of blood from kittens, weakness from iron-deficiency anemia or blood-loss anemia may be noted. melena and diarrhea may also be recognized. signs are uncommon in adult cats. identification and treatment of hookworm infections are similar to that for roundworm infections (see table - ) . hookworm larvae are not as hardy as roundworm eggs. soil contamination may be a temporary problem in areas that experience a hard frost. hookworm larvae will not develop in temperatures less then ° c or greater than ° c. frequent, appropriate disposal of feces, cleaning surfaces with a % bleach solution, and deterring hunting may prevent infections. migration through the skin of persons coming into contact with the larvae of a. braziliense is the most common cause of cutaneous larval migrans, particularly in the southeastern united states. this is an erythematous, pruritic skin eruption often found on the soles of the feet of infected children. the tapeworms most commonly found in cats are dipylidium caninum and taenia taeniaeformis. diphyllobothrium latum, spirometra spp., and echinococcus multilocularis occasionally infect cats. the latter is important, because it can lead to alveolar echinococcosis in humans. spirometra tapeworms are found in north america (s. mansonoides) and far-east asia (s. mansoni and erinacei), while d. latum prefers temperate climates. unnoticed, but the cat may cough or experience hemoptysis. diagnosis involves demonstration of fluke ova in the feces. although therapy may be unnecessary, praziquantel or epsiprantel are effective in eliminating the intestinal population of the fluke. platynosomum spp. are flukes living in the gall bladder, bile ducts, and pancreatic ducts. these flukes are most prevalent in the southeast united states and caribbean islands and require two intermediate hosts. the first host is a snail, while the second intermediate host is a lizard, toad, gecko, or skink. cats become infected with this fluke after ingesting an infected second intermediate host. the prepatent period for the fluke is weeks. most infections are subclinical. if clinical signs do occur, they may include weight loss, vomiting, diarrhea, icterus, hepatomegaly, or abdominal distention. diagnosis involves identification of ova shed in the feces using a fecal sedimentation method or by finding adult flukes in the gall bladder or bile ducts during abdominal surgery. treatment involves administering praziquantel ( mg/kg, q h, po for to days) and/or surgical removal of the flukes. two species of coccidians are the most common to infect cats, isospora felis and isospora rivolta (figure - ) . the genus isospora may be renamed cystoisospora. these are epsiprantel are safe and effective. fenbendazole is effective against t. taeniaeformis, but not d. caninum. without controlling exposure to intermediate hosts, tapeworm infections are difficult to eliminate. flea control is imperative in eradicating infections with d. caninum. controlling predation helps prevent ingestion of t. taeniaeformis-infected rodents. infection with d. caninum occurs in young children who are most likely to eat fleas. infection results in only minimal signs of illness. the larval stage of t. taeniaeformis is of little zoonotic importance. although cats are uncommonly infected with echinococcus multilocularis, potentially life-threatening alveolar damage occurs in north american humans infected with this tapeworm. plerocercoids of spirometra spp. can penetrate the mucous membranes or open skin wounds of humans and migrate around the subcutaneous connective tissue, forming nodules, a condition called sparganosis. megaloblastic anemia, as a result of vitamin b deficiency, may occur in humans infected with d. latum or spirometra spp. tapeworms. alaria marcianae flukes reside in the intestinal tract of cats and the mammary glands of lactating queens. miracidia hatch underwater from ova shed in the feces and penetrate the skin of a snail. after further development, cercariae penetrate the skin of leopard frog tadpoles and are able to survive the metamorphosis to the adult frog. if the tadpole is eaten by a snake, bird, or mammal, the parasite enters the host's tissues but does not undergo further development. after a male or nonlactating female cat ingests the infected intermediate host, the parasite penetrates the wall of the small intestine, passes through the diaphragm, and enters the lungs for further development. finally, the parasite is coughed up and swallowed to complete maturation and reproduce in the small intestine. if, however, an infected host is ingested by a lactating queen, the parasite migrates through the tissues to the mammary glands, rather than the lungs. once shed in the milk, the parasites develop into mature adults in the kittens. some of the mesocercariae remain in the mammary glands to infect future litters. clinical signs associated with worms in the small intestine are uncommon. migration through the lungs often goes species-specific obligate intracellular parasites. , they are able to survive in the environment for months. a detailed description of the coccidial life cycle can be found elsewhere. , simply put, direct transmission is by ingesting oocyst-contaminated food or water or by grooming contaminated body parts. indirect transmission occurs after ingesting a mechanical vector or the infected tissues of paratenic hosts. after ingestion by a cat, the oocyst excysts in the small intestine and enters the enterocyte where further development occurs. the parasite may also migrate through the intestinal wall to form cysts in mesenteric lymph nodes. these cysts may serve as a source for reinfection. , the prepatent period is to days and the shed oocyst becomes infective after several days of exposure to warmth and moisture. infection with isospora spp. is usually subclinical. signs, if they occur, range from mild, transient watery diarrhea to severe mucohemorrhagic diarrhea with vomiting and resultant dehydration and weight loss. , signs are most commonly recognized in severely infected neonatal kittens, particularly those with concurrent illness, and arise because of small intestinal congestion, mucosal erosion, or villus atrophy. signs may also be noted in immunosuppressed adult cats. isospora species are readily found in fecal floatation or wet-mount examinations. shedding can be intermittent, but most cats with diarrhea caused by coccidial infection shed large numbers of oocsyts. fortunately, in most cats, the diarrhea from isospora spp. infection is self-limiting. in fact, if a kitten is persistently shedding oocysts despite appropriate treatment or the parasite is identified in an adult cat with chronic diarrhea, attempts should be made to identify co-infections or other diseases that may cause diarrhea. anticoccidial drugs are either coccidiostatic or coccidiocidal (table - ) . coccidiostatic drugs are the most commonly used drugs for individual pet cats. trimethoprim-augmented sulfadiazine (tribrissen; intervet/schering-plough animal health, summit, nj) or another sulfa-containing antibiotic, sulfadimethoxine (albon; pfizer animal health, madison, nj), can be used. supportive care for severely affected kittens, such as parenteral rehydration, should be used as needed. coccidiocidal drugs are often reserved for use in densely populated situations such as catteries or shelters. however, many veterinarians are now using them as a first-line defense against isospora spp. infection. ponazuril (marquis oral paste; bayer animal health, shawnee mission, kan.), formulated for horses, is effective and can be safely administered to cats. for more on the use of ponazuril in cats, see chapter . a related drug, diclazuril, is also available and may be administered once at mg/kg po. while not available in north america, toltrazuril (baycox, bayer animal health) may be administered once at mg/kg po or mg/kg po once daily for days. a a second course of therapy days later may be required to completely eliminate the oocysts. sanitation is very important, because the oocyst requires several days to become infective. frequent removal of feces, preferably daily, is recommended to prevent re-infection and transmission to other cats. controlling a cat's ability to hunt reduces the chance of ingesting an isospora-infected rodent. control of mechanical vectors, such as cockroaches and flies, is also useful. since a cat can become infected after grooming an infected cat's perineum, consideration should be given to treating all cats in contact with the patient. in addition, catteries and shelters should ensure all food is well cooked, litter boxes are cleaned daily, and surfaces are well cleaned with steam or % ammonia. where recurrent isospora spp. infections are a problem, prophylactic treatment of all -to -week-old kittens with ponazuril should be considered. despite all wellintentioned efforts at hygiene and treatment, isospora spp. infection can still be transmitted to other cats. because these are species-specific parasites, transmission of i. felis and i. rivolta from cats to humans does not occur. the flagellated protozoal parasite, giardia duodenalis, has seven microscopically indistinguishable genotypes or assemblages. assemblages a and b infect humans, while assemblage f is harbored by cats. cats will occasionally harbor assemblages a and b. infection with g. duodenalis occurs after ingesting cystcontaminated feces, by grooming an infected cat or from contaminated fomites. re-infection may occur by selfgrooming. only a small number of cysts need be ingested to establish an infection. in humans as few as cysts are required to cause infection. after ingestion of infective cysts, trophozoites begin to excyst in the stomach. this process is completed in the proximal duodenum. the trophozoites adhere to enterocytes along the length of the small intestine using the ventral suction disk. intermittent shedding of immediately infective cysts begins to days after infection. proteins released during encystment of the trophozoites are detected by the fecal antigen tests. cysts may adhere to the perianal region, facilitating re-infection by self-grooming. occasionally, trophozoites are found in examinations of fresh, watery feces. these do not survive for long and are not infective. the mechanisms of disease induced by g. duodenalis are still unclear. after the trophozoite attaches to the brush border of the enterocyte, the tight junction between cells is disrupted, increasing intestinal permeability. the brush border becomes attenuated, further exacerbating malabsorption of water, electrolytes, and other nutrients. the alteration in intercellular adhesion results in t-lymphocyte activation and mucosal cell injury. infection also promotes mucosal cell apoptosis (preprogrammed cell death). in addition, small intestinal bacterial overgrowth may accompany g. duodenalis infections, resulting in more severe clinical signs. fortunately, most cats infected with g. duodenalis show no clinical signs. , the most common sign is acute, transient, small bowel diarrhea without systemic illness, such as fever or vomiting. less commonly, a cat might have profuse, watery malodorous diarrhea with mucus. also possible, but uncommon, is weight loss , or abdominal pain. the severity of clinical signs exhibited in an individual cat depends on the age and general health of the cat. cats co-infected with cryptosporidium felis or tritrichomonas foetus may have more severe diarrhea that is more difficult to control, as will the presence of bacterial overgrowth. the diagnosis of g. duodenalis requires demonstration of trophozoites or cysts in a fecal examination, or detection of encystment proteins or giardial dna in a fecal sample. a reliable diagnosis may be difficult to obtain for several reasons. cysts are small, easily missed, and must be differentiated from plant debris or yeast. trophozoites are short lived outside the body and can only be found in very fresh, watery feces or, better yet, in diarrheic feces collected directly from the cat's rectum. shedding of cysts is usually intermittent, and the intensity of shedding varies greatly. , because of these difficulties, the absence of the organism in a fecal sample does not eliminate it as the cause of diarrhea. it is often necessary to test multiple fecal samples, using at least two different techniques in order to find the organism. , the easiest test to perform is a fecal smear or wet mount examination to identify trophozoites or cysts (figures - and - ). the sample examined should be very fresh, warm, diarrheic feces. one drop of feces is placed on a slide along with a drop of . % saline or lugol iodine. trophozoites are identified by their characteristic structure (table - ) . the motile trophozoites have a motion described as appearing like the back and forth rolling motion of a falling leaf. since lugol iodine stain kills the trophozoite, there will be no motion to detect. this test is not very sensitive; however, with trained examiners, the test has a high specificity. increased sensitivity can be gained by performing a centrifugal flotation using zinc sulfate. the sample should be warm, fresh feces or feces refrigerated for no more than days. the processed sample is examined for the same structures as the wet mount. the sensitivity of examining one sample is % and increases as more samples are examined. the sensitivity of looking at three samples is % , ; therefore the test is not considered negative until three specimens have been found free of the organism. a fecal antigen test that identifies the encystment protein is available. the snap giardia antigen test (idexx laboratories) uses fresh or frozen feces, or feces refrigerated for less than days. since the antigen is continuously shed, this test avoids the problem of intermittent shedding of the whole organism. the sensitivity of the test is %, with a specificity of %. by combining the antigen test with a zinc sulfate fecal centrifugal flotation, the sensitivity improves to . %. it is unknown how long the antigen remains in the feces after treatment. thus a zinc sulfate centrifugal flotation examination should be used to evaluate therapeutic efficacy. , the use of this test in cats without diarrhea is controversial, because these cats are unlikely to shed cysts. the zoonotic significance of a positive antigen test in a cat not shedding cysts is unknown and may cause confusion. polymerase chain reaction detection of giardia dna is available, but the test has not been standardized across all diagnostic laboratories. one needs to ensure the laboratory performing the test has validated it for assemblage f. the test may also be used to identify cats harboring the zoonotic assemblages a and b. the sensitivity of this test is unknown. two commonly available drugs are used most frequently to treat infections with g. duodenalis (see table - ) . fenbendazole may be effective and can be used in pregnant queens and in cats co-infected with roundworms, hookworms, and taenia spp. tapeworms. however, in one small study, only four of eight cats infected with both g. duodenalis and cryptosporidium felis stopped shedding giardia permanently after receiving fenbendazole. febantel, in the combination product drontal plus (bayer animal health), is converted to fenbendazole. when six experimentally infected cats received . mg/ kg of febantel q h po for days, four of them stopped shedding g. duodenalis cysts. metronidazole has been the traditional drug used to treat g. duodenalis in pets. the drug is also useful for treating concurrent small intestinal bacterial overgrowth and clostridial infections. the administration of metronidazole may eliminate shedding in % of cats. neurologic side effects may occur at the dose recommended for treatment of giardia (see above, therapeutics for vomiting and diarrhea). the use of a giardia vaccine was ineffective in clearing infection by itself. the combination of fenbendazole and metronidazole has been suggested as the initial treatment of choice for g. duodenalis infections. although controlled studies are lacking, they may work synergistically by acting on two different targets within the parasite. febantel would be expected to have the same synergism with metronidazole. drug therapy may not be necessary in cats without diarrhea that are infected with g. duodenalis, because it is uncommon for a cat to carry the assemblages required to infect humans. the veterinarian may be obligated to treat a healthy cat if the owner wants to treat, the owner is immunocompromised, or the goal is eradication of an infection from a multicat home or prevention of parasite transmission to giardia-naïve cats is attempted. what may appear to be treatment failure is more likely to be re-infection. in addition to drug therapy, steps should be taken to prevent re-infection. all cats with diarrhea positive for g. duodenalis should be treated along with their housemates. sanitation is imperative in the fight against re-infection and transmission of g. duodenalis. dispose of old litter pans and scoops and use disposable litter boxes during treatment. when the infection is eliminated, not just controlled, new litter boxes and scoops may be purchased. bathe all cats during treatment to remove cysts from the hair coat. since giardia spp. cysts are susceptible to desiccation, blowdry all cats using a warm air blower, paying particular attention to the perineal area. disinfect bowls, housing, and other utensils with bleach. in addition to antiprotozoal drugs and sanitation, supportive care may become necessary. probiotics and a highly digestible, bland diet may be offered to cats with small bowel diarrhea, while a high-fiber diet may be useful for those few cats with large bowel diarrhea. where required, hydration and electrolyte imbalances must be corrected and antiemetics used to control vomiting. therapy can be evaluated by retesting feces with a zinc sulfate centrifugal flotation examination to days after the end of treatment and again weeks later. a positive test immediately posttreatment is most likely because of therapeutic failure. if the cat is negative immediately after treatment ends, but is positive weeks later, re-infection is likely. since the fecal antigen test may remain positive long after the infection is eradicated, this test is inappropriate for evaluating therapy. , re-treatment of fecal flotation-positive, recovered cats may be handled in a manner similar to the positive healthy cat mentioned above. cats with diarrhea that continue to shed cysts may be re-treated for g. duodenalis infection along with dietary modification and empirical treatment for other common intestinal parasites. however, serious consideration should be given to investigation into other potential causes of diarrhea. the giardia vaccine has been found to be ineffective in preventing infection and production has been discontinued. this means prevention of giardia infection involves avoiding exposure, stress and re-infections. providing a clean environment, feeding only processed foods, and controlling potential transport hosts will help reduce the chances of exposure. isolation of cats with diarrhea may be important, too. municipal sanitation control is difficult as the cyst survives for weeks in cool, moist environments. cysts are also able to survive water treatment and can pass through attempts at water filtration. giardiasis is associated with debilitating diarrhea in some humans, particularly those who are immunocompromised. however, cats do not commonly carry the assemblages needed to infect humans. transmission of g. duodenalis from cats to humans is rare and unproven. still, it seems prudent to consider the owner's health when contemplating management of giardial infections in cats. to avoid human health risks, cats with diarrhea that test positive for g. duodenalis should be treated with the goal of controlling the diarrhea. since no treatment for g. duodenalis is completely effective or % safe, treatment of positive cats without diarrhea should only begin after a discussion of the benefits and risks of the treatment with the owner. tritrichomonas foetus is best known for causing bovine reproductive infections. it is an obligate anaerobic parasite that also colonizes the lower intestinal tract of cats. there are enough differences between the two isolates that the feline isolate does not cause disease in heifers and vice versa. the parasite depends on the host's normal intestinal flora and secretions for obtaining nutrition. a report from the united states of purebred cats tested at an international cat show found t. foetus in of the cats tested, a prevalence of %. this parasite seems to have a higher prevalence in purebred cats than nonpurebred cats. a study of pet cats visiting veterinary hospitals across the united states reported of purebred cats were positive for t. foetus, while only of nonpurebred cats were positive. in this same study, of the positive tests were from purebred cats. a study from the united kingdom of diarrheic fecal samples sent to a veterinary diagnostic laboratory reported similar results. purebred cats represented of the cats testing positive for t. foetus. the u.k. study also found the siamese and bengal breeds each represented of positive cats; only two other breeds tested positive. transmission like most other protozoal parasites, t. foetus is transmitted by ingestion of the parasite, in this case, the trophozoite. unlike most of the other parasites, t. foetus does not form cysts and only survives up to days outside the body in moist feces. a cat becomes infected through the use of a shared litter box with an infected cat. after walking into the box, the parasite is transferred from the infected feces of one cat to the paws of the other. infection then occurs through ingestion of the trophozoites during grooming. after infection, t. foetus colonizes the distal ileum and colon, followed by shedding of infective trophozoites to days later. there are several mechanisms by which t. foetus causes diarrhea. these include alteration of the cat's normal bacterial flora population, increases in local inflammatory cytokine concentrations, production of enzymes, and direct mucosal injury. the resulting injury leads to plasmacytic-lymphocytic and neutrophilic colitis. although most infections involve only the mucosa of the colon, one study reported two of seven cats with diarrhea and t. foetus infections as having trophozoites in deeper layers of the colonic wall. co-infection with cryptosporidium felis or giardia duodenalis can be associated with increased numbers of t. foetus trophozoites and increased severity of diarrhea. signs of infection are most frequent in kittens and young cats, although infections without clinical signs can occur. adult cats, however, may also show signs of t. foetus infection. the most common sign is a foulsmelling large bowel diarrhea with increased frequency of defecation, mucus, blood, and flatulence. the consistency of the diarrhea may wax and wane, but the presence of diarrhea does not. cats with diarrhea are otherwise in good health and maintain their body condition. , severe diarrhea can result in anal swelling and fecal incontinence. diarrhea may respond to the use of antibiotics because of changes in the cat's intestinal microbial flora. however, it always returns at the cessation of therapy. , many cats experience a spontaneous resolution of the diarrhea within years of diagnosis. , since t. foetus causes reproductive infections in heifers and bulls, there is speculation the parasite also infects the reproductive tract in cats. tritrichomonas foetus was found in the uterus of a queen with pyometra. however, in a study of breeding male and female cats from catteries, no cytologic or molecular evidence of t. foetus was found in the reproductive tract. the authors reported colonic infection with t. foetus in of the cats representing of the catteries. detection of the trophozoites in a sample of feces is the most expedient means of diagnosing an infection with t. foetus (figure - ). an index of suspicion is required, because the clinical presentation of t. foetus infection is often mistaken for infection with giardia duodenalis. if a cat is not responding to treatment for that parasite, consider t. foetus as a cause of the diarrhea. the sample required for the diagnosis of t. foetus is a fresh, nonrefrigerated sample of watery feces. refrigeration kills the trophozoites, and they are not found in normal feces. the sample may be freshly passed diarrhea, feces collected using a wire loop passed into the colon, or collected by a colonic flush using a red rubber catheter and ml of saline. a wet mount or smear examination of the feces should be performed on all cats with diarrhea. examination of multiple samples may be required to find the t. foetus trophozoites with this technique because it is insensitive. the trophozoites must be differentiated from giardia duodenalis based on structural differences and motility patterns (see table - ). the trophozoites of t. foetus can be cultured using the inpouch tf system (biomed diagnostics). this test is more sensitive than the fecal wet mount examination and detects trophozoites per sample. the number of parasites shed by a cat with diarrhea is high enough to be routinely detected with this method. the test should be performed in-house, because the parasite is unlikely to survive the trip to the laboratory. the test pouch is inoculated with µg of freshly collected feces, about the size of a peppercorn. any more than this increases the chances of bacterial overgrowth. the pouch is incubated at ° c and examined under the microscope for motile trophozoites every other day for days. the pouch should be tapped gently to dislodge the parasites, which tend to collect along the seams. the test is considered negative if parasites are not found after days. one benefit of this system is that it does not support growth of giardia duodenalis or pentatrichomonas hominis. if a fecal wet mount examination and culture are both negative and infection with t. foetus is still under consideration, a pcr test can be performed. this test detects dna from live or dead trophozoites, but is more expensive than other diagnostic methods. this test is more sensitive than the other two methods and can detect parasites per sample. the sample size is mg of feces not contaminated by litter preserved in to ml of rubbing alcohol shipped at room temperature. trophozoites of t. foetus are sometimes found in colonic biopsy samples adhered to the surface or in the lumen of crypts. the most effective drug for the treatment of t. foetus in cats is ronidazole. the drug has a bitter taste and should be compounded into capsules. veterinary staff and owners should use gloves when handling ronidazole. if a confirmed relapse occurs, another course of treatment may eliminate the parasite. diarrhea may take several weeks to resolve after elimination of the parasite, because significant colitis is often present. effectiveness of treatment can be evaluated by performing fecal pcr tests and weeks after the end of treatment. apparent treatment failures may occur because of re-infection, co-infection with giardia duodenalis or cryptosporidium felis, or the presence of another concurrent diarrhea-causing disorder. a more worrisome cause for treatment failure is a recent report of parasite resistance to ronidazole in two cats. fortunately, diarrhea ultimately resolved in both cats despite the continued presence of the parasite. if the cat retests negative and the diarrhea is not improving after weeks, consider the possibility that another disease may exist. nonspecific treatment for diarrhea is unhelpful and may prolong the duration of diarrhea. diarrhea may respond to antibiotics as they alter the intestinal flora population; however, once treatment is stopped, the diarrhea will return. an important and potentially serious adverse effect of ronidazole administration in cats is a reversible neurotoxicity. onset of signs often begins within week of the onset of therapy and may last between and weeks after cessation of therapy. these signs can include depression, ataxia, seizures, behavioral changes, weakness, hyperesthesia, and trembling. neurotoxicosis usually requires only supportive care along with discontinuation of the drug. the neurologically affected cat should be retested for the parasite, because it may have been eliminated. because of the potential for neurotoxicity, the use of ronidazole should be restricted to cats with confirmed infections with t. foetus. crowded conditions should be avoided, because transmission of t. foetus trophozoites is more efficient in these settings. cats testing positive should be isolated from other cats during treatment. providing a clean environment will help prevent transmission of trophozoites. although there is a report of an infection in one immunocompromised person, transmission of t. foetus trophozoites from cats to healthy humans has not been reported. still, prudence dictates handling feces infected with t. foetus trophozoites carefully. recent genetic evaluations have shown that most feline infections with cryptosporidium spp. are with c. felis; not, as previously thought, with c. parvum. cryptosporidium parvum seems to be limited to farm animals. cryptosporidium felis is an obligate intracellular parasite infecting the small intestine. infective oocysts are ingested from contaminated feces during self-grooming of contaminated body parts and from contaminated food and water. , after infection, the parasite attaches to the brush border of the enterocyte. the prepatent period is to days, and the oocysts are infective as soon as they are shed, making this a very contagious disease. like most intestinal parasites, shedding is often intermittent. the pathogenic effects of c. felis infections are not well understood. direct cytotoxicity and inflammation causes villus atrophy and decreased surface area for absorption of water, electrolytes, and other nutrients. , apoptosis (preprogrammed cell death) of the mucosal cells may be accelerated, adding to the malabsorption. most infections with c. felis are subclinical. signs, if present, range from a mild, self-limiting small bowel diarrhea to chronic intermittent small bowel diarrhea. severe diarrhea with weight loss and anorexia may also occur. , clinically apparent infections are most common in kittens, adult cats with concurrent gastrointestinal diseases, and cats co-infected with giardia duodenalis or tritrichomonas foetus. cats with co-infections may experience more severe clinical signs. a fecal flotation, which should be performed on all cats with diarrhea, may reveal c. felis if there are large numbers of oocysts (figure - ) . the fecal floatation test, however, is often negative because of intermittent shedding. the parasite is small and floats in a higher plane than helminth ova; the high-power lens and appropriate adjustment of the microscope stage is required to find the parasite. the small size of the oocyst makes identification difficult, particularly if the examiner is not specifically looking for them. a modified ziehl-neelsen stain of a thin fecal smear may help in the identification of the oocysts. this technique works well in humans with large numbers of oocysts. once signs resolve or the oocyst numbers decline, a single examination of a stained smear becomes insensitive. when only one sample is available, testing for c. felis antigen is a good choice. the prospect microplate assay (alexon biomedical, sunnyvale, calif.) is more sensitive and specific for the diagnosis of c. felis than is the examination of a stained smear. immunofluorescent antibody testing is available from some laboratories. fecal c. felis dna can be detected using pcr testing. this test is available at many veterinary diagnostic laboratories; however, at present, there is no test standardization among laboratories. the clinical and zoonotic significance of a positive pcr test combined with an oocyst negative test is unknown. therefore a positive pcr test in a cat without diarrhea presents a confusing situation for the attending veterinarian with regard to recommendations for the owner. unfortunately, there are no completely effective and safe treatment protocols available for c. felis. , a concerted attempt to find other causes of diarrhea should take place prior to convicting a cat of having diarrhea solely from c. felis infection. most reports on therapy for c. felis are uncontrolled and anecdotal. a number of drugs have been discussed. azithromycin for at least days appears safe but produces variable results. paromomycin, an oral aminoglycoside, may be effective. however, one study reported acute renal failure in of cats receiving the drug. deafness also occurred in three of those four cats. nitazoxanide is a drug approved for treating humans with diarrhea caused by cryptosporidium spp. infections. the administration of nitazoxanide to cats at mg/kg q h po for at least days up to days may be effective. however, nitazoxanide is a gastrointestinal irritant and commonly results in vomiting and foul-smelling diarrhea. co-infections with giardia duodenalis and/or tritrichomonas foetus are more difficult to control. if diarrhea from c. felis infection improves but does not resolve at the end of therapy, the duration of treatment may be prolonged. additional diagnostic testing should also be performed to ensure the only cause of the diarrhea is infection with c. felis. environmental control of c. felis is difficult, because it is extremely hardy. it is resistant to chlorination and most disinfectants. oocysts remain viable at temperatures above freezing up to ° c. the parasite is difficult to filter and survives treatment at municipal water treatment facilities. steam-cleaned housing and utensils may be beneficial in controlling parasite numbers, and they are susceptible to % ammonia solutions; however, the required contact time is hours. cryptosporidium spp. are relatively species specific, and there are no reports of waterborne outbreaks of human cryptosporidiosis associated with c. felis. cryptosporidiosis can cause life-threatening diarrhea in hivpositive persons. fortunately, humans are rarely infected with c. felis. in fact, the zoonotic species most commonly found in humans (often veterinary students), is c. parvum found in young heifers. regardless of a person's health, feces from a cat with diarrhea should be handled carefully. if a cat infected with cryptosporidium spp. is owned by an immunocompromised person, a pcr test may be useful in determining the species of the parasite and its zoonotic risk. like other coccidians, toxoplasma gondii is an obligate intracellular parasite. domestic cats and other felids are the only animals that shed oocysts. any warmblooded animal, including humans, can be infected with this parasite. toxoplasma gondii can be transmitted by ingestion of infective oocysts in fecally contaminated food or water after ingestion of tissue cysts through carnivorism, or by transplacental or trans-mammary transmission of the parasite. the parasite enters into one of two cycles, depending on the host species. the enteroepithelial cycle only occurs in cats and results in shedding of oocysts after sexual reproduction of the parasite. after a cat ingests an infective oocyst or a tissue cyst, the parasite enters the mucosal cells of the small intestine, where it may undergo development and sexual reproduction, after which oocysts are shed. the prepatent period after ingesting an infective oocyst is to days, while shedding after ingesting tissue cysts starts in to days. fecal shedding, which occurs only after initial infection, lasts for to weeks , and the oocysts become infective to days after they are shed. the extraintestinal cycle occurs in any animal, including cats. after ingestion, the parasite penetrates the cells of the small intestine and rapidly replicates in the enterocytes and associated lymph nodes into tachyzoites. after hematogenous and lymphatic spread, tachyzoites infect cells in all tissues of the body. tissues most commonly infected include the brain, liver, pancreas, and lungs. if a pregnant queen becomes infected, tachyzoites cause placentitis, after which they infect the fetus. in weeks, the host's immune response slows parasite replication, and the resultant bradyzoites form tissue cysts in the brain, striated muscle, and liver, and they remain viable for the life of the animal. immunosuppressive drugs or disease may dull the suppression of parasite division by the host immune system and allow the slowly dividing bradyzoites in tissue cysts to begin rapid division, thereby reactivating the infection with tachyzoites. none of the forms of t. gondii produces a toxin. rapid replication of tachyzoites within a cell leads to rupture of the cell and necrosis of the tissue in which they are located. the most commonly injured tissues are the brain, lungs, liver, and pancreas. prenatal infection leads to more severe illness, because the immature immune system is unable to slow down replication by tachyzoites, allowing continued damage to tissues. prenatal infection is more likely to result in ocular infections, and neonatal death is usually caused by pulmonary or hepatic infection. type ii and iv hypersensitivities may be involved in the pathogenesis of chronic disease from bradyzoites in tissue cysts. kittens infected perinatally can be stillborn or die shortly after birth. they may also suffer from hepatomegaly and ascites, central nervous system signs resulting from encephalitis, respiratory distress, or uveitis. , clinical signs of infection in healthy adult cats are uncommon (box - ). diarrhea from enteroepithelial development of the parasite is rare. cats that develop clinical disease often have an episodic course with vague signs that depend on the body system affected. onset of illness may be acute or chronic, and the most commonly affected organs include the brain, lungs, liver, heart, pancreas, and the eyes. signs are the result of spread of tachyzoites after initial infection or after reactivation of tissue cysts. cats suffering from uveitis may develop lens luxation and glaucoma. the best way to identify a cat shedding t. gondii oocysts is to demonstrate them with a centrifugal fecal flotation technique using sheather sugar solution. the oocysts are about a quarter of the size of isospora felis oocysts ( figure - ). oocysts of t. gondii are morphologically indistinguishable from hammondia or besnoitia spp. oocysts. detection of fecal t. gondii dna using a pcr test can be used to definitively differentiate t. gondii oocysts from similar coccidians. it is probably best, however, to assume suspicious oocysts are those of t. gondii until proven otherwise. proving infection with t. gondii is responsible for a cat's systemic illness is also difficult. finding tachyzoites in cytology samples is uncommon. they are most likely to be identified from body cavity effusions. the most common method of identifying an infected cat is by detecting t. gondii-associated immunoglobulins using immunofluorescent antibody or elisa techniques. since cats are infected for life, a seropositive cat has been infected at some point in its life. however, use of serology alone is insufficient to diagnose an active t. gondii infection. serum immunoglobulin m (igm) is produced within to weeks after infection, but increased igm titers may persist for months to years. serum immunoglobulin g (igg) begins to rise later; in some cats, igg may not be detectable for to weeks. by the time igg is detectable, shedding will have ceased. maternally acquired igg persists in kittens for to weeks. a rising igg titer is associated with an active infection, but the degree of increase is not associated with the severity of the clinical signs. if a cat becomes seronegative, it is more likely the titer has fallen below the sensitivity of the test rather than the parasite has been eliminated from the body. because of the vague nature of the clinical signs, many cats are presented later in the course of the disease. by this time, they may have switched from igm to igg production or passed the time of maximal igg production. thus a negative igm titer or a lack of rising igg titer does not rule out t. gondii infection. also, reactivation of tissue cysts is rarely associated with rising igg titers. ultimately, the diagnosis of an active systemic t. gondii infection requires demonstration of an igm titer greater than : or a fourfold increase in igg titers over a -to -week period along with signs consistent with toxoplasmosis, the exclusion of other disorders that may cause the clinical signs, and response to appropriate anti-t. gondii treatment. although serum igm titers may be increased in otherwise healthy cats, increased igm titers in cerebrospinal fluid or aqueous humor only occurs in cats with active cns or ocular infections. the goals of treating a cat infected with t. gondii are to reduce shedding of oocysts and to control the clinical signs in sick cats. shedding can be reduced by using ponazuril, toltrazuril, or high doses of clindamycin. the drug options for treating a sick cat include clindamycin, trimethoprim-augmented sulfadiazine, or azithromycin for at least weeks (see table - ) . recurrences are more common if the cat is treated for less than weeks. , the antifolate drug pyrimethamine may be more effective than trimethoprim, but megaloblastic anemia develops in many cats. supplementation with folinic acid ( mg/cat, once daily, po) or brewer's yeast ( mg/kg, once daily, po) may prevent or reverse the anemia. no drug clears all of the tissue cysts; so, cats remain infected for life. if uveitis is also present, use appropriate topical, oral, or parenteral corticosteroids. for a cat with proven t. gondii-associated uveitis alone, a topical ocular glucocorticosteroid is the only required treatment; no antibiotics are necessary unless the uveitis is persistent or recurrent. • wash hands after handling cats, especially if you are pregnant or immunocompromised. • remove fecal material from the home environment daily, since shed oocysts require a minimum of hours to become infective. • do not have immunocompromised persons clean the litter box. if they must clean the litter box, they should wear gloves and wash hands thoroughly when finished. • use litter box liners, and periodically wash the litter box with scalding water and detergent. • wear gloves when gardening, and wash hands thoroughly when finished. • cover children's sandboxes when not in use to avoid fecal contamination by outdoor cats. • only feed cats cooked or commercially processed food. • control potential transport hosts, such as flies and cockroaches, that may bring the organism into the home. • filter or boil water from sources in the environment. • cook meat for human consumption to ° c for minutes minimum (because of uneven heating, microwave cooking does not kill all t. gondii ). • freeze meat at − ° c for hours. • wear gloves when handling meat, and wash hands thoroughly with soap and water when finished. clinical signs such as malaise, fever, and muscle pain should begin to resolve in to days. if there is no response within days, switch to or add another drug. if there is still no response, search for another condition that may cause the observed clinical signs. however, ocular and cns signs resolve more slowly and thoracic radiographic changes may take weeks to resolve. some cns changes may never completely resolve. cats co-infected with feline immunodeficiency virus (fiv) do not respond to anti-t. gondii treatment as well as fivnegative cats respond. feeding cats commercially processed cat food and avoiding undercooked or raw meat can prevent exposure to t. gondii. controlling hunting reduces access to paratenic hosts with infective tissue cysts. access to mechanical carriers of t. gondii, such as earthworms or cockroaches, should be minimized. human infection with t. gondii is common, more so in warm, humid climates where the prevalence of t. gondii seropositive persons approaches %. the number of persons seropositive for t. gondii is estimated to be around , , worldwide. infective oocysts are hardy and may remain viable in the environment for up to months. human infection most often occurs after eating raw or undercooked meat infected with tissue cysts or by transplacental infection. seropositive cats are finished shedding and are unlikely to resume shedding even if the infection becomes reactivated. cats found to be shedding oocysts should be quarantined at a veterinary hospital until shedding ends. oocysts of t. gondii have not been found on the hair coat ; so, transmission of toxoplasmosis does not occur after touching a cat. pregnant women infected with t. gondii for the first time, or chronically infected women who are also hiv positive, can transmit the parasite to their unborn child. transplacental infection can result in stillbirths, cns, or ocular disease. more severe fetal disease may occur if the infection happens in the first half of the woman's pregnancy. toxoplasma gondii infection of immunocompetent humans usually results in a self-limiting fever and malaise. steps useful in preventing transmission of t. gondii to humans can be found in box - . pancreatitis refers to inflammation of the pancreas only, with no implication of the underlying cause or pathology. for example, acute necrotizing pancreatitis (anp) with pancreatic auto-digestion, requiring predominantly supportive care by maintaining fluid and electrolyte balances and pain relief, must not be confused with chronic pancreatitis (cp) caused by lymphocytic infiltration, and commonly associated with lymphocytic inflammatory bowel disease (ibd), and often requires corticosteroids to manage. these two conditions (and others) can only be definitively distinguished histologically. in many cases, the clinical signs of cats with acute pancreatitis will resolve with supportive care before a precise diagnosis is reached and will thus remain undiagnosed. there are no formal classifications for feline pancreatitis, but most authors , , use the terms • acute pancreatitis • acute necrotizing pancreatitis, characterized by severe peri-pancreatic fat necrosis • acute suppurative pancreatitis, characterized by neutrophilic infiltration • chronic pancreatitis, characterized by lymphocytic infiltration the exact prevalence of feline pancreatitis is unknown. necropsy studies from the s to s reported prevalence of feline pancreatitis ranging from . % to . %. , a more recent study found % of cats had evidence of pancreatitis. however, this included pancreatic pathology in % of apparently healthy cats, which suggests that mild pathology is unlikely to cause clinical signs. these studies all show lymphocytic pancreatitis to be significantly more prevalent than acute pancreatitis. this may underestimate the true prevalence of acute pancreatitis, since it is understood that no permanent histopathologic changes are present after resolution of acute pancreatitis. it is also possible that studies assessing pathology in necropsy cases do not reflect clinical practice. there are no specific age, breed, or sex predispositions. although one study reported siamese cats to be at increased risk of acute pancreatitis, subsequent studies have recognized the majority of cases are domestic shorthair cats, suggesting no specific breed predispositions. , , , most studies have indicated older cats ( to years of age) are more likely to be affected, , , , but these studies most likely underrepresent cats with less severe clinical disease for which definitive diagnosis may not be reached and which may be younger. no association has been made with a high-fat diet or obesity. in most cases of both acute and chronic pancreatitis, no specific cause is found, and the disease is primarily considered to be idiopathic. , there are, however, some specific underlying causes that are sporadically recognized. these include infections with herpesvirus, calicivirus, , feline infectious peritonitis (fip), liver fluke and pancreatic fluke, , and toxoplasmosis. however, a recent paper found no association between serum feline pancreatic lipase immunoreactivity (fpli) concentrations and toxoplasma gondii serology. pancreatitis has also been recognized subsequent to trauma and organophosphate poisoning. the association of pancreatitis with inflammatory bowel disease and cholangitis is frequently mentioned (triaditis) but poorly described in the literature. one study found % of ibd cases to have histologic evidence of pancreatic involvement, and another found fpli concentrations were elevated in % of cases with histologically confirmed ibd. it is the author's experience that many cases of pancreatitis recognized with ibd have no specific clinical signs attributable to pancreatitis and should therefore be diagnosed and treated as intestinal disease. diabetes mellitus is a recognized co-morbidity of pancreatitis in cats. a recent study found fpli concentrations were significantly higher in diabetic cats compared with non-diabetics. no association could be made between fpli concentrations and the degree of diabetic control. one study found of cats ( %) histologically diagnosed with hepatic lipidosis were also histologically diagnosed with acute pancreatitis. it is not known if pancreatitis is a cause, consequence, or coincident disease of hepatic lipidosis. for example, anorexia associated with acute pancreatitis could predispose to fatty infiltration of the liver. however, the high rate of concurrent disease has important implications for ensuring cats with pancreatitis receive adequate caloric intake. ongoing or recurrent pancreatitis may lead to pancreatic cysts or exocrine pancreatic insufficiency, which are both covered later in this chapter. although pancreatitis has been experimentally induced in cats, , , the pathophysiology of spontaneous pancreatitis remains unknown. acute pancreatitis is initiated by an increase in secretion of pancreatic enzymes that leads to inappropriate cellular activation of trypsin and subsequently other digestive zymogens. these activated digestive enzymes lead to local effects including inflammation, hemorrhage, acinar cell necrosis, and peripancreatic fat necrosis. , , chronic pancreatitis may result from any of several underlying processes: ongoing, low-grade acute pancreatitis episodes may instigate chronicity; chronic pancreatitis, with a predominance of lymphocytic inflammation has been induced experimentally within weeks by narrowing the main pancreatic duct to approximately % of its normal diameter ; and the association with ibd may suggest an immune-mediated cause. the clinical signs of pancreatitis in cats are nonspecific. a review of eight prior series totaling cases of acute pancreatitis in cats found anorexia ( % of cases) and lethargy ( %) to be the most common historical findings. vomiting was recognized in % of cases, diarrhea in %, and weight loss in %. physical examination findings were similarly nonspecific with dehydration ( %) being the major finding; fever was recognized in only % of cases and abdominal pain in %. it is important to note that vomiting and abdominal pain, key features of pancreatitis in dogs, are not consistently recognized in cats. similar, nonspecific findings indistinguishable from ibd are recognized in cats with chronic pancreatitis. , diagnosis because the presenting signs and physical examination findings are nonspecific, the diagnosis of pancreatitis can be challenging, requiring not only clinical suspicion but a combination of diagnostic modalities. for the most part, hematology and plasma biochemistry findings are unremarkable, although a combination of findings may increase clinical suspicion. for example, moderate elevations in liver enzymes, bilirubin, and glucose are present in approximately % of cases and hypocalcemia in approximately two of three of cases; hypocalcemia infers a poorer prognosis. hypoalbuminemia is seen in approximately one of three of cases and has important implications for fluid therapy. amylase and lipase elevations are not reflective of pancreatitis in cats. feline trypsinlike immunoreactivity (ftli) is the diagnostic test of choice of exocrine pancreatic insufficiency, but elevations in pancreatitis are not seen consistently enough to warrant use of this test for this purpose. , , the biggest recent advance in feline pancreatic diagnostics has been the characterization of feline pancreatic lipase, leading to the development of a radioimmunoassay for the measurement of feline pancreatic lipase immunoreactivity (fpli). it must be remembered, however, that an increase in fpli only tells the clinician that pancreatic pathology is present, but not the cause of pathology, which may be, for example, neutrophilic or lymphocytic pancreatitis or neoplasia, and it may or may not involve the intestines or liver. fpli should therefore be used as a screening test, with elevated results not suggesting a diagnostic end point. further, the high interassay variability of this test would suggest that mild cases may be missed as shown in one study and that the test may not be appropriate for serial monitoring. fpli is currently available as "spec fpl" from commercial laboratories and has a sensitivity of % and a specificity of % when . µg/l is used as the diagnostic cut off compared with . µg/l, which is the listed reference range high point. in an acutely unwell cat (less than days) with only mild to moderate signs of disease, further diagnostics may not be warranted, and many cats will improve with supportive therapy of balancing fluid and electrolytes, pain relief, and antinausea/vomiting therapy. cats with chronic duration of signs and acutely unwell cats that do not improve with supportive therapy warrant further diagnostics. the underlying disease process cannot be assumed from an elevated fpli; in one study of cases, acute necrotizing pancreatitis could not be distinguished from chronic nonsuppurative pancreatitis by signalment, duration of signs, or clinical findings. the major utility of diagnostic imaging is to rule out other differential diagnoses, such as an intestinal foreign body, and perhaps confirm that the pancreas is affected. radiography is non-specific for diagnosis of pancreatitis, but findings may include decreased abdominal detail (sometimes associated with ascites), soft tissue density in the right cranial quadrant of the abdomen, hepatomegaly, or gas-filled intestines , , (see . additionally, thoracic radiographs may show pleural effusion. one study found of cats with pancreatic necrosis had such a change ; the mechanisms resulting in pleural effusion are not precisely defined. ultrasonography has high specificity (> %) but low sensitivity (< %) for recognizing pancreatitis in cats, , , , with findings dependent on operator skills, quality of equipment, and severity of lesions. typical findings are hypoechogenicity of the pancreas, which may be enlarged or irregular; hyperechogenicity of the peripancreatic fat; the possible presence of abdominal effusion; and abnormal findings with other organs, such as liver or intestine, may add to the clinical picture , , , . one study indicated that contrast-enhanced doppler ultrasonography can provide further diagnostic insights. a recent study suggested that endosonography may be useful in cases where transabdominal ultrasonography is difficult, for example, because of obesity, hyperechoic mesentery, or excessive intestinal gas. for more than years, computed tomography (ct) has been a commonly used modality to confirm pancreatitis in humans, but this reliability has not been demonstrated in cats, where sensitivity may be as low as %. , definitive diagnosis of pancreatitis, including differentiation of the inflammatory process, can only be made by cytologic assessment of pancreatic tissue. in most cases, ultrasound-guided fine-needle aspiration (fna) of the pancreas is technically difficult because of the small dimension of the feline pancreas; there appears to be no assessment of feline pancreatic fna findings in the literature. gross inspection of the pancreas and samples for histologic assessment can be obtained during laparotomy , (see or laparoscopy. , because pancreatitis often occurs concurrently with pathology of other organs, thorough evaluation of the abdomen by ultrasonography or gross inspection is recommended, as are multiple biopsies of, for example, intestines, liver, and mesenteric lymph nodes, where appropriate. clinicians may be reluctant to biopsy the pancreas because of perceived risks of deleterious effects. studies of pancreatic biopsy in healthy cats dispel the concern that the pancreas is unforgiving to mild manipulation and biopsy , a and the author's clinical experience is consistent with these findings. supportive care comprising correction of fluid/ electrolyte imbalances, pain management, and nutritional support are the mainstay of therapy for cats with figure - gross appearance of pancreas at laparotomy; this was histologically diagnosed as chronic pancreatitis (i.e., lymphocytic infiltration was recognized). gross appearance of pancreas at laparotomy; this pancreas was found to be histologically normal. it does look smaller than is typically seen; pancreatic atrophy can look similar to this, grossly. pancreatitis. , , specific underlying causes, when diagnosed, should be managed, as should concurrent diseases. follow-up evaluation is determined on a case-by-case basis; reduction or resolution of clinical signs is the main criterion for success of therapy. serial fpli values may be monitored when initial results are extremely high but are of limited value for mild increases because of assay variability. dehydration, acid-base and electrolyte abnormalities should be corrected during the first to hours. hypocalcemia, if present, should be treated with a calcium gluconate infusion of to mg/kg during to hours, with continued assessment of plasma calcium concentrations. plasma transfusions can be considered in cats with hypoalbuminemia. , , although abdominal pain is not commonly described in cats with pancreatitis, it is likely to be present in most cases and may contribute to anorexia. historical concern about exacerbation of pancreatitis with opioids is no longer accepted, and this class of drugs is considered appropriate. meperidine ( to mg/kg sc or im) every to hours, butorphanol ( . to . mg/kg sc) every hours, or sustained-release buprenorphine ( µg/kg sc) every hours are alternatives. , , the author uses one dose of methadone ( . to . mg/kg sc, im, or iv) initially and places a fentanyl patch for longer-term pain management. the traditional recommendation for management of pancreatitis across all species has been nil per os for several days. this recommendation is appropriate for cats with severe vomiting, but there is no evidence to support this approach in cats that are not vomiting and that are eating normally. further, nutritional support is vital for those cats with concurrent hepatic lipidosis. if the cat is not eating voluntarily, nutritional support by tube feeding is often warranted. , , a recent paper found nasogastric tube feeding of cats with pancreatitis was tolerated well and resulted in few clinically significant complications. other reported nutritional strategies for cats with pancreatitis incorporate partial parenteral nutrition (ppn; . % amino acids, % lipids), or total parenteral nutrition (tpn; % amino acids, % lipids, % dextrose), or both instead of enteral feeding. , , cats do not seem to benefit from feeding of specially formulated low-fat diets; commercially available, veterinary liquefied diets appear to be well tolerated despite their high-fat contents. other therapy may be appropriate in individual cases. all cats with pancreatitis that are vomiting should be treated with antiemetics. examples of drugs that can be used are -ht antagonists, such as dolasetron ( . to . mg/kg iv or po, once to twice daily); ondansetron ( . to . mg/kg iv every to hours); and maropitant, an nk -inhibitor ( . to . mg/kg sc once daily). these drugs are covered in detail earlier in this chapter under therapeutics for vomiting and diarrhea. dopaminergic antagonists, such as metoclopramide, are less effective antiemetic agents in cats than the other choices mentioned. , in most cases, pancreatitis begins as a sterile process, and antibiotic therapy is controversial. pancreatic necrosis and inflammation may predispose to bacterial colonization of the pancreas as demonstrated in experimental models. , this has not been demonstrated in spontaneous disease, and no comparison of outcomes has been made of cats with pancreatitis treated with or without antibiotics. cefotaxime ( to mg/kg iv, im) has been used to prevent bacterial colonization in experimental models. other broad-spectrum cephalosporins or ampicillin may act similarly. antibiotic considerations are possibly more important for acute pancreatitis than for treatment of chronic disease. cats with demonstrated lymphocytic pancreatitis, with or without concurrent ibd or lymphocytic cholangitis, should be treated with corticosteroids (e.g., prednisolone, to mg/kg once to twice daily) with tapering to the lowest effective dose. there is no justification for use of corticosteroids in cats with acute necrotizing or acute suppurative pancreatitis, or cats for which the cause of pancreatitis has not been diagnosed histologically. use of corticosteroids in cats with pancreatic disease creates a risk of iatrogenic diabetes mellitus. surgical intervention is warranted to relieve any bile duct obstruction that may result or for the débridement of pancreatic abscesses or necrotic tissue; in many cases, cats will survive multiple years after such corrective surgery. pancreatic cysts, pseudocysts, and bladders have been described sporadically in cats.* pancreatic cysts are lined by a single layer of cuboidal epithelium and do not communicate with the pancreatic duct; pseudocysts are enclosed by a wall of fibrous tissue, lacking the epithelial lining characteristic of true cysts and can form secondary to pancreatic inflammation; cystic dilations of the pancreatic duct are referred to as pancreatic bladder. true pancreatic cysts have been described in three cats , , ; a congenital pancreatic cyst with associated inflammation was described as an incidental finding in an adult cat ; multiple pancreatic cysts were described in a cat with concurrent polycystic disease in the kidney and liver ; and a another cat had multiple recurrent pancreatic cysts with concurrent mild pancreatic inflammation and atrophy associated a with rapid clinical course resulting in diabetes mellitus. cysts, pseudocysts, and bladders may be identified ultrasonographically or by ct. they may be benign, but the associated pancreatic inflammation and other sequelae, such as diabetes mellitus, may need to be managed. pancreatic bladders may result in biliary obstruction, and surgical correction may be required. pancreatic nodular hyperplasia is recognized quite frequently as an incidental finding in older cats or at necropsy. neoplasia of the exocrine pancreas is rare in cats. its frequency was assessed in the s when one study estimated . cases per , patients per year at risk, and another found pancreatic tumors in of feline necropsies. a more recent study recognized, from , feline admissions over a -year study period, only two cats with pancreatic adenomas ( . % of admissions) and eight with pancreatic adenocarcinomas ( . % of admissions). adenomas appear as small, solitary or multifocal nodules and are not typically associated with adjacent pancreatic inflammation. they do not cause clinical signs, unless large, when any clinical signs result from the physical size and are usually an incidental finding. , few generalities can be made about the presentation for pancreatic adenocarcinoma. the age range is large ( to years), there is no sex predisposition, and no clear breed predispositions are present. , only cytology or histopathology can distinguish pancreatitis from pancreatic carcinoma in cats antemortem, yet it is important to differentiate the two conditions, because, in contrast to adenomas, pancreatic adenocarcinoma is associated with a grave prognosis. the presence of lesions consistent with metastases on radiography or ultrasonography may suggest malignancy, but one study could not distinguish neoplasia from pancreatic nodular hyperplasia ultrasonographically based on the appearance of the pancreas alone (figures - and - ) . pancreatic adenocarcinomas in cats can result in a paraneoplastic dermatologic condition consisting of nonpruritic, symmetric alopecia affecting the face, ventral body, and medial aspect of the limbs of cats. the skin is usually glistening but not fragile, and there can be crusty lesions on the footpads.* the pathogenesis of this dermatologic disease is unknown. in one case, surgical excision of the pancreatic carcinoma resulted in resolution of dermatologic disease, indicating that the process is reversible (although signs recurred as the tumor re-emerged). diabetes mellitus is a recognized complication of pancreatic adenocarcinoma. the mechanism is unknown and may simply be secondary to compression or invasion of islet cells by the tumor. in some cats, diabetes is recognized ahead of pancreatic neoplasia. , , obstructive jaundice has also been described with pancreatic adenocarcinoma. most cases of pancreatic adenocarcinoma in cats have metastasized by the time of diagnosis, and most reported cases die or are euthanized within days of diagnosis. surgical excision is a potential option if neoplasia is confined to one limb of the pancreas, but recurrence is possible even if there is no evidence of metastasis and excision seems complete at the time of surgery. exocrine pancreatic insufficiency (epi) is a condition caused by insufficient synthesis and secretion of pancreatic digestive enzymes from the exocrine portion of the pancreas. in humans it has been reported that % of pancreatic acinar cells must be lost before clinical signs of epi are seen. epi is considered rare in cats but is perhaps being recognized more frequently because of increased awareness. there are less than fifty cases described in the veterinary literature* with one of these papers describing only cases from five institutions, with prevalence described as . % to . % of cats seen over a -year period. in contrast to this, the gastrointestinal laboratory at texas a&m university recognized samples with serum ftli concentrations at or less than . µg/l, which is diagnostic for epi, out of , submissions, which equates to . % of cats with known or suspected gastrointestinal disease. all studies indicate a wide age range of cats can be affected, from kittens less than months of age to cats more than years old, with a median age of approximately years. there is no apparent breed predisposition. , , one paper recognized of ( . %) cats to be male, and another recognized of ( %) male cats, suggesting a possible sex predisposition. chronic pancreatitis is believed to be the most common cause of epi in cats, acinar atrophy (paa) is recognized as the most common cause of epi in dogs, and has been definitively described in two feline cases and mentioned as a cause for three other cases. other potential causes of epi include disruption of pancreatic enzyme flow at the duodenal papilla following duodenal resection and pancreatic fluke infection (eurytrema procyonis), , and amyloid deposition and neoplasia are other possible causes of pancreatic cell damage that have not definitively been described in cats. congenital pancreatic hypoplasia or aplasia has not definitively been reported in cats, but reports of epi in cats as young as months of age , suggest this possibility. since chronic pancreatitis is a common cause of epi and chronic pancreatitis has a strong association with ibd, many cats may have concurrent lymphocytic pancreatitis and enteritis. , , therefore cats failing to respond to therapy for epi may require further diagnostics and management of an underlying condition. further, destruction of functional exocrine pancreatic tissue can also affect pancreatic endocrine tissue, resulting in concurrent diabetes mellitus. several studies have indicated that all cats with epi will have weight loss when diagnosed, unless a kitten, in which case ill-thrift is recognized. , diarrhea is not necessarily present, being described in % to % of cats; the nature of feces can vary from voluminous, malodorous stools that can be discolored (yellow or pale), sometimes with steatorrhea, to normal feces in other cats. increased frequency of defecation and the presence of mucus in the feces of some cats can lead to the diarrhea being characterized as large bowel. only about % to % of cats are polyphagic, some described as having a ravenous appetite; conversely, some cats present with anorexia. vomiting has also been described. since cats with epi often have concurrent disorders, such as ibd, the clinical signs recognized may reflect the concurrent disease and not necessarily epi alone. physical examination findings are similarly nonspecific, with thin/ emaciated body condition being the most common finding. hematologic findings are non-specific, but a mild nonregenerative, normocytic, normochromic anemia may be recognized as well as lymphopenia or neutrophilia. plasma biochemistry results may show a mild to moderate increase in alanine aminotransferase (alt) and a mild increase in alkaline phosphatase in some cats. mild to moderate hyperglycemia may be seen, as may mild hypoglycemia or normoglycemia. , , hypocobalaminemia is recognized in nearly all cats with epi. , , , , this may be because of insufficient production of intrinsic factor, a cobalamin-binding protein only produced by the pancreas in cats and necessary for ileal absorption of cobalamin ; it may also be because of failure of pancreatic enzymes to liberate cobalamin from binding by r protein in the duodenum or small intestinal bacterial overgrowth (sibo), not yet specifically described in cats. folate concentrations may be reduced (because of concurrent intestinal malab sorption), normal, , or increased, which may relate to reduced pancreatic bicarbonate secretion, secondary to severe hypocobalaminemia, or associated with sibo. none of these presenting complaints, physical examination findings, or routine testing results are specific to epi. therefore epi requires a degree of clinical suspicion and/or thorough diagnostics to ensure the diagnosis is not missed. a low level of serum ftli is diagnostic for epi. , , samples can be sent to the gastrointestinal laboratory at texas a&m university from anywhere worldwide (with instructions about sample handling requirements on their website: http://vetmed.tamu.edu/gilab/). the reference range for serum ftli is to µg/l, with concentrations at or less than . µg/l diagnostic for epi. since the clinical signs and routine laboratory findings are nonspecific for epi, it is ideal to test serum for ftli in any cat with weight loss or ill-thrift. the texas a & m gastrointestinal panel also includes testing for levels of cobalamin, folate, and fpli, ensuring concurrent hypocobalaminemia will not be missed and potentially providing indications of other gastrointestinal disease. conversely, although a low level of serum ftli confirms a diagnosis of epi, it is not necessarily a diagnostic end point, since epi is so often recognized concurrently with other gastrointestinal disease. failure to respond to therapy should prompt the clinician to consider and investigate further for concurrent processes. most cats with epi can be successfully managed with dietary supplementation of pancreatic enzymes. commercial products (e.g., viokase [axcan pharma, birmingham, ala.], pancrezyme [virbac, fort worth, tex.], and creon [abbott laboratories, abbott park, ill.]) are available, and powder is considered more effective than tablets or capsules (some capsules can be opened and the contents sprinkled onto food, like powder). the required dose can vary quite substantially from cat to cat. it is appropriate to start with one teaspoon of powder with food twice daily, and adjustments can be made depending on the response; most cats accept the powder readily if it is mixed thoroughly through canned food, but other flavors (e.g., fish oil or brine from canned tuna) can be used to disguise the taste if necessary. raw pancreas (e.g., from beef or pork) may also be used, with to g twice daily an appropriate starting dose. since most cats with epi are hypocobalaminemic, supplementation by subcutaneous injection is required (oral supplementation is not effective since cobalamin deficiency leads to cobalamin malabsorption). an appropriate dose for most cats is µg, and it is usually given weekly for weeks, then every second week for a further six doses; it is appropriate to continue dosing every month beyond that. owners can be taught to inject their cats at home (as owners of diabetic animals are taught to do with insulin). because some cats may have sibo, antibiotics such as metronidazole ( to mg/kg po every hours for days) may be warranted. an elevation of folate may arouse suspicion of sibo, but it is appropriate to try antibiotics in a cat failing to respond to enzyme and cobalamin supplementation. concurrent diseases, such as lymphocytic, chronic pancreatitis, or ibd may need to be managed with corticosteroids, or diabetes mellitus with insulin. no studies have assessed specific dietary requirements in cats with epi. most cats respond to appropriate treatment, with a return to normal weight and normal feces. with ongoing therapy, cats can lead normal lives for a full life span. the feline liver is a large, complex organ involved in a variety of essential metabolic, functional, and detoxification processes that can be affected, individually or collectively, by disease or dysfunction. cats have a unique set of liver diseases that occur more commonly in this species compared with the typical diseases that occur in dogs, and these include hepatic lipidosis, feline cholangitis syndrome, and infectious hepatopathies (e.g., fip, flukes, histoplasmosis, toxoplasmosis). , , , , nevertheless, these conditions often present with characteristic clinical, laboratory, and histopathologic changes that are necessary for proper diagnosis and management. the goal of this section is to review the interpretation of clinical and laboratory changes that occur in these feline liver diseases, provide an approach for separating the more common diseases by their clinical footprint, and then discuss therapy of each liver disease based on our current level of understanding of hepatoprotectants, antioxidants, and drugs used for specific therapeutic purposes. the clinical signs of liver disease in cats are often vague and nonspecific; however, recognition of certain clinical and laboratory abnormalities and their association with liver disease can greatly aid the diagnostic process. the most common early clinical signs observed in cats with liver disease are anorexia, lethargy, and weight loss, which are signs present in many (if not most!) feline diseases. , because these early indicators of disease do not point specifically toward liver disease, a delay in diagnosis will occur unless the clinician carefully considers all possibilities and performs other tests to further evaluate the situation. for example, feline hepatic lipidosis is the most common form of liver disease in cats in the united states, united kingdom, japan, and western europe, occurring with a prevalence of nearly % in one study. however, the most common, and often only, clinical sign associated with onset of this condition is anorexia; the signs of serious hepatic disease (especially jaundice and vomiting) do not occur until later (days or weeks) in the course of the disease. , recognition that anorexia in a cat, even for a few days, is a risk factor for development of hepatic lipidosis is essential, and this risk is increased in obese cats. , further, the clinical signs of liver failure develop much more slowly; many cats with hepatic lipidosis present alert and responsive until much later in the course of the disease, thus delaying onset of appropriate therapy. a similar clinical situation exists for the second most common form of liver disease in cats, feline cholangitis syndrome. , , this complex of diseases in the cat can be associated with signs ranging from anorexia and lethargy to vomiting and jaundice, and these signs can vary in severity and prevalence. the key point is that except for development of jaundice, there is no constellation of clinical signs that are classic clinical indicators of liver disease in cats. , as with many feline diseases, the subtle clinical signs of anorexia, lethargy, or inactivity are often the only signs of illness and should be further investigated. there are few changes that occur in the complete blood count that are specific indicators of primary liver disease in cats. the most common finding is the presence of poikilocytes, which are red blood cells with an irregular shape, speculated to be caused by changes in membrane lipids as a result of liver dysfunction. other abnormalities may occur, such as anemia of chronic disease or neutrophilia, but these findings are nonspecific and occur with variable frequency. perhaps the most important reason for obtaining a hemogram is in icteric cats, because this test is essential to help rule out hemolysis as the cause of the hyperbilirubinemia. the serum chemistry profile can be very helpful, but there are several critical points in interpretation of these values that are important to review. the hepatic transaminases (alanine aminotransferase [alt] and aspartate aminotransferase [ast]) are leakage enzymes but do not discriminate among hepatobiliary disorders, nor do they provide an indicator of severity or disease origin. thus although increases in alt may be noted in cats with liver disease, they are also present in a variety of other systemic infectious, inflammatory, neoplastic, and and protein-losing nephropathies can also cause loss of albumin and affect cholesterol, it is essential to evaluate the cat for these problems when interpreting these results. finally, bilirubin metabolism is a critical function of the liver, but interpretation of hyperbilirubinemia requires a careful consideration of bilirubin disposition. hyperbilirubinemia develps because of one of three possible causes: ( ) excessive hemolysis of red blood cells (rbc) (also known as prehepatic icterus)-high bilirubin in the blood stream occurs because of an overload of the mononuclear/phagocyte system with heme pigments from rbc destruction, ( ) hepatic parenchymal disease or insufficiency (also known as hepatic icterus)-resulting in lack of normal bilirubin metabolism in hepatocytes and regurgitation of the pigments into the blood stream when they are not taken up into cells and excreted in bile, and ( ) disease of gall bladder, biliary tract, or pancreatic duct (also known as posthepatic icterus)resulting in obstruction of the bile ducts or loss of bile into the abdomen (duct or gall bladder rupture and bile peritonitis). the bottom line is that in any cat with hyperbilirubinemia, an assessment of the packed cell volume and rbc morphology should be completed to determine whether icterus is caused by hemolysis. once hemolysis is ruled out, then assessment of primary endocrine diseases, including hyperthyroidism, feline heartworm disease, fip, and neoplasia.* alternatively, the cholestatic membrane-associated enzymes alkaline phosphatase (alp) and gamma glutamyltransferase (ggt) are especially useful for recognizing disorders involving biliary or pancreatic ductal components. unlike the dog, these enzymes will only increase modestly in cats, even in severe disease, and there is no glucocorticosteroid or drug induction of the enzymes to influence interpretation. , thus increases in alp in the adult cat represent a release of enzyme from the hepatobiliary tree and should be considered clinically important. both alp and ggt are produced in other tissues than the liver, with the highest ggt activity present in the kidney and pancreas; however, sources other than the liver do not contribute to the activity of these enzymes in health. recent studies of the effects on these enzymes in cats with pancreatitis, cholangitis, extrahepatic bile duct obstruction (ehbdo), and hepatic lipidosis reveal some important characteristics in interpreting increases in these enzymes. first, both alp and ggt are increased in cats with pancreatitis, cholangitis, or ehbdo, because inflammation in the biliary tree also affects the pancreatic ducts (and vice versa, figure - ) , and if the fold increases in these enzymes are similar, the diagnosis is likely one of the three. conversely, in cats with hepatic lipidosis (without concurrent inflammatory disease of the biliary or pancreatic duct system), large increases in alp are observed, but ggt will remain normal or only slightly increased. thus if the increase in alp is to times, while ggt is not increased or is only increased to times, then the likely diagnosis is hepatic lipidosis. [ ] [ ] [ ] other than enzymes on the biochemistry panel, which are of limited value for assessing liver function, there are several key tests that can be used to help assess liver function cats with elevated liver enzymes. these five tests found on most routine biochemistry panels are helpful functional indicators: cholesterol, bilirubin, glucose, albumin, and urea nitrogen (bun). however, none are immune to outside influences on their interpretation, including bilirubin and cholesterol, which are the most liver specific. in cats with severe liver disease or failure, bilirubin levels tend to be quite elevated, while bun, albumin, cholesterol, and glucose concentrations tend to be significantly decreased, reflecting inability to metabolize urea (lack of arginine), inability to produce albumin or cholesterol, and abnormal metabolization of glucose. however, these changes represent severe loss of liver function and thus are not sensitive indicators of liver function because the changes occur quite late in the course of the disease. nevertheless, in cats with elevated liver enzymes and clinical signs of liver disease, these values should be carefully assessed. because gi disease of hepatic failure. in nonicteric cats with severe liver disease or in young cats suspected of having a portosystemic shunt, serum bile acids are the more reliable indicator of hepatic insufficiency. the measurement of serum bile acid concentrations, preprandially and postprandially, is the most reliable, readily available, and sensitive test of hepatic function in nonicteric cats. , that being said, although increases in bile acids are accurate indicators of hepatic insufficiency, the levels cannot be used to assess severity of disease or the type of dysfunction. further, bile acid assays are most effective when paired samples (preprandial-and postprandial) are compared, because single, fasting, or random bile acid samples can result in a falsenegative (normal) result. however, cats will often not eat in the hospital or when they are sick, and this prevents collection of a postprandial sample. however, this does not invalidate the results, because if the result of the single bile acid sample is abnormal, it does reliably indicate liver dysfunction. an alternative to using serum for testing bile acids in cats is urine bile acid analysis. healthy cats excrete a small percentage of conjugated bile acids in the urine ; however, in cats with liver disorders that cause increased serum bile acids (and especially cholestatic liver diseases) a significant increase in urine bile acid excretion occurs. when urine bile acids (uba) were collected to hours after a meal and measured (normalizing the value with urine creatinine: uba/ucr) and compared with serum bile acids in a study of cats with hepatic disease, cats with nonhepatic disease, and normal cats, the results were highly correlated. the utility of the urine bile acid test is that it does not require a paired sample (postprandial test), and it is not as affected as the serum test is by hemolysis or lipemia of the blood sample. normal cats will have an uba/ucr of less than . µmol/mg, while values greater than . are considered evidence of significant hepatic dysfunction. it is well known that the liver plays a central role in coagulation homeostasis and is the single site of synthesis of many coagulation proteins, anticoagulant proteins, and fibrinolytic factors. vitamin k is one of the most common factors found to be inactive or deficient in cats with liver dysfunction, and it is essential for normal functioning of factors ii, vii, ix, and x; protein c and s; and thrombin. insufficient or inactive vitamin k can occur for a variety of reasons, including dietary restriction (e.g., anorexia or diet deficiency), disruption of the enteric microflora that synthesize vitamin k (e.g., chronic antibiotic therapy), diseases causing fat malabsorption (e.g., ibd, exocrine pancreatic insufficiency), ingestion of vitamin k antagonists, or liver dysfunction. for example, in cats with hepatic lipidosis, approximately % will have an increased prothrombin time (pt), % will have an increased partial thromboplastin time (ptt), but % of cats will have increased pivka parenchymal disease versus disease of the biliary tree is completed by evaluating the clinical presentation, laboratory values, and imaging of the biliary tree and abdomen for possible evidence of biliary or pancreatic disease. a urinalysis is also an important part of the minimum database, and it is no different in a sick cat with suspected liver disease. in cats the presence of hyperbilirubinuria is abnormal at any urine concentration, because they do not conjugate bilirubin in their renal tubules. however, like bilirubinemia, presence of bilirubin in the urine can occur because of any of the three possible causes of hyperbilirubinemia: prehepatic, hepatic, and posthepatic; thus further evaluation is necessary once bilirubin is detected. ammonium biurate crystalluria suggests the presence of hyperammonemia, which in the cat is either because of a congenital portosystemic shunt (less common in cats than in dogs) or because of severe, end-stage liver disease resulting in portal hypertension, which is typically caused by cirrhosis or advanced polycystic liver disease. , the most common feline liver diseases are hepatic lipidosis and feline cholangitis syndrome, which are two diseases that often result in development of clinical or biochemical icterus. thus because hyperbilirubinemia is a more sensitive indicator of liver function than bile acids or other liver function tests, the need for further testing is moot. however, there will be circumstances when further assessment of liver function is indicated, and for this, serum bile acids, blood ammonia levels, and urine bile acids may be needed. there are several situations where liver function testing may be indicated, but the most common indications for additional testing would be a cat with persistently elevated liver enzymes of unknown origin, a cat that develops urethral obstruction because of urate stones (suggestive of portosystemic shunting) or a cat with possible polycystic liver disease. one of the oldest tests of liver function, because of its association with development of hepatoencephalopathy, is measurement of blood ammonia levels. however, although this test is the only practical way to diagnose hepatoencephalopathy in dogs, the test has a number of limitations, including differences in ammonia levels between arterial and venous (lower) samples and significant sample handling issues (ammonia is labile and results are affected by improper sample handling or lack of immediate measurement) that make its use difficult in practice. in cats hyperammonemia is even less common than in dogs likely because of their highfunctioning urea cycle pathways ; the assays have not been validated for feline blood in most laboratories, and as such, the test is not recommended as the sole indicator uncommon in cats, the most common causes are neoplasia (primarily of the pancreas, but cholangiocarcinomas can occur) or chronic pancreatitis, which can occur concurrently with cholangitis in cats, resulting in both intrahepatic and extrahepatic cholestasis in some cats. , the bile ducts are affected in cats with chronic pancreatitis, because the feline biliary system and pancreatic duct system merge at the level of the pancreas to form a single duct that empties into the duodenum. thus in cats with either pancreatitis or biliary disease, recent evidence has shown that the inflammation affects both organs. , further, in chronic pancreatitis, either persistent inflammation or development of fibrosis can result in dilation or obstruction of the common bile duct. in cats with chronic ehbdo, the common bile duct will become widely dilated and tortuous, a finding easily seen on abdominal ultrasonography but a problem not easily managed (figures - and - ) . interestingly, the gallbladder is often not enlarged, and may in fact be small in cats with this condition, because the remaining fluid in the gallbladder is white bile (highly concentrated mucinous bile from which the pigment has been resorbed). in addition, variable filling of the gall bladder is a normal phenomenon; thus gallbladder size is not an indicator of ehbdo. (proteins induced by vitamin k antagonists or absence). nevertheless, although pivka is a very sensitive test for abnormalities of vitamin k function, most cats with liver disease that have a normal pt/ptt, but abnormal pivka do not represent clinical evidence of bleeding. in any case, abnormalities in the clotting cascade related to vitamin k deficiency in cats with liver disease are common, whether or not they show evidence of active bleeding. and because the balance of the coagulation system in a cat with liver disease can be disrupted by a procedure that initiates small amounts of bleeding (e.g., a biopsy), all cats with liver disease should be given vitamin k as a precautionary measure before and after invasive procedures, even if the clotting times (pt and ptt) are normal. this may be especially important in cats with hepatic lipidosis, because their vitamin k clotting status is likely to be even more affected by the concurrent anorexia and disruption of enteric microflora. the dose of vitamin k (phytonadione, aquame-phyton [merck, west point, pa.]) used prophylactically is . mg sc, im, or po q h for to days, then weekly until recovered. see box - for a summary of the causes of icterus. cholestasis is the reduction of bile flow, which can occur at any point along the biliary tree; bile production occurs in hepatocytes, and flow is connected to the distal concentrating components (gallbladder and common bile duct) by the bile ductules. thus cholestasis can occur inside the liver's biliary tree (intrahepatic cholestasis) or outside the liver in the gallbladder and common bile duct (extrahepatic cholestasis). intrahepatic cholestasis most often occurs in diseases involving hepatocellular damage, leakage, or swelling, such as infections (e.g., bacterial cholangiohepatitis, toxoplasmosis, fip, or other diseases causing inflammation), infiltrative diseases (e.g., lymphoma), metabolic diseases (e.g., hepatic lipidosis), or diseases causing disruption of architecture (e.g., cirrhosis or severe polycystic disease). intrahepatic cholestasis occurs in zone of the liver lobules (periportal zone); at the level of hepatocytes, canaliculi or bile ductules; and is damaging to cells because of the emulsifying properties of lipid on membrane lipids. however, because the liver has a large reserve capacity, clinical icterus (e.g., jaundice) only occurs in the most severe cases when the liver is affected diffusely. thus severe or persistent intrahepatic cholestasis can serve to perpetuate the inflammation and cell damage if it is not corrected. extrahepatic cholestasis or extrahepatic bile duct obstruction (ehbdo) is less common than intrahepatic cholestasis and is most commonly associated with obstruction of the common bile duct. since gallstones are icterus is the result of cholestasis, and the underlying cause can be either hemolysis or hepatobiliary disease, for which further clinical examination will be needed to determine if rbc destruction or liver disease is occurring. in most hepatobiliary diseases of cats, cholestasis is occurring, but there may be no clinically apparent icterus because the degree of hyperbilirubinemia must be at least to times greater than the normal values to exceed the capacity of the liver to process the excess bilirubin. in cats with hyperbilirubinemia not caused by hemolysis, whether it is clinical or subclinical, there is no need for further evaluation of liver function (e.g., bile acid assays), because bilirubin is a more sensitive indicator of liver function than bile acids. the degree of hyperbilirubinemia does not suggest differentiation of intrahepatic versus extrahepatic cholestasis; however, the presence of acholic feces (white feces) is diagnostic for extrahepatic bile duct obstruction (ehbdo), because lack of stercobilinogen (the brown/black pigment in feces) is only found in cats with complete obstruction of the bile duct. finally, the presence of intrahepatic cholestasis and clinical icterus in a cat indicates a diffuse hepatobiliary disease, such as cholangitis or hepatic lipidosis, as focal liver disease, even if severe, will not cause clinical hyperbilirubinemia because of the tremendous reserve capacity of the liver for bilirubin uptake. portal hypertension is an abnormally high venous pressure in the portal system and is typically caused by increased resistance to portal blood flow. there are potentially three regional causes of portal hypertension: prehepatic (disease in the portal vein itself), hepatic (intrahepatic diseases causing compression or decreased flow), and posthepatic (diseases of the caudal vena cava, right heart or pulmonary vasculature). the most common cause of portal hypertension in the cat is cirrhosis or portal venous thrombosis, because portal vein hypoplasia (formerly known as microvascular dysplasia) is known to occur only in the dog, and the other causes of portal hypertension (budd-chiari syndrome, heartworm caval syndrome, pulmonary hypertension) are rare and more likely to occur in the dog. , in any case, the clinically recognizable effects of portal hypertension are development of ascites (unusual in the cat), acquired portosystemic shunting (reported in cats), and development of hepatic encephalopathy (less common in cats than in dogs, because of their profound ability to handle protein wastes). , , most cats and dogs that develop hepatic encephalopathy (he) secondarily to portal hypertension do so because of reduced liver function (because of portosystemic vascular shunting [pss] or cirrhosis and the acquired shunting that develops). cats can develop another form of chronic he because of hepatic lipidosis, but this is believed to be because of the combination of liver failure and prolonged fasting, resulting in arginine deficiency and impaired ammonia detoxification. portosystemic vascular anomalies, also called portosystemic shunts or portovenous shunts (pss), although less common than in dogs, also occur in cats. these vascular anomalies can be either congenital or acquired, single or multiple in number, and occur as extrahepatic vascular shunts or within the liver itself (intrahepatic shunts). the shunting of blood around the liver is the cause of hepatic atrophy and reduced hepatic function that results in an accumulation of toxins, particularly ammonia that leads to the development of hepatoencephalopathy. the two most common veins that serve as the connection point for the shunting portal venous blood are the caudal vena cava and the azygous. in cats a single, extrahepatic, portocaval shunt is the most commonly reported form, and occurs in % of cats with pss. as in dogs, specific breeds of cats may have pss more commonly, and these include domestic shorthair cats, burmese, siamese, persian, and himalayan breeds. in contrast to dogs, males may be more predisposed to pss than females, but the clinical signs relate to the three body systems most affected: the central nervous system, gi tract, and urinary tract. the most common presenting complaints in cats are weight loss or poor/stunted growth, and dull, bizarre or lethargic behavior, especially after eating. signs of gi disease common in dogs, such as vomiting, diarrhea, or inappetence, are less common in cats, but in one report, % of cats with pss drooled. finally, cats with pss often present with signs of lower urinary tract disease (e.g., hematuria, stranguria, or even obstruction) because of the development of urate uroliths (which are radiolucent, thus difficult to detect). because the most common signs of he are apathy, listlessness, and decreased mental alertness, they are often not recognized specifically as indicative of brain dysfunction but as part of the constellation of signs of the liver disease. however, with progression of the has not been reported. the clinical presentation is typically nonspecific (the most common signs are vomiting, lethargy, and anorexia), and there are no laboratory changes that are suggestive of hepatic neoplasia. thus the diagnosis must be made by identification of disease, other signs will develop, including ataxia, salivation, stupor, or coma. the best and only practical diagnostic test for he is plasma measurement of ammonia levels. however, as previously noted, the test has many technical issues that make its clinical utility in the practice setting difficult at best, and there are few laboratories that have validated ammonia measurement in the cat. cancer of the liver can occur as a primary disease (table - ) or as a result of metastasis of neoplastic disease occurring elsewhere and, most typically, the abdominal cavity. the most common neoplastic infiltration of the liver that is not a primary liver tumor is lymphoma (figures - and - ), followed by visceral mastocytosis. as with many other types of cancer, hepatobiliary neoplasia is most common in middle-aged to older cats, and it is relatively rare, with a reported incidence of . % to . %. benign tumors, such as biliary cystadenoma ( figure - ) , carry a good prognosis if they are amenable to surgical resection. the incidence of metastatic neoplasia (including lymphoma and mast cell tumors) the most common clinical signs are related to spontaneous rupture of the enlarged and friable liver. affected cats may present with lethargy, anorexia, pale mucous membranes, and a heart murmur secondary to anemia. clinical signs of liver disease are usually absent. hepatomegaly and hypotension may also be found. results of routine laboratory testing (mild to marked increases in alt and globulins while alp and ggt are typically normal) and ultrasonographic examination (hepatomegaly, generalized increase in hepatic parenchymal echogenicity) a of the liver may be supportive, but definitive diagnosis relies on histopathologic examination of a liver biopsy. fna of the liver is not helpful because amyloid is rarely detected with this method. hemostasis should be evaluated carefully before any biopsy procedure is planned. the most important differential diagnoses are fip, hepatic lipidosis, and hepatic lymphoma. scintigraphic imaging using i- serum amyloid p component has potential as a noninvasive test. a there is no specific treatment for amyloidosis in cats, so therapy is primarily supportive care (antioxidants, vitamin k, blood transfusion). attention should be paid to identification and control of any underlying chronic inflammatory disease. unfortunately, the long-term prognosis is poor as most affected cats die of intra-abdominal bleeding. survey abdominal radiography is the simplest and most readily available imaging modality to assess structures in the abdominal cavity. radiographs are most useful to assess liver size, will reveal large hepatic masses, and provide evidence of radiopaque masses or other abnormalities in the abdomen. however, the preferred imaging modality used to assess hepatic structures in cats with suspected liver disease is abdominal ultrasonography (aus). the reasons why ultrasonography is a more useful tool for assessment of the liver in cats are numerous, but because feline liver diseases are primarily diffuse, infiltrative, or metabolic diseases that also affect the biliary tree, ultrasonography is the only imaging tool that will give reliable diagnostic information. this widely available diagnostic tool can be helpful in determining liver size and parenchymal echogenicity, in identifying mass lesions, evaluating the biliary tree and gallbladder, quantifying flow (doppler techniques), and identifying vascular anomalies. as with all diagnostic modalities, the skill and experience of the operator is vital to accurate procurement and interpretation of the images. further, it is important to remember that although ultrasonographic images are extremely useful in the clinical evaluation of a cat with possible liver disease, the images themselves do not represent a histologic diagnosis. structural abnormalities by hepatobiliary imaging and subsequent examination of the tissue either by fna or biopsy techniques. historically, amyloidosis has been recognized as primarily a renal disease, especially in abyssinian cats. more recently, cases of hepatic amyloidosis without renal involvement have been diagnosed in siamese and related breeds, as well as in nonpedigreed cats. a, a, a the majority of cases have been described in australia, the united kingdom, and europe. amyloid a is deposited in the liver, probably in response to chronic inflammation in another organ. in the siamese breed, a genetic component may contribute. a the amyloid a protein occurring in the siamese breed differs from that known in the abyssinian breed. a contraindicated in cats, because they may cause a lethal shock reaction. a similar reaction may be seen with penetration of the larger bile ducts or gallbladder with a large-bore biopsy needle, because these tissues have a significant autonomic innervation in the cat that may result in bradycardia and shock following the procedure. , , it is particularly important to recognize this as a risk in cats with ehdbo or dilated bile ducts, and this risk factor reiterates the need for ultrasound examination of the liver prior to making biopsy decisions. nonetheless, owners should be informed of these potential risks, in addition to the risk of bleeding from biopsy sites in any cat undergoing liver sampling. , biopsy techniques liver biopsies, whether they are obtained by needle, laparoscopy, or surgical means, should be taken from a location that represents the primary liver pathology, handled appropriately to ensure accurate interpretation of the sample, and the histopathologic description should be interpreted according to the guidelines set by the wsava standards for clinical and histologic diagnosis of canine and feline liver disease. , guidelines for obtaining and handling surgical biopsies of the liver are reviewed elsewhere and will not be further discussed. because needle aspirates/biopsies, tru-cuttype biopsies, and laparoscopic biopsies are commonly used to obtain liver tissue in cats, the benefits and limitations of each of these techniques will be discussed. as a general rule, the more tissue that can be obtained, the better the pathologist's interpretation of the tissue abnormalities will be. for example, most pathologists believe that at least six portal areas are necessary to make a diagnosis of inflammation liver disease in cats. this will require either a -or -gauge needle size or larger piece of tissue than is obtained with smaller needles or an aspirate. the amount of tissues required to view at least six portal areas is approximately mg, and mg will be required for culture of the tissue. if other analyses of the tissues are considered (e.g., metal analysis), approximately to mg of liver is needed. a typical laparoscopic cup biopsy forceps will provide mg of liver tissue, a -g tru-cut-type biopsy needle provides to mg, and an -g needle biopsy provides only to mg of liver tissue. thus, depending on the clinical circumstances and considered differentials, the best approach for obtaining the needed tissue must be considered prior to planning the procedure. fine-needle aspiration to obtain liver tissue for cytologic examination is commonly performed in cats with liver disease for good reason. the procedure is inexpensive, easy to do, is relatively low risk, and often requires only sedation to complete. further, samples obtained by this method can be diagnostic for hepatic lipidosis, hepatic lymphoma or other round cell tumors, and in for the most common liver diseases of cats (hepatic lipidosis, feline cholangitis syndrome, and neoplasia/ lymphoma), aus examination provides a useful means of obtaining clinical clues and tissue to support or refute the differentials. for example, in cats with hepatic lipidosis, the liver is quite enlarged and typically diffusely hyperechoic, while in cholangitis or other inflammatory diseases, the liver is more often diffusely hypochoic. however, these sonographic findings are very nonspecific and can easily lead to errors in diagnosis if the tissue is not subsequently sampled for confirmation. , thus one of the most important utilities of the aus is the ability to obtain liver tissue (either by aspiration or guided-needle biopsy) and for aspiration of the gallbladder to obtain bile for culture. , these techniques alone have made the aus an extremely important diagnostic tool in the evaluation of liver disease in cats. the diagnosis of most liver diseases requires a histopathologic sample of liver tissue, and this is particularly true in the most common feline liver diseases, which tend to be diffuse diseases affecting the entire liver. cats with one of these diffuse diseases can be sampled randomly using any one of these commonly employed techniques: ultrasound-guided fine-needle aspirates (fna), ultrasound-guided needle biopsy, laparoscopic biopsies, or biopsies obtained surgically. some types of neoplasia (particularly round cell tumors) and vacuolar hepatopathies (hepatic lipidosis) can often be diagnosed by cytology using fna techniques. however, differentiation of liver cell tumors (adenomas and carcinomas) and inflammatory diseases of the liver cannot be diagnosed without a larger sample of tissue and histopathologic examination. , further, even in cats with classic hepatic lipidosis changes, concurrent diseases such as cholangitis or lymphoma can be missed if only fna techniques are employed. thus it is essential to consider that in many liver diseases the lesions, although typically diffuse, may also have focal components; for example, inflammation may be throughout the liver, but fibrosis will be present only in focal areas. thus the results of fna or tru-cut needle biopsies should always be considered in the light of the clinical, laboratory, and ultrasonographic evidence. prior to scheduling a cat for a biopsy, the risk-tobenefit ratio of performing a liver biopsy should always be considered. this is primarily because heavy sedation or anesthesia will be essential in most cats undergoing a liver fna, and for all cats undergoing a liver biopsy (needle or otherwise). in addition to anesthesia risks, the use of automatic spring-loaded biopsy guns to obtain ultrasound-guided biopsies of liver tissue is equipment, the interested reader is referred to several recent reviews on the subject. , to maximize the histopathologic accuracy, biopsies taken at laparoscopy or surgically should be taken from both normal-appearing and abnormal areas in the liver. further, if there is a need to obtain samples from the deeper tissues, the laparoscope can be used to direct a tru-cut needle biopsy to the best location for sampling. one of the major advantages of the laparoscopic technique is that it allows the operator to observe the biopsy sites for excessive bleeding, which is unusual, but if observed can be staunched by using pressure on the site, gelatin coagulation material placement, or electrocautery. with experienced operators, the complication rate for laparoscopy is very low (less than %), and most complications were because of anesthesia, bleeding, or air embolism. finally, although not necessary to have direct visualization to obtain an aspirate of gallbladder bile, laparoscopy allows easy sampling of bile for culture, which is important in all cats with suspected inflammatory liver disease or hepatobiliary disease. once a diagnosis of liver disease is made in the cat, specific therapy for the cause (if available) should be instituted; however, for many feline liver diseases, no specific therapy is available, and thus hepatoprotective therapy is used concurrently to aid in the recovery of the liver from the insult. in this section, therapy of two of the most common diseases of the feline liver will be considered, with a special emphasis on nutritional aspects of treatment, nutraceutical therapy, and the unique needs of cats. the most common liver disease of cats is idiopathic hepatic lipidosis (figures - and - ) , a disease that results in liver failure because of a combination of factors including hepatic lipid accumulation, insulin resistance, fasting, and protein (especially arginine) deficiency. , , , thus, unlike many diseases of the liver, the primary focus of therapy and the essential component for recovery is nutritional support. as in any patient with serious liver disease, initial therapy is always aimed at correction of any fluid or electrolyte abnormalities that may exist, because these may be profound if the cat has been vomiting. in addition, normalization of electrolytes is particularly important in cats that have been anorexic for an especially long time ( to weeks), because refeeding syndrome may be triggered with the initiation of feeding, resulting in sudden drops in potassium, phosphate, and magnesium. although this phenomenon is less common and usually less profound in cats fed enterally versus areas where appropriate, definitive diagnosis of certain infectious diseases (e.g., histoplasmosis). however, even with these relatively straightforward diseases, fna of liver tissue has significant limitations, the most important of which is the failure to accurately identify the primary disease. for example, although it is easy to make a diagnosis of hepatic lipidosis using this technique, a paper recently showed four cats that were incorrectly diagnosed with hepatic lipidosis instead of lymphoma because the fna samples were obtained from areas that did not have lymphoma infiltration. in another study, reviewing the agreement between fna cytologic samples of liver and the histopathologic diagnosis, only % of the cases had overall agreement. thus although cytology of fna samples of liver tissue in cats with diffuse hepatic disease remains a useful first step, it is important for the clinician to carefully interpret the results and discuss the potential limitations of this technique with owners. there are several needle biopsy techniques available for sampling liver tissue, but not all are suitable or safe for use in cats. the menghini technique is one such approach that is not suitable for use in cats, because it is a blind procedure using a large-bore needle that cannot be used with ultrasound guidance. the second option among the needle biopsy techniques that is not recommended for cats is the biopsy gun device. tru-cut biopsy guns are operated by a triggering device that can result in the induction of a lethal vagotonic shock reaction in the cat immediately following the procedure. for most ultrasound-guided liver biopsy procedures, either the manual or, preferably, the semiautomatic tru-cut device is recommended for use in obtaining needle biopsies from cats. as a general rule, the tru-cut device will advance into the liver to a depth of cm; so, it is essential to carefully note the amount of liver tissue available during the ultrasound assessment before advancing the needle for tissue collection. properly obtained tru-cut needle biopsies are a valuable technique for obtaining a representative sample of liver tissue ; however, because of the risk for bleeding or liver fracture with any movement, it is essential that cats be anesthetized for this procedure. laparoscopy is an intermediate step between needle biopsy and surgical laparotomy for obtaining liver tissue for histopathology in cats. , this technique is becoming more widely used as more specialists are trained for this procedure that allows visualization of tissues to be biopsied without opening the entire abdomen. although this technique does require general anesthesia, the limited degree of invasiveness, the large biopsy sample size, and rapid patient recovery make laparoscopy a valuable tool for obtaining liver tissue, and it can be used to obtain biopsies from the spleen, pancreas, kidneys, lymph nodes, or to aspirate the gallbladder. for a detailed discussion of laparoscopic techniques and lipidosis. the echogenicity of the parenchyma is uniformly increased, which is more apparent when compared with other ultrasonographic images presented in this chapter. additionally, the gall bladder is distended. hepatic lipidosis in this cat was secondary to anorexia associated with primary intestinal disease. (courtesy dr. randolph baral.) liver gb figure - gross appearance of liver from a cat with hepatic lipidosis. note the pale tan and exaggerated reticular pattern. in most cases, the edges appear more rounded than is evident here. hepatic lipidosis in this cat was secondary to anorexia associated with primary intestinal disease (same cat as in figure - ) . (courtesy dr. randolph baral.) those started on intravenous nutrition, it can be a significant source of morbidity if electrolyte replacement and monitoring are not carefully attended. once the cat is hemodynamically stable, the next step in treatment planning in cats with hepatic lipidosis is re-introduction of nutrition, which must include placement of a feeding tube (box - ). however, because many of these cats are extremely ill and are not good candidates for anesthesia, placement of a nasoesophageal (ne) tube to allow initiation of enteral feeding is often the most appropriate step for the first few days. when administering food through a feeding tube, there are several important points: . the food should be room temperature (not too hot or cold). . the tube should be flushed with water following feeding, to remove any particles of food or medication that may cause the tube to clog. . if the cat is volume sensitive, it is important to carefully calculate how much water is used for flushing the tube, because a significant volume of fluid can be infused, creating a potential fluid overload. if the cat is fluid sensitive, the total amount of fluids (amount in the food, amount added to food if blenderized, and amount of flush) must be determined, and the amount of fluid used in flushes or food preparation may have to be reduced. force feeding is to be strongly discouraged in these sick cats for several reasons: • it is highly stressful and will further increase the stress response and insulin resistance phenomena that are perpetuating the hepatic lipidosis. • it can be dangerous to the cat (aspiration) or operator (scratches/biting). • it is rarely able to meet the necessary nutritional goals set for the patient. • it may induce food aversion, a phenomenon unique to cats, but creating a profound aversion to the chosen food that can be lifelong. although ne tubes are excellent choices for short-term feeding of cats unwilling to eat, there are several disadvantages to their long-term use, including the nasal irritation that occurs, the relative ease with which cats can (and will) remove them, and the need to use liquid enteral diets. thus once the cat is deemed stable enough for general anesthesia, a long-term feeding tube solution is needed, and this typically is either an esophageal (e) tube ( figure - ) or percutaneous endoscopic gastrostomy (peg) tube. , both feeding options are generally well tolerated methods for providing long-term feeding, but e tubes have the advantage of being placed without the need for any specialized equipment, and if complications occur, they are generally easily addressed, because the most common complications are infection at the tube site or premature removal of the tube by the cat. placement of a peg tube, although relatively easy to learn to place, requires having the appropriate endoscopic equipment, and if complications occur as a result of infection or tube removal, more significant morbidity can result. because there is no advantage to placement of peg tubes easiest to use, and are an acceptable choice in most situations. finally, because many cat stomachs are volume sensitive with initiation of feeding, it is very important to start conservatively with small-volume feeding on a more frequent schedule. with prolonged fasting, the stomach volume of a cat with hepatic lipidosis may be reduced dramatically, preventing normal expansion and limiting intake to as little as % of normal. thus to avoid vomiting when feeding, the starting volume may have to be as small as to ml every to hours. a good rule of thumb is to start with estimation of resting energy requirement (rer) ( to kcal/kg is a good estimate of rer), and then attempt to meet % of rer the first day. if no problems are encountered, increase the amount to % of rer the second day, and so on, but during this period, keep the frequency as high as possible (feed four to six meals per day) so that the volume remains relatively small at each meal. once full rer has been achieved with multiple meals per day, the frequency of feeding can be gradually reduced to three to four meals per day. most cats will eventually tolerate three meals per day well, and some can tolerate two meals per day, but this is quite variable and should not be attempted during the first weeks of feeding. in general, most cats with hepatic lipidosis will require tube feeding for a minimum of to weeks before they will show interest in food and begin eating again on their own. the tube should be retained until the cat has been eating on his or her own for at least week or longer and can be maintained for a longer duration if it is being used to administer medications, because cats can eat normally with the e tube in place. the other therapeutic considerations for cats diagnosed with hepatic lipidosis are directed toward dealing with the complications of the disease and reducing the oxidative stress on the liver with hepatoprotective therapy (table - ) . in cats that are vomiting, in cats versus e tubes, placement of e tubes is advocated as the best approach for most practice situations. interested readers are referred to several recent reviews on tube placement for specific details on each method and to chapter . , diet selection is the next step in treatment planning for cats with hepatic lipidosis. in contrast to the belief that animals in liver failure need lower quantities of protein to reduce the workload on the liver, cats with hepatic lipidosis actually need protein to recover. in fact, the work of biourge and coworkers showed that protein was the essential nutrient in reducing hepatic lipid accumulation, was essential to eliminate the negative nitrogen balance, and also appeared to minimize muscle catabolism. further, diets high in protein can improve insulin sensitivity and assist weight loss in recovery from obesity. , conversely, although carbohydrates are a readily available energy source, they are often associated with gastrointestinal distress (diarrhea, abdominal cramping) and hyperglycemia (secondary to the insulin resistance in place as a result of obesity and hepatic lipidosis). thus diets selected for cats with hepatic lipidosis should ideally be high in protein (> % metabolizable energy [me]) and have lower amounts of carbohydrates (< % me), with the remaining calories coming from fat. the diets that best fit this profile are the diets formulated for diabetic cats; however, kitten food, many adult cat foods, and some of the enteral recovery diets have this high protein/low carbohydrate profile. many of the intestinal diets are not higher protein and are higher in carbohydrates, and so would not be the ideal choice. the key to using any of the foods that are not designed for use in a feeding tube is to blenderize them (and if necessary, strain the food) so that the food will easily go through the -or -g feeding tube without clogging it. enteral diets designed for use in feeding tubes are the because the primary starting point of the inflammatory disease in cats is the bile ducts (cholangitis), with inflammation extending to the hepatic parenchyma (cholangiohepatitis) only with time and severity, the term cholangitis syndrome has become the preferred terminology. the disease syndrome has been further classified by the wsava liver diseases group into one of three primary types: neutrophilic or suppurative, chronic lymphoplasmacytic (figures - and - ) , and lymphocytic (non-suppurative). each of the forms appears to behave quite differently clinically as well as in their progression and outcome. in general, cats with the suppurative form of cch typically have an acute onset of illness, which often includes fever, anorexia, and vomiting, and they may become icteric quite quickly (figure - ) . , the nonsuppurative form of cch (lymphocytic form) tends to be a more chronic condition, with affected cats showing nonspecific signs of illness that may include partial anorexia and lethargy, but the signs may wax and wane or are non-progressive. , because of the feline pancreatic and bile duct anatomy, it is common for cats with cch to have pancreatitis and vice versa, and in some cases, cats will also have concurrent ibd; the constellation of the three conditions occurring together is called triaditis. this combination is increasingly recognized in cats, and recent reports suggest from % to % of cats with one syndrome have all three diseases. , , , at this time, the etiology of each of these syndromes and the pathogenesis is not well understood; however, the enteric microflora are presumed to play an important role in the suppurative form, and immune mechanisms are presumed to be the cause of the chronic inflammation found in the chronic nonsuppurative antiemetic therapy is beneficial, because it is imperative that the cat continues to receive some food, and vomiting will complicate this. metoclopramide is often used in cats because of its ready availability and low cost, but it is a very weak antiemetic in cats and thus may not be the best choice. in most cats, the novel nk- receptor antagonist maropitant has been a safe and effective choice. the most commonly used antiemetics in the author's feline practice are maropitant ( mg/kg iv, sc or through the e tube q h), ondansetron ( . mg/kg iv q - h), or dolasetron ( . mg/kg iv, sc q h). in addition to control of vomiting, all cats with hepatic lipidosis should be given vitamin k ( . mg/cat po, sc) daily for a week, then weekly until the cat has recovered, and vitamin b (cobalamin) ( µg/cat sc) weekly for weeks, then monthly until blood values are normal. other vitamins may become deficient, such as some of the b vitamins and vitamin e; however, feeding is likely to rapidly replenish these deficiencies if they exist. this is also likely true of amino acid deficiencies, but supplementation of l-carnitine ( mg/day po) may be beneficial by improving fatty acid oxidation. finally, hepatoprotectant and antioxidant therapy with s-adenosylmethionine (same) ( mg/kg po q h) has been advocated to increase glutathione and may be beneficial in cats with hepatic lipidosis. , , it is important to note that if same is given through the tube (and thus the tablets must be crushed), the dose must be increased by approximately % to allow for the loss of absorption from loss of the enteric coating. because drug metabolism is often impaired in cats with hepatic lipidosis, appetite stimulants, such as mirtazapine, cyproheptadine, and clonazepam, should not be used in cats because dosing and side effects can be unpredictable. benzodiazepine agonist drugs (e.g., diazepam) should be completely avoided in cats with possible lipidosis-induced hepatoencephalopathy, because they will exacerbate the signs and may cause fulminant liver failure. , fortunately, most cats with idiopathic hepatic lipidosis that receive immediate and aggressive therapy and feeding for their disease will recover completely. cats that develop hepatic lipidosis secondary to other serious diseases (e.g., lymphoma) have a much lower chance of complete recovery and often die of their disease or its complications. the most common inflammatory liver disease in the cat is a complex syndrome with multiple subgroups of disease previously termed cholangitis/cholan giohepatitis complex (cch) but currently recognized under the terminology feline cholangitis syndrome. this disease is quite variable in both its presentation and severity, and it may occur as a primary process or secondary to/ concurrent with other diseases (e.g., pancreatitis, ibd). ultrasonographic appearance of liver with lymphocytic/plasmacytic inflammation. note the varying echogenicity throughout the hepatic parenchyma; areas of hypoechogenicity likely reflect inflammatory cell infiltration. the gall bladder is distended; its shape is distorted by pressure from the transducer. (courtesy dr. randolph baral.) liver gb [ mg/kg po q h]), and if pancreatitis is concurrent, pain control with opioid pain relievers (e.g., buprenorphine . to . mg/kg po, sq q - h). if culture is not possible, combination therapy with enrofloxacin ( mg/kg po q h) and metronidazole ( mg/kg po q h) is reasonable. in cats with chronic lymphoplasmacytic forms of cholangitis, management must be tailored to the individual situation and often requires therapy with either immunosuppressive doses of prednisolone ( to mg/kg po q h) or chlorambucil ( mg/m po q d), along with the hepatoprotectants and cholerectics, and concurrent treatment of other diseases (pancreatitis or ibd) that may be occurring. the lymphocytic or lymphoplasmacytic forms of cholangitis may wax and wane in intensity over time, and may require long-term continuous or intermittent therapy to control the disease. there is no specific diet that is recommended for cats with inflammatory liver disease, but protein restriction should not be initiated unless the cat has clear evidence of severe hepatoencephalopathy. the diet should be selected based on other conditions (such as ibd), for which the diet may be more critical in the management. monitoring of serum chemistry values (especially glucose), clotting times, cobalamin levels, and pli/tli concentrations are recommended every few months, as well as careful monitoring of the cbc for all cats on chlorambucil. in all cats with chronic inflammatory liver disease, prior to initiation of immunosuppressive therapy, a careful assessment of the cat for other possible causes of inflammation should be completed (box - ). as in dogs, if a cat with pss can have surgical closure of the shunting vessel (ligation, placement of an ameroid constrictor, intravenous coiling), the long-term forms. however, whether or not these syndromes are related, a continuum of disease or completely different diseases remains undetermined. once a definitive diagnosis is obtained by histopathology of the liver tissue and culture of bile, treatment can be tailored to needs of the cat. cats with the more aggressive suppurative form of cholangitis often require intravenous fluid therapy, antibiotic therapy (based on results of culture whenever possible), and supportive therapy (antiemetics, vitamin k , hepatoprotectants such as same [ important antioxidant and stabilizes membrane functions • n-acetylcysteine-a precursor to glutathione and antioxidant, also improves tissues oxygen delivery • ursodeoxycholic acid (tertiary bile acid)-used to replace hepatotoxic, hydrophobic bile acids and increase bile flow • silymarin (milk thistle)-a free radical scavenger and anti-inflammatory/antifibrotic agent • vitamin e-an antioxidant and antiinflammatory vitamin* although few clinical trials of these nutraceuticals have been performed in feline liver disease, a few studies have recently appeared showing that same, ursodeoxycholic acid, silymarin, and n-acetylcysteine all are hepatoprotective, have few adverse side effects, and may be beneficial in many types of liver disease in cats. , , , , feline liver disease is a common problem that requires careful consideration of the presenting complaint, clinicopathologic findings, imaging results, and, if available, histopathologic interpretation to be able to provide an accurate diagnostic and therapeutic plan. a variety of insults can be responsible for liver dysfunction or failure, but hepatic lipidosis and feline cholangitis syndrome remain the most common reasons for cats to present prognosis for function and quality of life is generally very good. however, even if surgical correction is anticipated, and especially if surgical correction is impossible or not completely successful, medical management of he is indicated. see table - for the basic therapeutic approach to medical management of cats with he resulting from pss. because hepatocytes, by their position in the body between the gi tract and rest of the body, as well as their critical role in metabolism and detoxification, are uniquely susceptible to oxidative injury and reactive intermediates of metabolism, they must be able to protect themselves. the natural defenses of the liver include superoxide dismutase and glutathione, free-radical scavengers such as vitamin e and ascorbate, and other prosurvival signaling pathways that are controlled by hormones and growth factors. however, in injury or overwhelming infection or inflammation, the natural defenses of the liver can be overwhelmed, and then it is essential for medicines and nutraceutical therapy to be included in the treatment plan to help reduce inflammation and fibrosis, protect against oxidant injury, and enhance bile flow. the cytoprotective agents most commonly used in liver diseases to assist in these processes (table - ) are: • s-adenosylmethionine (same)-a precursor in the synthesis of glutathione and an important methyl donor to dna and proteins, is an with icterus or liver failure. therapy must be tailored to the individual, but nutritional support is critical in the management of hepatic lipidosis, and appropriate supportive therapy with hepatoprotectants may be crucial to treatment success. resorption from that space. effusion accumulation is therefore correlated to increased capillary hydrostatic pressure, widening of the oncotic pressure gradient, increased endothelial permeability, increased interstitial hydrostatic pressure, or loss of effective lymphatic drainage or a combination of these factors. , , peritonitis of any cause results in vascular dilation, increased capillary permeability, and the migration of inflammatory cells into the peritoneum in response to immunomodulatory mediators. the inflamed peritoneum becomes a freely diffusible membrane, allowing a massive outpouring of fluid and plasma proteins from the circulation. , ascites is not commonly seen in practice; one study recognized ascites in only three cats out of admissions to an american veterinary teaching hospital, but the prevalence may be greater in primary care practice. in that study, dilated cardiomyopathy (dcm) was the most common disease associated with peritoneal effusion; however, dcm was diagnosed in most of these cats before , when taurine deficiency was found to be a primary cause of this form of cardiomyopathy in cats. neoplasia was the most common cause after . feline infectious peritonitis (fip) was by far the most common cause of feline ascites diagnosed over a -year period at the feline centre at the university of bristol, comprising % of all cats with recognized ascites. cats with ascites usually present with nonspecific clinical signs, such as anorexia or lethargy. the owners may present the cat because they recognize abdominal enlargement ( figure - ), but in many cases, owners perceive this as weight gain. clinicians should be aware that sudden weight gain in a chronically underweight cat may be because of fluid accumulation (which can be intrathoracic fluid if ascites is not present), particularly if muscle mass seems reduced. ascitic cats presenting subsequent to trauma may have intraabdominal hemorrhage or urinary tract rupture. fever in a young ascitic cat will often suggest fip, and cats with fip may or may not be jaundiced. presence of jugular distention or even a jugular pulse can suggest right-sided heart failure. a palpable fluid thrill can help to distinguish ascites from other causes of abdominal enlargement, such as organomegaly, abdominal masses, bladder distention, abdominal wall weakness, obesity or, occasionally, accumulations of gas within the abdominal cavity (table - ) . recognizing a fluid thrill involves gently tapping one side of the abdominal wall with the fingers of one hand while feeling for a sensation of fluid movement the peritoneum is the serous membrane lining the abdominal cavity, as well as covering the organs of the abdomen. it comprises a single layer of squamous mesothelial cells resting on a deeper layer of loose connective tissue. the layer of peritoneum that lines the inner surface of the abdomen is called parietal peritoneum; the abdominal organs are lined by visceral peritoneum. the total surface area of the peritoneum is one to one-and-ahalf times that of the total cutaneous area of the body. , the peritoneal cavity contains a small amount of fluid (less than ml/kg body weight) that reduces friction between the abdominal organs as they slide over each other. the fluid is a pure transudate and contains solutes in the same concentration as serum (box - ). this fluid is absorbed from the abdominal cavity predominantly through lymphatic vessels lying beneath the mesothelial basement membrane on the surface of the diaphragm. lymphatic drainage occurs predominantly to the sternal lymph nodes. , ascites is the abnormal effusion and accumulation of fluid in the abdominal cavity. fluid exchange across the capillary bed is determined by starling forces, that is, the balance between hydrostatic pressure, which causes transudation of fluid out of blood vessels, and the colloid osmotic pressure, which acts to retain fluid within blood vessels. the amount of peritoneal fluid is therefore determined by the balance of these, as well as vascular permeability, with excess fluid drained by the lymphatic system. accumulation of fluid within a body cavity results when the rate of filtration of fluid into a space is greater than the rate of fluid routine laboratory findings are usually nonspecific but may provide clues to the underlying cause of ascites. for example, neutrophilia may point towards septic peritonitis but can also occur with fip; most cats with hemoperitoneum are anemic at presentation ; uroperitoneum often results in azotemia and electrolyte abnormalities; hypoglycemia may reflect sepsis with septic peritonitis, and a recent study recognized % of cases of septic peritonitis had ionized hypocalcemia ; elevated liver enzymes may be associated with inflammatory, infectious or neoplastic hepatopathies including fip; elevation of serum globulins occurs in many cats with fip but can also be associated with neoplasia or septic peritonitis; and a finding of hypoalbuminemia (which can cause a pure transudate) should prompt for an assessment of urine protein : creatinine ratio to assess if there is renal protein loss. imaging may be required to confirm the presence of fluid as well as to aid in diagnosis of the underlying cause. radiographic findings can vary greatly depending on the amount of abdominal fluid present and the underlying etiology. loss of normal detail or presence of a "ground glass" appearance to the abdominal cavity is suggestive of the presence of fluid (figures - and - ). very young, thin or dehydrated cats may also have a loss of detail that can mimic the presence of fluid. ultrasonography of the abdomen (figures - and if a large volume effusion causes discomfort because of abdominal distention, a three-way stopcock may be used so large volumes can be drained from one puncture (figure - ) . however, removal of large volumes of ascitic fluid can be detrimental, because it may prevent the subsequent reabsorption of valuable protein and/or red blood cells; the resulting reduction in intraabdominal pressure may encourage further accumulation of fluid; and rapid removal of large volumes can lead to fluid shifts causing cardiovascular collapse. fluid can be collected into ethylenediaminetetraacetic acid (edta) tubes (for total nucleated cell count, packed cell volume, total protein, and cytology), serum tubes (for biochemistry, such as albumin, bilirubin, creatinine, potassium, triglyceride, glucose, lactate, and lipase), sterile tubes for culture, and/or other tubes for effusion-specific tests such as pcr. samples should be prioritized according to the volume of fluid available and to the suspected underlying disease process. initial assessment of fluid retrieved is made on the basis of color and protein concentration, and much information can be gleaned from this simple assessment, even before cell numbers and types are assessed. although this brief, initial assessment is useful to refine the differential diagnoses, a thorough assessment based on underlying etiology and pathophysiology is required for definitive diagnosis and therefore appropriate management (table - ) . ascitic fluid, classified according to its pathophysiologic cause, can be divided into transudates, modified transudates, exudates (septic or nonseptic), or effusions (chylous or hemorrhagic). , can allow the detection of even very small volumes of fluid. it also enables evaluation of the size and structure of intraabdominal organs, such as the liver and spleen, which can help determine the underlying cause of ascites. abdominocentesis confirms the presence of abdominal fluid (in cases of low-volume effusion) and assessment of the fluid is required to diagnose the underlying cause of ascites. most cats tolerate abdominocentesis without sedation and the cat can be held in a standing position or in lateral recumbency (whichever is more comfortable for the cat and familiar to the clinician). the abdomen is clipped and aseptically prepared. a -to -gauge butterfly needle may be used with a -to -ml syringe. in cases with low-volume effusion, ultrasonography can help to guide fine needle aspiration from small pockets of abdominal fluid. diagnostic peritoneal lavage can be used if ultrasound-guided aspiration is unsuccessful. for this procedure, to ml/kg of warmed, sterile fluid is infused into the abdomen over to minutes after aseptic preparation of the site. the cat is gently rolled from side to side or allowed to stand; gentle massage of the abdomen also helps distribute the fluid. the fluid is allowed to dwell for a minimum of to minutes before aseptic preparation is repeated before paracentesis. no attempt is made to remove all the fluid. it must be remembered that, since the recovered fluid has been diluted by this procedure, cell counts and biochemical analyses will be affected. transudates are a consequence of altered fluid dynamics. protein-poor transudates (commonly referred to as pure transudates) form predominantly as a result of severe hypoalbuminemia, which causes a lowered colloid osmotic pressure. since there is no change in endothelial or mesothelial permeability, as fluid accumulates, there is no concurrent cell leakage; so, there is a decrease in the cell count through a dilutional effect. consequently, transudative effusions are typically clear and colorless. , , other pathologic causes of proteinpoor transudates include cirrhosis, lymphatic obstruction, and noncirrhotic portal hypertension (presinusoidal and sinusoidal). since hypoalbuminemia is the most common cause of transudates, serum albumin concentrations must be measured to guide further diagnostics. if the serum albumin concentration is normal (or only minimally decreased), then radiographs, abdominal ultrasonography, and/or echocardiography are indicated to assess cardiac function and for urinary bladder rupture. one review of feline ascitic cases found % of effusions were protein-poor transudates, of which % were the result of hepatic failure or primary renal disease. modified transudates can result from increased hydrostatic pressure within the postsinusoidal vessels of the liver secondary to right-sided congestive heart failure (e.g., tricuspid insufficiency) or potentially from mass lesions (such as neoplastic masses) obstructing blood flow from the hepatic vein or caudal vena cava into the right side of the heart. the increase in hydrostatic pressure within the vessels of the liver causes a protein-rich fluid to leach out of the liver into the abdominal cavity. since cell membrane permeability does not change, cells do not accumulate in the effusion. modified transudates can also result from increased vascular permeability in the early stages of an inflammatory process, in which case cellularity will be increased. modified transudates were described as the most common type of ascitic effusion identified in cats in one study, with most being resulting from neoplasia and congestive cardiac failure; however, this study partially included cases prior to , when right-sided heart failure associated with dilated cardiomyopathy (dcm) was prevalent. the recognition of the role of taurine deficiency in this condition and the subsequent addition of this amino acid to feline diets now means that right-sided heart failure is only rarely encountered as a cause of ascites in cats. exudates are a consequence of altered mesothelial and/ or endothelial permeability. this permeability results from a cytokine-mediated inflammatory response of any underlying cause (e.g., infectious, neoplastic, immune mediated). exudates have high protein and moderate to high cell concentrations and are classified as nonseptic or septic. exudates are often primarily composed of neutrophils. nondegenerate neutrophils (and the absence of organisms) points to a nonseptic exudate (mostly fip but also neoplasia). fip is the most common cause of exudative effusion in cats and was the most common cause of feline ascites diagnosed over a -year period at the feline centre at the university of bristol. the presence of neoplastic cells rules in neoplasia, but the absence of such cells does not rule out this diagnosis since many cases of neoplastic ascites are not associated with exfoliated neoplastic cells. other causes of nonseptic exudates include pancreatitis, lymphocytic cholangitis, and viscus rupture, such as the gall bladder or urinary bladder. degenerate neutrophils typify septic exudates (i.e., septic peritonitis), and their presence should instigate investigation for causes of infection (mostly leakage of gastrointestinal contents). chylous effusions appear as milky or pink opaque fluid, and small mature lymphocytes initially predominate in cell counts. after drainage, more macrophages and nondegenerate neutrophils may be found. chyle is typically classified as an exudate, but its physical characteristics can be consistent with a modified transudate (protein content between and g/l); biochemical analysis of triglyceride and cholesterol levels in the fluid are required to confirm the diagnosis. pseudochylous effusions resemble true chyle both in appearance and cytology but do not contain fat. similar conditions result in both chylous and pseudochylous effusions. chylous abdominal effusions are rarely reported in the cat and only accounted for % of cases of ascites in one study. the described causes of chylous ascites in cats are predominantly neoplastic. in a series of nine cats, chylous ascites was associated with nonresectable abdominal neoplasia in four cases (i.e., hemangiosarcoma and paraganglioma), with intestinal and mesenteric lymphoma in two cases and lymphangiosarcoma of the abdominal wall in another. one described case in a -year-old cat was thought to be because of fip. figure - shows an ultrasonographic image of a cat with chylous abdominal effusion associated with pancreatitis. other potential causes include right-sided congestive cardiac failure, steatitis (inflammation of fat), biliary cirrhosis, and lymphangiectasia. hemoperitoneum in companion animals is categorized as traumatic or spontaneous. traumatic hemoperitoneum is further divided into blunt causes of trauma (i.e., motor vehicle accidents and high-rise falls) and penetrating trauma (i.e., gunshot wounds and bite wounds). , inadvertent splenic aspiration, venipuncture, or acute severe hemorrhage should be suspected if the cytology is consistent with peripheral blood including platelets but without erythrophagocytosis or if the blood clots readily. when there is no history of trauma, coagulopathy or spontaneous rupture of a vascular neoplasm should be considered. in one study of feline cases of spontaneous hemoperitoneum, cases ( %) were associated with hepatic pathology such as neoplasia, necrosis, and amyloidosis. in another study of cases of spontaneous hemoperitoneum, % ( of ) of cats had abdominal neoplasia, and % ( of ) had non-neoplastic conditions. cats with neoplasia were significantly older and had significantly lower packed cell volumes (pcvs) than cats with non-neoplastic disease. hemangiosarcoma was the most often diagnosed neoplasm ( of , %), and the spleen was the most common location for neoplasia ( of , %). coagulopathies ( of , %) and hepatic necrosis ( of , %) were the most common causes of non-neoplastic hemoperitoneum. other nonneoplastic causes of hemoperitoneum include ruptured bladder, hepatic rupture secondary to hepatic amyloidosis, gastric/duodenal ulcer, hepatic hematoma, hepatitis, perinephric pseudocyst, feline infectious peritonitisinduced liver rupture, and feline infectious peritonitisinduced nephritis. , the prognosis of cats with spontaneous hemoperitoneum is poor. in two studies, only approximately % of cases survived to be discharged from hospital. , median survival time for cats that were discharged in one of those studies was days (range, to days). feline infectious peritonitis (fip) comprised % of cats with recognized ascites over a -year period at the feline centre at the university of bristol, and, as a rule of thumb, when ascites is recognized in a younger cat, fip should be considered the major rule-out. the abdominal effusion found with fip is typically straw to golden yellow (although the color can be very variable, for example, chyle may be present), may contain fibrin clots, and has a high protein concentration. the total protein content is greater than g/l and often greater than g/l, with globulins comprising % or more. one study described an effusion with total protein greater than g/l as % specific, % sensitive, and having a . positive predictive value to diagnose fip. the rivalta test evaluates the fluid's globulin content, and was found to be very sensitive but only % specific; this test is performed by adding one drop of acetic acid ( %) to ml of distilled water. this fluid is mixed thoroughly, and then one drop of effusion is gently placed on the surface of the mixture. if the drop stays at the top of the fluid or slowly floats to the bottom, the test is considered to be positive. this test can give inaccurate results if inappropriate technique is used or if there is a significant temperature difference between the fluid sample and the acetic acid solution. a positive rivalta test can result from lymphosarcoma, septic, or fip effusions; these can be distinguished by cytology and culture. immunofluorescence staining of coronavirus antigen in macrophages had a positive predictive value of . but a negative predictive value of . . the potential clinical presentations, diagnosis, and management of fip are covered in detail in chapter . one study found neoplasia to be the most common cause of ascites in cats, and neoplasia should be considered the major rule-out in older cats with ascites. the effusion from cats with ascites resulting from neoplasia may be a modified transudate, resulting from compression of hepatic veins or the caudal vena cava, or metastases to the peritoneum; hemorrhage from neoplasia can cause hemoperitoneum; chylous effusions may result from reduced lymphatic drainage or rupture of lymphatic vessels; and raised vascular permeability caused by neoplastic infiltration can result in an exudative effusion. carcinomas, mesotheliomas, and discrete (round) cell neoplasms (e.g., lymphoma, mast cell tumors, malignant histiocytosis) exfoliate cells into effusions more readily than sarcomas, and of these, lymphosarcoma is the most common malignancy of cats. cytology of ascitic fluid reveals neoplastic cells in less than a quarter of cases; so the absence of such cells does not rule out a diagnosis of neoplasia. in these circumstances, the diagnosis may be achieved by ultrasound-guided fine-needle aspiration of affected organs, or even biopsy samples obtained at laparotomy. the specific approaches will depend on the specific neoplasia diagnosed. exudates caused by septic inflammation usually result from bacterial contamination of the peritoneal cavity secondary to gastrointestinal tract leakage or penetrating wounds associated with trauma. gastrointestinal tract leakage may occur as a result of ulceration associated with neoplasia or inflammatory disease or as a result of penetration of a sharp object ingested (such as a toothpick), it can also occur subsequent to prior abdominal surgery. , , , primary septic peritonitis in which no apparent cause can be identified has also been described in cats. septic exudates are usually yellow to tan in color, with yellow particulate matter and are foul-smelling. microscopically, the fluid is characterized by the presence of degenerate neutrophils and bacteria. bacteria are often seen intracellularly within neutrophils. the condition is associated with high morbidity and mortality rates, with survival rates reported between % and %. , , , , the history and clinical signs are often vague and nonspecific but can include abdominal pain, vomiting, lethargy/depression, and anorexia. abdominal pain is an inconsistent finding, being recognized in only % of cats in one study and % in another. some cats may have an inappropriately low heart rate. , hematologic and serum biochemistry findings are also inconsistent; neutrophilia with a left shift may be present, as may neutropenia or a normal neutrophil count. similarly, cats may be hypoglycemic, hyperglycemic, or normoglycemic, and they may be hypoalbuminemic. , , one study recognized ionized hypocalcemia in % of cats with septic peritonitis at the time of diagnosis, and another suggested hyperlactatemia, when present, may be associated with a poorer prognosis. radiographic findings are usually typical of ascites of any cause, but presence of pneumoperitoneum in a cat that has not undergone recent surgery may suggest the presence of gas-forming bacteria or rupture of an abdominal viscus and warrants immediate surgical intervention. ultrasonography does not directly aid the diagnosis of septic peritonitis. exploratory laparotomy to determine and correct an underlying problem, such as full-thickness gastrointestinal perforation (often requiring partial resection) is required, as is copious abdominal lavage with sterile, warmed fluids ( figure - ). there are no statistically significant survival differences between postsurgical primary closure, open peritoneal drainage, or closed suction drainage postsurgical lavage; however, a trend toward a higher survival rate has been seen in cats treated with primary closure. , treatment also involves antibiotics, initially parenterally, based on culture and sensitivity findings. consistent with intestinal contents, most bacteria recognized are gram-negative aerobes, such as e. coli or enterobacter spp., but mixed infections are usually found. , anaerobes seem more common in cats with primary septic peritonitis, which perhaps suggests these cases may result from healed over-bite wounds into the abdomen. amoxicillin/ clavulanate would be an appropriate empirical choice of figure - fulminant peritonitis associated with gastrointestinal perforation. in this case, the effusion volume was low but the high degree of serosal inflammation is evident. antibiotics while awaiting sensitivity results. there are no definitive guidelines for duration of antibiotic treatment; the author uses extended treatment courses of to weeks. supportive care with intravenous fluids to maintain fluid and electrolyte balances is also required perioperatively. bile peritonitis is infrequently reported in cats but has been recognized in association with gunshot or motor vehicle trauma, with biliary obstruction from gall stones , and subsequent to percutaneous ultrasoundguided cholecystocentesis in a cat with infectious cholangitis. concurrent bacterial infection was recognized in each case; this increases severity of inflammation and worsens the prognosis, although full recovery was achieved in most reported cases. , , bile peritonitis has the potential to result in small-volume effusions; so, if abdominocentesis does not yield a sample of effusion but bile peritonitis is high on the differential list, then diagnostic peritoneal lavage is appropriate. since repair of or removal of the gall bladder and abdominal lavage are required, exploratory laparotomy is an appropriate means to diagnose this condition. management should be considered as for septic peritonitis of other causes. trauma, including blunt abdominal trauma, urethral catheterization, and bladder expression, is the most common cause of uroperitoneum in cats. it is also recognized as a complication of ureteral surgery. the bladder is the most frequent site of urine leakage after blunt abdominal trauma, whereas the urethra is most commonly injured following catheterization. cats with ruptured bladders may still have a palpable bladder and the ability to urinate. common historical complaints are anuria ( . %) and vomiting ( %). azotemia is a common finding, and hyperkalemia is seen in around % of cases. drainage of urine from the peritoneal cavity seems to improve patient stabilization. morbidity and mortality depended largely on the severity of associated injuries. regardless of the site of injury or the cause of uroabdomen, the first goal of treatment is patient stabilization. isotonic replacement fluids are used for initial resuscitation. treatment of hypovolemic shock, if present, is the first order of fluid therapy. after fluid resuscitation, drainage of urine from the abdomen should be established. continuous passive drainage of the urine is necessary for stabilization and allows effective diuresis to occur. indwelling catheterization of the urinary bladder is recommended to keep the bladder decompressed and reduce urine flow into the abdominal cavity in patients with bladder and proximal urethral injury. if the urethra is traumatized and a catheter cannot be placed, prepubic tube cystostomy may be necessary to achieve temporary urinary diversion. the decision to treat the uroabdomen patient surgically or conservatively should be based on the location and severity of the underlying injury, the condition of the patient at presentation, and the patient's response to initial stabilization. , congestive heart failure has become an uncommon cause of ascites in cats since the late s/early s, from which time dilated cardiomyopathy has been largely eradicated. , ascites does still result from rightsided congestive heart failure in conditions such as tricuspid insufficiency, arrhythmogenic right ventricular cardiomyopathy, myocardial fibrofatty infiltration, or restrictive cardiomyopathy. , concurrent pleural effusion or pulmonary edema is often, but not necessarily, present with cardiac induced ascites. a heart murmur is not necessarily noted. noting a jugular pulse or thrill is helpful diagnostically, if present. the ascitic fluid is typically a modified transudate, but a chylous effusion is also possible. cardiac diseases are covered in chapter . in some cases, hepatic lipidosis has been reported to cause ascites, particularly in association with pancreatitis. these cats are often hypoalbuminemic, with the possibility of intravenous fluid therapy contributing to the ascites by raising hydrostatic pressure. other liver diseases which can result in ascites include lymphocytic cholangitis, , neutrophilic cholangitis, cirrhosis, necrosis, neoplasia, and suppurative cholangiohepatitis. portosystemic shunts in cats rarely result in ascites, compared with dogs. hypoalbuminemia and hepatic failure result in transudates; portal hypertension and cirrhosis cause higher protein ascites because of raised capillary hydrostatic pressure causing leakage of high protein 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transudates. adapted from tasker s, gunn-moore d: differential diagnosis of ascites in cats, in practice : , . key: cord- -nx kwn authors: ehrenpreis, eli d.; kruchko, david h. title: rapid review: nonsteroidal anti-inflammatory agents and aminosalicylates in covid- infections date: - - journal: j clin gastroenterol doi: . /mcg. sha: doc_id: cord_uid: nx kwn in the current covid- pandemic, there has been concern regarding the use of ibuprofen and other nonsteroidal anti-inflammatory agents by covid- infected patients. aminosalicylates ( -asas) are structurally similar and have anti-inflammatory functions that resemble those of nonsteroidal anti-inflammatory agents. since -asas are a mainstay treatment for inflammatory bowel disease, the authors review the pharmacology of both classes of drugs and discuss the potential relevance of -asas in the ongoing discussion of medication use in patients infected with covid- . t here has been recent controversy regarding the risk that ibuprofen (and possibly other nonsteroidal antiinflammatory drugs, nsaids) may worsen the clinical course of patients that are infected with covid- . aminosalicylates ( -aminosalicylic acids or -asas) such as mesalamine and sulfasalazine are amino-analogs of salicylate, and therefore their chemical structure and mechanisms of action are related to nsaids (fig. ) . although the primary effects of -asas occur directly in the gastrointestinal tract, -asas are absorbed systemically. it is therefore the purpose of this brief review to inform practitioners treating patients with -asas about the potential relationships between -asas and nsaids. at the time of this writing, both the world health organization (who) and the food and drug administration (fda) have not recommended the avoidance of ibuprofen in patients with known or suspected covid- infections. most recently, the international organization for the study of inflammatory bowel disease released a summary of recommendations with pointed statements in regard to the risk of developing covid- in the inflammatory bowel disease (ibd) patient population. statement states, " -asa does not increase the risk of covid- " however this statement is not based data collected in patients with covid- . because these types of recommendations have the potential for changes as the pandemic progresses, it is the author's belief that the information provided in this review can serve as a tool for discussions of potential risks of -asa usage if patients with ibd become infected with covid- . nsaids reduce inflammation by reversibly and nonselectively inhibiting the cyclooxygenase (cox) enzymes cox- and cox- . cox- and cox- synthesize prostanoids from arachidonic acid. in ex vivo studies, single and repeated doses of nsaids significantly inhibited cox- products more so than cox- products. prostanoids consist of prostaglandins (pg)e , pgd , pgf (alpha), pgi , and thromboxane (tx). prostanoids are responsible for many biological effects in several organ systems and have hematologic, pulmonary, renal, and cardiovascular effects. pge and pgi (also known as prostacyclin) are the primary proinflammatory prostanoids that increase inflammation via increasing vascular permeability with subsequent edema formation and promotion of leukocyte infiltration. pge also mediates fevers (pyresis) and is synthesized in the hypothalamus once systemic cytokines are activated by leukocytes in response to inflammation. pgd is produced by mast cells and contributes to inflammation in allergic responses, primarily in the lung. by inhibiting cox- and cox- , nsaids effectively reduce regional and systemic inflammation as well as functioning as an antipyretic agent. because of these antipyretic effects, nsaids are commonly prescribed in patients with upper respiratory infections (fig. ). interestingly, nsaids contribute to nonspecific inflammation, particularly within the gastrointestinal tract. this inflammation plays a role in the loss of protein and blood and there is endoscopic evidence of sharply demarcated ulcers in the colon that resolve after discontinuation of nsaids. the proinflammatory mechanisms are enhanced in patients with ibd as compared with the general population. nsaids also increase mucosal permeability, induce intracellular atp deficiency, and increase enterohepatic circulation. prostaglandins affect the colonic immune system and they help to maintain the integrity of mucosal barriers. therefore, by reducing prostaglandin levels with inhibition of cox- and cox- , colonic mucosal wall integrity weakens, and patients can consequently develop ibd flares. cox- is constitutively active, but cox- is only induced upon interaction with proinflammatory cytokines. in response to inflammatory stimuli, nuclear factor kappa-light-chain-enhancer of activated b cells produces cytokines such as il- and tumor necrosis factor-alpha. increases in these proinflammatory cytokines at local sites of inflammation lead to induction of cox- . in both animal and human models, cox- expression was found to be higher in affected intestinal and colonic mucosa, which correlated with ibd activity and played a role in mucosal healing. therefore, inhibiting cox- specifically can have detrimental effects on the healing of inflamed intestinal and colonic mucosa, which can be seen at both a pathologic level and an endoscopic visualizable level. it is standard practice for patients with ibd to avoid intake of nsaids, primarily because nsaids may worsen the natural disease course. -asa has anti-inflammatory and immunosuppressive properties via multifactorial mechanisms. one well understood anti-inflammatory mechanism of -asa is similar to nsaids. both classes of drugs inhibit the synthesis of prostaglandins and leukotrienes. high levels of pge are found in the stools of patients with ulcerative colitis and prostaglandins are known entities in the pathogenesis of ibd. furthermore, pgi and tx have been found to increase in the serum of patients with ibd resulting in stimulation of bowel motility and mucous production. by inhibiting the cox enzymes, -asas reduce the production of inflammatory prostaglandins in the gastrointestinal tract and this inhibition results in remission of clinical symptoms in patients with ibd. interestingly, laboratory studies demonstrate that nsaids reduce inflammation by the same mechanism, but rather than having a role in treating ibd, they are known to exacerbate ulcerative colitis and crohn's disease. in addition to inhibition of cox- , -asas block the aggregation of leukotrienes, such as ltb (which stimulates neutrophil accumulation and degranulation), in the colonic mucosa. altering neutrophil chemotaxis and macrophage activity reduces cell damage and subsequent inflammation. because -asas work primarily as topical gastrointestinal agents, orally administered forms of -asa are designed for the release of active drug release at their site(s) of action in the small and large intestine. the amount of ingested -asa, n-ac- asa and prodrugs that appear in the urine and feces is the most common method of determining how much of the drug is absorbed. a systematic review of the pharmacokinetic profiles of a variety of -asa preparations included data for urinary -asa excretion for a variety of -asa preparations including sulfasalazine, olsalazine, balasalazide, asacol, and pentasa. mean -asa excretion ranged from % to %, indicating that significant absorption of these drugs occurs. it may therefore be assumed that there is enough absorption of -asa to produce systemic antiinflammatory effects. this is borne out by their use in the treatment of rheumatologic diseases. on march , , the who sent a message in a tweet that "based on currently available information, who does not recommend against the use of ibuprofen." the original concern about the risk of nsaids use worsening the severity of covid- infections arose in a short review in the lancet published on march . fang noted that coronaviruses that cause human disease such as the severe acute respiratory syndrome coronaviruses (sars-cov and sars-cov- ) bind to their target cells through angiotensin-converting enzyme (ace- ). since ace- expression is increased in patients treated with ace inhibitors and angiotensin ii type-i receptor blockers, and potentially ibuprofen, the authors speculated that these drugs may predispose to more severe manifestations of covid- infection. figure shows a schematic of the hypothetical mechanism by which nsaids could promote covid- infection. on march , the french health minister olivier véran, tweeted that "taking anti-inflammatory drugs [ibuprofen (or) cortisone] could be an aggravating factor for the infection. if you have a fever, take paracetamol." his comments may stem from an account of a french infectious disease specialist that described otherwise healthy young patients severely infected with covid- infections. the severity of their disease course was attributed to their use of nsaids during the early stage of the infection. the argument that ibuprofen might increase the severity of covid- infections was bolstered by a statement by paul little, a professor of primary care research at the university of southampton. on march th, dr little commented in an article published online by the science media centre that "the finding in randomized trials that advised to use ibuprofen results in more severe illness or complications, helps confirm that the association seen in observational studies is indeed likely to be causal. advice to use paracetamol is also less likely to result in complications." an analysis of the quoted prospective studies suggests that these studies provide little evidence that nsaids increase the severity of upper respiratory infection. the first, by little and colleagues involved patients that were randomized to use several forms of advice administered to them in a sealed envelope. advice consisted of the type of analgesic/antipyretic to take (eg, , take paracetamol; , ibuprofen; or , both paracetamol and ibuprofen), the regimen of analgesia use (eg, use drugs regularly times a day for at least days then ad lib or them as required), and the use or avoidance of steam inhalation. results of symptom diaries on the second to the fourth day of illness were compared between the groups. the study actually demonstrated no difference in symptom severity between the analgesia groups, although the subgroup of patients with lower respiratory infection had close to % reduction of symptoms when advised to use ibuprofen alone (reduction of . ; % confidence interval − . to − . ) and those that used ibuprofen and paracetamol (reduction of . ; % confidence interval − . to − . ). the conclusion that patients were harmed by taking ibuprofen was based on a secondary outcome analysis of the study data, where consultation for the combination of recurring symptoms or complications was statistically higher in the groups receiving ibuprofen alone or in combination with paracetamol compared with paracetamol alone. the other trial by little and colleagues was designed to assess the use of an interactive website with tailored advice for managing patients with symptoms of upper respiratory infections. patients were randomized to either use the website or receive standard care. patients in the website group completed a checklist of symptoms and could request personalized advice or care. if patients wanted to use medications, they were advised to optimize their use of overthe-counter medications which included paracetamol and ibuprofen. the finding of their study was that patients in the website intervention group had increased contact with national health services personnel (intervention / , or . %; vs. control / , or . %), but deceased direct physician contact ( / , or . %; vs. / , or . %), the apparent goals of the study. there were paradoxical findings of the study. the first of these was that patients in the website intervention group reported a nonstatistically significant increase in illness duration ( . vs. . d, respectively); multivariate estimate . days longer (− . to . , p = . ). they also had a nonclinically significant increase in illness severity, defined as more days of illness rated moderately bad or worse illness ( . d; . to . , p = . ). in an attempt to explain why the intervention group appeared to have a longer duration of moderately bad or worse illness compared with the control group, the study authors performed a post hoc analysis of the data. they found that by controlling for whether individuals used ibuprofen from the pages on the website, they found a reduced difference in the length of illness ( . , − . to . , p = . ) and the occurrence of moderately bad or worse symptoms ( . , − . to . , p = . ). they then concluded that it was the encouragement of the use of ibuprofen was the cause of the reported difference in duration of symptoms. there has been much attention on the public stage regarding the possibility that ibuprofen (and potentially other nsaids) may worsen the clinical course in patients with covid- infection. because nsaids have structural and mechanistic similarities to -asas, their effect on patients that are infected with covid- may be questioned. in this review of the data, we have questioned the correctness of the suggestion that nsaids play a role in upper respiratory tract infections. as the beneficial role of anti-inflammatory agents in management of covid- infections evolves, further data on -asas in the process may emerge. our review of the circumstances surrounding concern about nsaids and covid- leads us to the suggestion that patients taking -asas may continue their treatment. however, patients on immunomodulatory and immunosuppressive agents for ibd should be included in the group of patients at high risk for complications of covid- infection. are patients with hypertension and diabetes mellitus at increased risk for covid- infection? the lancet fda advises patients on use of non-steroidal anti-inflammatory drugs (nsaids) for covid- . u.s. food & drug administration; . available at: www.fda.gov/drugs/drug-safety-and-availability/fdaadvises-patients-use-non-steroidal-anti-inflammatory-drugs-nsa ids-covid- ioibd update on covid for patients with crohn's disease and ulcerative colitis; . available at: www.ioibd.org/ioibd-update-on-covid -for-patients-with-crohnsdisease-and-ulcerative-colitis pharmgkb summary: ibuprofen pathways prostanoids in health and disease clinical and endoscopic features of nonsteroidal anti-inflammatory druginduced colonic ulcerations non-steroidal anti-inflammatory drugs and inflammatory bowel disease chemistry, pharmacology, pharmacokinetics, and clinical applications of mesalamine for the treatment of inflammatory bowel disease systematic review: the pharmacokinetic profiles of oral mesalazine formulations and mesalazine pro-drugs used in the management of ulcerative colitis a review of its pharmacological properties and therapeutic efficacy in the treatment of rheumatoid arthritis french health minister stirs up questions about ibuprofen, covid- available at: www.managedhealthcareexecutive.com/ news/french-health-minister-stirs-questions-about-ibuprofen-cov id- covid- : ibuprofen should not be used for managing symptoms, say doctors and scientists available at: www.sciencemediacentre.org/expert-reaction-to-reportsthat-the-french-health-minister-recommended-use-of-paracetamolfor-fever-from-covid- -rather-than-ibuprofen-or-cortisone ibuprofen, paracetamol, and steam for patients with respiratory tract infections in primary care: pragmatic randomised factorial trial primary care randomised controlled trial of a tailored interactive website for the selfmanagement of respiratory infections (internet doctor) key: cord- -rrr x authors: wang, hong-gang; xie, rui; ma, tian-heng; yang, xiao-zhong title: excessive anxiety in ibd patients is unnecessary for covid- date: - - journal: clin res hepatol gastroenterol doi: . /j.clinre. . . sha: doc_id: cord_uid: rrr x nan available online at sciencedirect www.sciencedirect.com to the editor, currently, novel coronavirus (sars-cov- ) infection disease (covid- ) broke out in wuhan, china and spread worldwide [ , ] . due to the highly infectious and pathogenic nature of sars-cov- , the public has shown excessive anxiety and even panic. inflammatory bowel disease (ibd) is a chronic non-specific intestinal disease, including ulcerative colitis (uc) and crohn's disease (cd), and many of these patients have anxiety [ ] . the anxiety not only comes from the intestinal diseases that already exist, the current epidemic situation of covid- has also made their anxiety worse. emotions are an important part of the management of ibd patients, because a less optimistic attitude can make their bowel symptoms recur [ ] . to date, there have been no reports of a confirmed covid- in ibd patients. as known, the coronavirus binds to the angiotensin-converting enzyme (ace ) protein, enters the cells, leading to covid- with the contribution of transmembrane protease, serine (tmprss ) [ ] . in human small intestine and colonic epithelial cells, ace exhibits higher expression than lung [ ] . according to autopsy results, the bowel of patients with covid- appeared segmental narrowing and expansion [ ] . immune disorders in ibd patients, coupled with current research reports, are ibd patients more susceptible to covid- ? we could obtain some information from the public ibd database [ ] . after screening, a total of ucs, cds, and non-ibds were included in the analysis (table ) . each subject had paired tissue samples of rectal and terminal ileal biopsies. analysis of the transcriptome data of these samples, we found that the expression of ace in the terminal ileum was significantly higher than that in the rectal colon, with a fold change of . times (p < . ). the expression of ace in the uc rectum was not significantly higher than that in non-ibd. in the terminal ileum of cd, ace expression was also not higher than that of non-ibd. at the protein level, ace was not higher in uc and cd patients than in healthy controls [ ] . these results indicate that the expression of ace does not show a significant difference between ibd and non-ibd patients. in addition, tmprss expression is lower in the terminal ileum than in the rectum (p = . ). compared with non-ibd, there was no significant difference in tmprss expression in the rectum and terminal ileum of patients with ibd. therefore, we speculate that patients with ibd may not be susceptible to covid- . however, this is only a theoretical guess, and we need to further understand the actual risk of covid- in ibd patients. at least, in theory, ibd patients don't need to be overly worried and anxious. like everyone else, personal protection is an important measure to prevent sars-cov- infection. hong-gang wang and xiao-zhong yang designed the research; hong-gang wang and rui xie analyzed the data; hong-gang wang and tian-heng ma wrote the paper. clinical characteristics of coronavirus disease in china covid- : what is next for public health? quality of life in inflammatory bowel disease: a systematic review and meta-analyses-part i symptoms of depression and anxiety are independently associated with clinical recurrence of inflammatory bowel disease sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically-proven protease inhibitor diarrhea may be underestimated: a missing link in novel coronavirus anatomy of a novel coronavirus pneumonia death corpse system ibdmdb. the nih human microbiome project niddk u de quantitative proteomic analysis reveals the deregulation of nicotinamide adenine dinucleotide metabolism and cd in inflammatory bowel disease none. key: cord- - n f mf authors: gajendran, mahesh; perisetti, abhilash; aziz, muhammad; raghavapuram, saikiran; bansal, pardeep; tharian, benjamin; goyal, hemant title: inflammatory bowel disease amid the covid- pandemic: impact, management strategies, and lessons learned date: - - journal: ann gastroenterol doi: . /aog. . sha: doc_id: cord_uid: n f mf the current outbreak of covid- pandemic caused by sars-cov- has affected nearly countries. patients with severe covid- are more commonly elderly and suffer from comorbidities such as hypertension, diabetes mellitus, coronary artery disease, chronic pulmonary disease, obesity, and cancer. inflammatory bowel disease (ibd) affects as many as . million people globally, and a significant proportion of them are treated with immunosuppressants. hence, there is an ongoing concern over the impact of covid- on ibd patients and their susceptibility to it. so far, there are about ibd patients in the surveillance epidemiology of coronavirus under research exclusion (secure-ibd) registry reported to be infected with sars-cov- . there are many unique challenges and dilemmas that need to be taken into account when managing an ibd patient with covid- . the management of each patient should be individualized. the ibd societies and experts have strongly recommended that patients should not discontinue their ibd medications. if the patients have symptoms of covid- or ibd flare-up, they are recommended to call their ibd physician first to discuss their medication. in addition, ibd patients are urged to practice social distancing strictly to minimize the chances of infection. as covid- is rapidly evolving, our experience and understanding of its impact on the ibd population may potentially change in the near future. the current covid- pandemic, caused by sars-cov- , has affected nearly countries. this outbreak started as an epidemic in wuhan, china, and has now spread globally. as of july , , there were more than . million confirmed cases worldwide ( fig. ) , with a case fatality rate of . % [ ]. the united states (us) has now become the worst affected country in the world, with more than million reported cases and a case fatality rate of . % [ ] . the data suggest that older adults aged more than years and those with underlying comorbid conditions have a greater risk of severe covid- [ ] [ ] [ ] [ ] [ ] [ ] . inflammatory bowel disease (ibd) is a chronic relapsing inflammatory disorder of the gastrointestinal (gi) tract that comprises major forms: ulcerative colitis (uc) and crohn's disease (cd). in this article, we aim to provide an overview of the implications of the covid- pandemic for ibd patients and outline the available guidance for their management. there has been a proactive approach from several international ibd and gi societies, issuing recommendations to minimize the risk of sars-cov- infection and provide support to ibd patients. some of these measures include the issuance of expert recommendations for managing patients with ibd during the covid- outbreak, postponement of elective endoscopy and surgeries, provision of personal protective equipment (ppe), and availability of online consultancy by ibd specialists. the global prevalence of ibd was estimated to be . million in , with an increase over the last decades [ ] . ibd is most commonly diagnosed between the ages of a and , and the majority of ibd patients are in the workingage group [ ] [ ] [ ] . ibd patients are known to make greater use of outpatient services, hospital services, endoscopic and surgical services compared with non-ibd controls [ ] . furthermore, a significant proportion of ibd patients are treated with immunosuppressive medications. hence, there is ongoing concern about the susceptibility of ibd patients to sars-cov- infection and its impact on them. according to a recent study from northern california, ibd patients did not have a higher rate of sars-cov- positivity compared with the general population [ ] . ibd patients are more frequently assessed with both esophagogastroduodenoscopies (egds) and ileocolonoscopies compared with the non-ibd population [ ] . the identification of sars-cov- rna in the stool of patients with covid- has raised concerns about possible fecal-oral transmission [ ] [ ] [ ] . furthermore, egds pose an increased risk of transmission to healthcare workers, due to their increased exposure to air droplets and saliva during the procedure. patients with ibd have a similar life expectancy to the general population, but infections are overrepresented as a cause of death in the ibd patient population [ , ] . most of these infection-related deaths in ibd patients are related to the complications of severe underlying ibd itself, such as intra-abdominal sepsis, but non-disease-related infections are also responsible for a significant number of deaths [ ] . in a large retrospective database-based study of , patients with ibd and non-ibd controls, ibd patients had a . times greater risk of influenza infection compared with non-ibd patients ( % confidence interval [ci] . - . ) [ ] . in that study, the use of corticosteroids was found to be independently associated with an increased risk of influenza (odds ratio [or] . , %ci . - . ) [ ] . sars-cov- is a positive-sense ribonucleic acid (rna) virus that consists of a nucleocapsid core with nucleoprotein (n) and membrane proteins: spike glycoprotein (s), membrane protein (m), and envelope small membrane protein (e) [ ] . the viral surface membrane proteins facilitate embedding in the cell membrane of host cells, and n proteins play an essential role in replication and rna packaging [ ] . angiotensin-converting enzyme (ace ) has been identified as the receptor for sars-cov- to enter epithelial cells, in a similar fashion to sars-cov- [ ] . sars-cov- is mainly spread through direct exposure (droplet, person to person), and it is also presumed to spread through indirect contact (contaminated objects, airborne transmission) [ ] . a recent study found that sars-cov- was viable and infectious in aerosols for or more hours and on surfaces for up to days [ ] . it is essential to consider the potential transmission via aerosol when performing aerosolgenerating procedures such as egd, endotracheal intubation, noninvasive ventilation, bag-valve-mask ventilation, the suction of gi secretions, nebulizer treatment and insertion of a nasogastric tube. in a study by xiao et al, sars-cov- rna was detected in the stool in % of the patients hospitalized with covid- , raising concerns about potential fecal-oral transmission [ ] . in humans, the ace receptor is highly expressed in the gi system, including the upper esophagus, duodenum, jejunum, ileum, and colon [ , ] . the ace surface receptor directly interacts with the spike glycoprotein (s protein) of the coronavirus, which results in the entry of the virus into the cells (fig. ) and induces the reproduction of new virions intracellularly [ , ] . hence, the detection of viral rna in the feces suggests that the infectious virions are secreted from the virus-infected gi cells [ ] . it is still uncertain whether the presence of active bowel inflammation would increase the risk of sars-cov- infection. in a study by wang et al (preprint) , rna sequencing data from the patients with ibd showed that ace expression in the colonocytes was positively associated with viral entry, but negatively associated with viral transcription and protein translation [ ] . hence, the authors concluded that ace expression might play a dual role in mediating the susceptibility and immunity to sars-cov- infection [ ] . the overall detection rate of sars-cov- rna in stool specimens has been reported to be in the range of - % [ , ] . a recent study by zhao et al showed that about % of covid- patients had positive rectal swabs for sars-cov- rna at week after illness onset, which decreased in weeks - to %, . %, . %, . %, . %, and . % respectively [ ] . the authors also observed that the sars-cov- rna in fecal samples remained for an unexpected longer duration of time at a higher viral load than the paired respiratory samples [ ] . the duration of positive stool ranges from to days, and more than % of patients have positive stool rna even after the negative conversion of the viral rna in the respiratory tract [ , , ] . in a study by santarpia et al (preprint) , samples from the toilets in the infected patient room were positive in % of the rooms, with a mean concentration of . copies/µl, suggesting viral shedding during toileting [ ] . the endoscopic examination in these patients with positive stool rna showed normal mucosa with no significant damage to mucous epithelium with hematoxylin and eosin staining. however, numerous plasma cells and lymphocytes with interstitial edema were found in the lamina propria of the stomach, duodenum, and rectum. most importantly, patients who had positive sars-cov- in the stool also tested positive for ace and sars-cov- viral nucleocapsid protein staining in the glandular cells of the gi epithelium [ ] . these findings, along with the unremarkable endoscopic findings in infected patients, are reminiscent of sars-cov- tropism to the intestinal epithelium [ ] . innate immunity appears to play a vital role in combatting against sars-cov- infection, but only limited data are available so far. in a study by chen et al, among patients with covid- pneumonia, % had a high neutrophil count, % had low lymphocytes, and % had elevated c-reactive protein [ ] . in a prospective study by huang et al, among hospitalized covid- patients, patients admitted to the intensive care unit (icu) were found to have higher neutrophil and lower lymphocyte levels compared with non-icu patients [ ] . the patients in the icu group were also found to have higher plasma levels of many innate cytokines, including ip- , mcp- , mip- a, and tumor necrosis factor (tnf)-α. these findings suggest that a "cytokine storm" mediates disease severity in patients who have severe covid- , similarly to those with sars-cov- or mers-cov infection [ , ] . previous studies based on sars-cov- showed that the "cytokine storm" was strongly associated with viral sepsis, inflammation-induced lung injury, and acute respiratory distress syndrome (ards) [ , ] . tnf-α plays a vital role across key points of the cytokine cascade of the "cytokine storm" syndrome [ ] . anti-tnf therapy has been shown to significantly reduce levels of cytokines and inflammatory markers in animal studies, with a significant reduction in mortality [ ] [ ] [ ] . however, in previous clinical trials, the use of tnf-α antibodies did not show any mortality benefit in patients with severe sepsis [ , ] . currently, there is an ongoing trial to study the efficacy of anti-interleukin (il) drugs (anakinra, tocilizumab, siltuximab) in halting the cytokine storm and thereby preventing the progression from cytokine release syndrome to ards and multi-organ system failure [ ] . innate immunity against viral infection is dependent on the interferon (ifn) type i responses and its downstream cascade, which restricts the viral replication and induces an adaptive immune response [ ] . to mount an antiviral response, invasion of the virus should be first recognized by the innate immune cells via pathogen-associated molecular patterns (pamps) [ ] . for coronaviruses, viral rna or the intermediates during viral replication, such as dsrna, function as pamps. they are recognized by pattern recognition receptors such as tlr /tlr and the cytosolic rna sensor, rig-i/mda [ , ] . this recognition event activates the downstream signaling cascade, resulting in the expression of type i ifn and other proinflammatory cytokines, the first line of defense against the virus [ ] . sars-cov- and mers-cov were known to utilize dampening strategies to suppress the type inf response, closely associated with disease severity [ ] . given the close similarity of sars-cov- to sars-cov- , it has been speculated that sars-cov- could be employing similar strategies to evade innate immunity and also may have additional novel mechanisms [ ] . humoral immunity sars-cov- infection has a significant impact on both humoral and cellular immunity. humoral immunity is responsible for antibody production with protection against future infection. in a study by zhou et al, serology from patients with severe covid- pneumonia showed a peak in igm at day nine after disease onset and a switch to igg by week [ ] . a similar pattern of antibody production was also seen against the sars-cov- infection. the igm antibodies are the first-line antibodies during the acute or early convalescent period, while igg antibodies are antigen-specific antibodies that peak at week and are supposed to be protective against future infection [ ] . helper t cells play an essential role in the overall adaptive response, with cytotoxic t cells involved in the killing of the infected cells. the cytokine response generated by the antigenpresenting cells orchestrates the initial t cell response. patients with covid- were found to have a significant reduction in their peripheral cd + and cd + t cells, but these cells had a hyperactive status, as evidenced by high proportions of hla-dr and cd double-positive fractions [ ] . so far, there is no evidence to suggest that sars-cov- infection occurs more frequently in ibd patients compared with non-ibd individuals [ , ] . nor is there any known difference between cd and uc in terms of infection risk. it is still uncertain whether the presence of active bowel inflammation would increase the risk of sars-cov- infection. similar to the general population, ibd patients are at increased risk of infection if they travel to high-risk regions and if they have had close contact with a covid- patient. with regard to ibd-specific risk factors, it is speculated that patients on immunosuppressive agents, those with active ibd symptoms, malnutrition, and frequent visits to clinics or hospitals are at greater risk of acquiring sars-cov- infection [ ] . in an italian study of ibd patients, the risk factors associated with covid- -related mortality included age > years (or . , %ci . - . ), charlson comorbidity index > (or . , %ci . - . ), and active ibd (or . , %ci . - . ) [ ] . table outlines the risk factors for covid- infection and poor outcomes [ ] [ ] [ ] , , ] . recent evidence suggests that the use of proton pump inhibitors is associated with an increased risk of sars-cov- infection and also potentially increases the risk of poor outcomes, such as death and mechanical ventilation [ , ] . the international organization for the study of inflammatory bowel diseases (ioibd) maintains a registry for reporting covid- in ibd patients called secure-ibd registry. using the secure-ibd database, ibd physicians can report confirmed cases of covid- in their ibd patients, regardless of severity, including the asymptomatic patients detected through public health screening [ ] . as of june th , , there were ibd patients reported to be infected with covid- ( fig. ) in the secure-ibd database globally [ ] . table summarizes the latest data from the secure-ibd database as of june th , . brenner et al published the data from the secure-ibd registry with reported cases; currently, cases have been reported. based on that study, among the cases reported from countries, % were hospitalized, % had severe covid- , and % of the patients died [ ] . the most common symptoms in covid- are fever and respiratory symptoms, followed by gi symptoms such as diarrhea, vomiting, anorexia, dysgeusia, and abdominal pain [ , [ ] [ ] [ ] [ ] . based on the secure-ibd registry-based study, about % of the patients reported an increase in baseline ibd symptoms [ ] . among the ibd patients, diarrhea was the most common symptom ( . %), followed by abdominal pain ( . %), nausea ( . %), and vomiting ( . %) [ ] . in contrast, among non-ibd patients, studies have reported diarrhea in - %, nausea or vomiting in - % and abdominal pain in . - . % [ ] [ ] [ ] . the diagnosis of ibd flare-up is challenging in the context of covid- , as the typical presenting features of ibd, such as diarrhea, abdominal pain and fever, along with elevated inflammatory markers, may also be secondary to covid- [ , ] . however, rectal bleeding is not common in covid- [ ] . laboratory tests such as c-reactive protein and ferritin are elevated in both conditions, although occasionally ferritin can be low in uc. patients with covid- have lymphopenia, whereas in ibd patients, lymphocyte levels are usually normal or elevated. the red blood cell distribution width index has been found to be elevated in nearly half of patients with covid- and has been found to be associated with an increased risk of in-hospital mortality (adjusted or [aor] . , %ci . - . ) and septic shock (aor . , %ci . - . ) [ ] . in all cases, standard stool tests, such as stool culture and stool clostridium difficile (c. difficile) toxin, should also be performed. for patients with severe gi symptoms, abdominal computed tomography or endoscopic evaluation should be considered to assess the bowel inflammation to determine appropriate management. based on recommendations from the ibd societies, nonimmunosuppressive medications such as -aminosalicylates ([ -asa], mesalamine) and locally acting steroids • age > years [ , ] • cardiovascular disease [ ] • chronic pulmonary disease [ ] • males [ ] • hypertension [ ] • diabetes mellitus [ ] • obesity [ ] • cancer [ ] • on corticosteroids, especially prednisone ≥ mg per day [ , ] • active ibd [ , ] [ , ] . surprisingly, the study from the secure-ibd registry observed that patients on -asa/sulfasalazine treatment had times as great a risk (aor . , %ci . - . ) of poor outcomes such as icu admission, mechanical ventilation or death [ ] . this finding persisted even after controlling for age, comorbidities, ibd disease characteristics, corticosteroid use, and other factors. furthermore, in a direct comparison, patients treated with -asa/sulfasalazine had worse outcomes than those treated with tnf inhibitors [ ] . further studies are needed to confirm whether this association is real and to explore the biological mechanisms that may contribute to these poor outcomes. prednisone at doses of more than mg per day impairs the immune response [ ] [ ] [ ] . corticosteroids affect the immune system via multiple mechanisms, including inhibition of adhesion molecules, inducing apoptosis of activated lymphocyte, and decreasing the expression of inflammatory cytokines [ ] . based on a large international registry-based study, among ibd patients, those on systemic corticosteroids had . times (aor . , %ci . - . ) greater odds of a poor outcome such as icu care, mechanical ventilation or death [ ] . studies based on mers and sars-cov- have reported delayed viral clearance in patients receiving highdose corticosteroids [ ] . the use of short-term steroids for the treatment of ards and hyper-inflammation in covid- is controversial. one study by wu et al showed that in covid- patients with ards, treatment with methylprednisolone decreased the risk of mortality by % (hazard ratio . , %ci . - . ) [ ] . immunomodulators, such as azathioprine, -mercaptopurine ( -mp) and methotrexate, inhibit the body's immune response to viral infections. azathioprine and -mp interfere with dna and rna synthesis, thereby inhibiting the proliferation of t-and b-lymphocytes [ ] . overall, the thiopurines can reduce the number of activated t cells and also affect t-cell function [ ] . methotrexate inhibits dna synthesis, repair and cellular replication by interfering with folate metabolism [ ] . hence, actively proliferating cells are more susceptible to the effects of methotrexate. there are no data currently available on how the changes in the immune system by these immunomodulatory medications affect the course of ibd patients with covid- . biologics are the key immunosuppressive medication group used in the management of ibd. these medications include infliximab, adalimumab, golimumab, certolizumab pegol, ustekinumab, and vedolizumab. activated macrophages and t-cells secrete tnf, which plays an integral role in cellmediated immunity [ ] . tnf-α plays a vital role in inducing bowel inflammation by induction of ils, facilitating leukocyte migration, activation of neutrophils, and the induction of acute-phase reactants. thus, tnf inhibitors are very effective against the heightened inflammatory response seen in ibd. however, this impacts the effectiveness of the host immune system response against infectious organisms, especially intracellular pathogens, such as mycobacteria, fungi and viral infections [ ] . in a study by brenner et al, no association was found between tnf inhibitor use and severe covid- [ ] . in a prospective study of ibd patients from the university of miami, the use of tnf inhibitors such as vedolizumab and ustekinumab was associated with lower expression of ace in cd b-enriched cells [ ] . elevated serum tnf-α levels have been observed in patients with covid- infection, and were positively correlated with disease severity. even though anti-tnf treatment was explored as a potential treatment for covid- , there are no sufficient data to date [ ] . tocilizumab, an il- inhibitor, is currently under investigation for cytokine release syndrome triggered by covid- [ ] . tofacitinib is an fda-approved jak inhibitor for the treatment of moderate-to-severe uc in patients who are refractory to biologic agents. jak inhibitors are smaller sized molecules, with a rapid onset of action, that cause immunosuppression by blocking multiple pathways of inflammation to control the inflammation of the intestines. in a new york-based study on covid- patients receiving jak inhibitors for underlying inflammatory disorders, there was no increase in the rate of hospitalizations [ ] . however, it is important to consider the higher risk of blood clots in patients on tofacitinib treatment, since covid- infection is strongly associated with hypercoagulability and venous thromboembolism in up to one third of patients with severe infection [ , ] . interestingly, another jak inhibitor, baricitinib, is being explored as a potential medication in the management of sars-cov- infection through its inhibitory action on numb-associated kinase (nak), involved in the endocytosis of sars-cov- [ ] . tofacitinib does not inhibit nak. cyclosporine a is used in severe steroid-refractory uc patients. it is a polypeptide that suppresses t cell by inhibiting transcription of genes encoding il- [ ] . the primary molecular target of cyclosporine is a cytosolic binding protein called cyclophilin, also required by many viruses for replication, including the coronaviruses. it has been speculated that patients on cyclosporine treatment during the covid- pandemic may experience a beneficial effect [ ] . so far, no study has reported any benefits or risks of cyclosporine with covid- . the care of the ibd patients has to be individualized based on whether the patient is infected with sars-cov- , has symptomatic manifestations of covid- and shows underlying activity of the ibd. we are still in the phase where studies are being actively published; this will soon help us determine the outcomes in ibd patients, and will also give a better objective measure of which recommendations can be followed and what needs to change. however, as of now, we are still largely dependent on anecdotal evidence, expert opinion and guidance from the societies to manage ibd patients during this current pandemic (fig. ) . the societies that have issued guidance include the american gastroenterological association (aga), the british society of gastroenterology (bsg), crohn's and colitis canada, the european crohn's and colitis organization, and the ioibd [ , [ ] [ ] [ ] . the current evidence shows that ibd patients are not at a higher risk of developing sars-cov- infection compared with non-ibd patients [ , ] . hence, all the societies have recommended that patients continue their ibd medications to sustain remission, because the risk of disease flare-up outweighs the chance of contracting sars-cov- infection. furthermore, a relapse in ibd may require steroid therapy or hospitalization, known risk factors for sars-cov- infection [ ] . the emphasis should be on enforcing preventive strategies, such as social distancing and hand hygiene, and to avoid contact with infected individuals [ ] . the bsg has categorized ibd patients into categories based on their medications (table ) [ ] . the patients in the highest-risk group have been recommended to practice "shielding", the most stringent version of isolation. the patients in the moderate risk group should follow "stringent social distancing", and those in the lowest risk group should follow "social distancing" practices. in a study published from wuhan, proactive measures were taken by the medical team by sending educational materials and instructions on covid- prevention to all registered ibd patients (n= ). by enforcing these preventive measures, they were able to achieve a "zero infection" rate among their ibd patients [ ] . another concern was the risk of sars-cov- infection among ibd patients who are on infusion therapies such as infliximab, vedolizumab and ustekinumab, for which the patients need to go to the infusion centers. however, the consensus statement from the ioibd recommended continuing infusions in an infusion center, as long as the infusion centers have protocols to screen for covid- exposure and symptoms, including fever checks at the door [ ] . it is also recommended that the infusion centers provide a minimum of -meter spacing between patients' chairs, provide the patients with gloves, ensure mask and glove use by the providers, and carry out adequate deep cleaning after the patient's departure [ ] . other strategies cited by other societies include moving infusion centers to "clean sites" in order to keep ibd patients away from centers that admit or review patients with covid- . as the testing capability of sars-cov- becomes widespread, we will encounter patients who are asymptomatic but have had a positive test for sars-cov- infection. these patients should be closely monitored for the development of covid- for at least weeks. it has been recommended that -asa medications be continued in these patients, but it is unclear why patients on -asa had poor outcomes in the secure-ibd study (table ) [ , ] . for patients on prednisone, it should be tapered off if feasible, but if this is not possible, then at least the dose should be reduced below mg/d or transitioned to budesonide [ ] . immunosuppressive medications, such as azathioprine, -mercaptopurine and methotrexate, should be stopped temporarily [ ] . for monoclonal antibody therapies, such as anti-tnf agents, ustekinumab and vedolizumab, the dosing should be delayed for weeks and can be resumed after weeks if the patient has not developed symptoms of covid- [ ] . the current approach to monitoring these patients could change with the development of point-of-care tests and serologic antibodies [ ] . there are many unique challenges and dilemmas that need to be taken into account when managing an ibd patient with covid- . the management strategy will depend on multiple factors, such as the patient's age, the severity of the covid- infection, the clinical status of the ibd, and the presence of other comorbid conditions. the management of these patients should be individualized, with a -pronged approach to manage both covid- and ibd. adjustment to ibd medications is mainly focused on reducing the level of immunosuppression during active covid- , with the goal of preventing complications. table outlines the critical medication management recommendations from the ioibd expert panel consensus [ ] . in terms of ibd medications, it is safe to continue nonimmunosuppressive medications, such as topical, rectal therapy and antibiotics [ ] . oral budesonide or beclomethasone may be safely continued to maintain remission. however, if the patient is on systemic corticosteroids, such as prednisone, it should be tapered quickly whenever possible, because of concerns over the immunosuppressive effect of steroids, which could increase the risk of severe infection [ , , ] . in addition, observational studies from past epidemics (sars-cov- , mers-cov) showed delayed clearance of viral rna in patients on corticosteroid therapy [ , ] . the covid- should be treated with supportive care, and self-quarantine until the symptoms resolve. patients should be encouraged to contact their healthcare provider by phone for guidance about clinical management. telemedicine should be utilized in such patients to minimize the spread of infection. in patients with active gi symptoms suggestive of ibd flare-up, the workup should be targeted to find the cause for the relapse. the stool should be tested for c. difficile and other enteric pathogens. the presence of active inflammation can be assessed by testing c-reactive protein, fecal calprotectin or cross-sectional imaging. according to joint gi society recommendations, endoscopic procedures should be reserved only for urgent and emergent indications during the covid- pandemic [ ] . for mildly active disease, nonimmunosuppressive medications can be used. for moderate to severe disease, the risks and benefits of escalating ibd therapy must be carefully weighed against the severity of the covid- [ ] . if the patient has only mild covid- , then the ibd flare-up should be treated similarly to the treatment that would have been considered before the pandemic. however, it is still essential to avoid or limit the use of corticosteroids, given their association with poor outcomes. in patients hospitalized for severe ibd who have mild covid- , intravenous steroids should be limited to days, and consideration should be given to the use of either infliximab or a calcineurin inhibitor for induction of remission [ ] . in patients hospitalized for severe covid- , the priority should be given to the treatment of the covid- . in patients presenting with severe covid- along with severe ibd flare-up, the management becomes challenging. severe ibd, such as acute severe uc (asuc), is typically managed with high-dose parenteral corticosteroids [ ] . if these patients fail to respond adequately, then they are treated with rescue therapy, such as infliximab, along with steroids [ ] . according to the rand appropriateness panel on the management of asuc in patients with covid- pneumonia, there was uncertainty among the panel regarding the first-line therapy [ ] . however, among all the suggested treatments, intravenous steroids were given the highest score by the panel. at that time, the results from the recovery trial were not available [ ] . in the recovery trial, the dexamethasone group had a lower mortality rate compared with the usual care group in patients receiving mechanical ventilation ( . % vs. . %; rate ratio . , %ci . - . ) and among those on supplemental oxygen ( . % vs. . %; rate ratio . , %ci . - . ) [ ] . the dose of dexamethasone used in the recovery trial was mg q.d. for days. the usual intravenous steroids used in asuc include methylprednisolone ( mg t.i.d.) or hydrocortisone ( mg t.i.d.) [ ] . dexamethasone at a mg dose is equivalent to mg of methylprednisolone or mg of hydrocortisone [ ] . in addition, dexamethasone is long-acting ( - h), whereas methylprednisolone and hydrocortisone last for - h [ ] . finally, if the patient fails to improve despite rescue therapy or develops complications, then surgical intervention should be undertaken. it was deemed inappropriate to delay surgery, even in patients with covid- pneumonia. multiple societies have recommended suspending all routine non-urgent endoscopic procedures during the covid- pandemic [ ] . hence, only urgent and emergent endoscopic procedures are being done currently, which could potentially change a patient's management. some of the clinical scenarios that would need an endoscopic procedure are the new diagnosis of severe ibd, which needs tissue diagnosis for confirmation of diagnosis, to rule out cytomegalovirus infection in cases where a serum polymerase chain reaction was inconclusive, and when cancer is suspected [ ] . the aga recommends the uses of n (or n or papr) masks and double gloving as a part of ppe for the healthcare worker performing the endoscopic examination, regardless of the covid- status [ , ] . covid- is the latest threat to global health; it has developed into a public health emergency and is a reminder of the ongoing challenge of emerging infectious pathogens. the most important question is how to modify the medication regimen in ibd patients with sars-cov- infection, without increasing the risk of complications from either the covid- or the ibd itself. the data published so far have been reassuring and do not appear to indicate that ibd patients are at greater risk or severe covid- compared with the general population. most of the guidelines have reinforced the importance of avoiding systemic corticosteroids for managing ibd and maintaining remission by recommending compliance with maintenance medications. if an ibd patient does develop covid- , it is important for them to immediately contact the ibd physicians to determine whether to continue the immunosuppressive medications or withhold them until infection resolution. it is essential for the ibd community, including the patient, physicians and advanced practice providers, to recommend and implement stringent prevention strategies. as the sars-cov- virus is rapidly evolving, our experience and understanding of its impact on the ibd population may potentially change in the near future. hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease -covid-net, states baseline characteristics and outcomes of patients infected with sars-cov- admitted to icus of the lombardy region china medical treatment expert group for covid- . clinical characteristics of coronavirus disease in china clinical insights into the gastrointestinal manifestations of covid- case-fatality rate and characteristics of patients dying in relation to covid- in italy clinical 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society of gastroenterology guidelines on the management of acute severe uc in the context of the covid- pandemic: a rand appropriateness panel dexamethasone in hospitalized patients with covid- -preliminary report a comprehensive review and update on ulcerative colitis potency and duration of action of glucocorticoids: effects of hydrocortisone, prednisone and dexamethasone on human pituitary-adrenal function gastroenterology professional society guidance on endoscopic procedures during the covid- pandemic aga institute. electronic address: ewilson@gastro.org. aga rapid recommendations for gastrointestinal procedures during the covid- pandemic the outbreak of covid- : an overview characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention cancer patients in sars-cov- infection: a nationwide analysis in china key: cord- - cd v bt authors: sebastian, s; gonzalez, h a; peyrin-biroulet, l title: safety of drugs during previous and current coronavirus pandemics: lessons for ibd date: - - journal: j crohns colitis doi: . /ecco-jcc/jjaa sha: doc_id: cord_uid: cd v bt the coronavirus (covid- ) pandemic has posed challenges in the routine care of patients with inflammatory bowel disease. one of the key challenges needing addressing is the quantification of the risks of immunosuppressive and biologic therapies in ibd patients during the pandemic. the similarities and differences between the previous coronavirus outbreaks and the pathobiology of the infections can give useful information in understanding the risks, and perhaps potential beneficial aspects of drugs used in ibd. although clinical, immunological and pharmacological data from the experience with the previous coronavirus outbreaks cannot be automatically translated to predict the safety of ibd therapies during covid- pandemic, the signals so far from these outbreaks on ibd patients who are on immunomodulators and biologics are reassuring to patients and clinicians alike. the st century has seen the worldwide spread of three previously unrecognized coronaviruses, the severe acute respiratory syndrome coronavirus (sars-cov- ) and middle east respiratory syndrome coronavirus (mers-cov), and more recently sars -cov- ( ) . starting from in china, the sars-cov- produced, then unprecedented, nosocomial transmission resulting in nearly deaths across countries ( ) . exactly a decade later another coronavirus mers-cov emerged with laboratory positive cases with at least deaths over countries ( ) . the current pandemic sars-cov originated in hubei province in china, and was declared a pandemic by world health organisation on march ( ). understandable concerns have been raised on the safety of steroids, immunosuppressive drugs, and biologics used in patients for a variety of indications including immune mediated inflammatory disease such as inflammatory bowel diseases (ibd), which do increase the risk of opportunistic bacterial, viral and fungal infections ( ) . the magnitude of the infection-risk in general with the therapies used in ibd is small and vary based on the patient, disease, and drug characteristics ( ) . equally, another aspect needing consideration if patients discontinue their ibd therapies is the potential risk of ibd flares needing hospitalization which will increase the risk of acquiring sars-cov- . several consensus guidelines recommend continuation of ibd therapies, primarily with the aim of reducing the risk of a flare needing hospitalization or surgery ( , , ) . two studies recently showed that ibd patients are not at increased risk of being infected with covid- ( , ) . by contrast, there is scarce data so far on the risk of sars-cov- infection in ibd patients who are on therapies which potentially alter the immune response to pathogens ( ) . therefore, continuing concerns remain both from ibd patients and the a c c e p t e d m a n u s c r i p t clinicians managing them, regarding the potential of ibd related drugs causing more frequent infections by sars-cov , and increased risk of severe complications from covid- ( ) . in this review we discuss the immune-pathological aspects of previous and ongoing coronaviruses ( ). the s protein is responsible for viral entry and thus plays a pivotal functional role in viral entry to the host cells, and the non-structural proteins are the key players in viral replication ( ) . the sars-cov- virus enters cells via the same mechanism as sars-cov, which is by binding of the surface spike glycoprotein (s protein) on the surface of the virus to angiotensin-converting enzyme (ace ), a receptor on the cell surface controlling the cleavage of several peptides. ace is expressed in type pneumocytes in the lung, blood vessels, oropharyngeal mucosa, small intestine, colon, and kidneys ( , ) with remarkably high expression in the epithelial cells of the proximal and distal intestines, and this is also a portal for viral entry (figure ). single cell rna sequencing analysis showed that ace expression in colon cells was positively correlated with the regulation of viral infection and congenital cellular immunity and was negatively correlated with viral transcription, protein translation, phagocytosis, and complement activation [ ] . recently, garg et al. have described differences in the ace between inflamed and non-inflamed biopsies in patients a c c e p t e d m a n u s c r i p t with ibd, but the mucosal expression or activity was not associated with the use of therapies in ibd ( ) . furthermore, their study indicated that the level of circulating ace is upregulated in patients with ibd. in addition, a more recent elegant study maria abreu and colleagues ( ) reported that the expression of ace and tmprss , which are the entry portals for sars-cov- , are not increased in inflamed colon and ileum of patients with ibd and some medical therapies are associated with lower levels of ace . therefore, ace - overall, it is becoming increasingly apparent. that the tug of war between coronaviruses and host antiviral defences are at the core of the pathogenic potential of all coronaviruses determining the clinical course and outcome, and this gives insights on the relative risks of drugs and also opens a window for exploring therapeutic options ( ) (figure a c c e p t e d m a n u s c r i p t immunosuppressive drugs and severity of coronavirus infections: opportunity in the face of adversity? the impact of drugs, in particular, steroids, immunosuppressive and biologics were brought in to sharp focus during the three coronavirus pandemics despite the wide variation in the severity of the outbreaks and the mortality rates. these drugs remain the cornerstones of therapies in ibd and have revolutionised ibd care. the lack of prospective data specific to the sars-cov- infection to date means that our understanding of the genealogy and immunopathology need to be integrated to the pharmacological aspects of these drugs to shed some light into this challenging conundrum. corticosteroids are thought to have a divergent effect on viral infections including sars cov viruses; on one hand they inhibit host immune response acting on migration and chemokines production leading to impaired viral clearance and the resultant prolonged moreover, a prospective, randomized double-blinded, placebo-controlled trial compared early hydrocortisone treatment (before day seven of the illness) with a placebo and found that early hydrocortisone therapy was associated with a higher subsequent plasma viral load ( ) . in the setting of mers-cov, use of corticosteroids for critically ill patients did not improve the corticosteroids also impair the induction of anti-viral type-i interferon responses to a range of respiratory viruses ( ) . early corticosteroid use did not benefit critically ill patients with ards but was independently associated with higher mortality in the setting of severe influenza pneumonia ( ) . overall, the current data does not support the use of corticosteroids in the coronavirus infections ( ) and randomised controlled trials are needed before routine use. there is data suggesting that doses above mg of prednisolone are associated with increased risk of bacterial and viral infections in ibd and even with increased hospitalization suggesting a lower dosing strategy ( , ) . in a recent italian series of ibd patients with covid- , a trend towards adverse outcome with concomitant corticosteroids was reported ( ) . in a more recent series from new york, the use of steroids appears to predict covid- (or . , % ci . - . ) ( ) . locally acting steroids such as budesonide and beclomethasone theoretically have some advantages in relation to side effect profile ( , ) , but no data is available in the setting of coronaviruses. hence overall, while there is no data a c c e p t e d m a n u s c r i p t on whether current use of steroids increases the risk of severe covid- , it seems prudent to minimise the use of systemic steroids, think of alternatives to steroids, and if used, taper to the lowest possible dose quickly. broad immunosuppression has the potential to increase susceptibility, persistence, and reactivation of viral infections in patients ( ) . acute respiratory viruses implicated in causing severe disease in immunocompromised patients include respiratory syncytial virus (rsv), influenza viruses a and b, parainfluenza viruses and adenovirus, and immunocompromised patients are generally considered at increased risk of influenza and at higher risk of complicated infection ( , ) . there is also risk of opportunistic viral infection with thiopurines, but these are mainly dna viruses such as hepatitis b and c, epstein barr virus and human papilloma virus. ( ) partial assessment regarding whether immunosuppression is a relevant risk factor for covid- infection and severe course can be guided by the findings of sars-cov- and mers-cov outbreaks ( ) . on review of large published cohorts of sars-cov and mers-cov infections ( , , , , ) , the risk factors for both infections included advanced age and presence of one or more co-morbidities such as diabetes, heart disease ,hypertension, lung disease and obesity. although there were no specific studies on immunosuppressed individuals including ibd, no fatality was reported in patients undergoing chemotherapy or other immunosuppressive treatments, at any age ( , , ) . although transplant patients were expected to have poorer outcomes following acquisition of sars cov , at the end of the outbreak no mortality or graft loss had been recorded ( , , ) a c c e p t e d m a n u s c r i p t atypical presentation of mers-cov was reported from korea in patients on immunosuppressants, all of whom made full recovery without the need for invasive ventilation ( ) . in another series of patients with serious mers-cov infection, there was a single patient who was on prolonged immunosuppression who also made an uneventful recovery ( ) . in a hospital outbreak of mers-cov in jordan, immunosuppressive individuals did not have additional risks ( ) . in an analysis of cases from the epicentre of mers in saudi arabia from - , immunosuppressant use was not associated with increased risk for mers-cov infection ( ) . in another study, with a cohort of patients, one of the patients was on immunosuppressants for double transplant (kidney and liver) and, while requiring intensive unit care, did not die ( ) . in a retrospective cohort study of host susceptibility in south korean mers outbreak, patients of solid organ transplantation were included but none developed mers-cov infection, but out of the patients with autologous cell transplantation did, without any mortality ( ) . in an animal model of mers-cov using immunosuppressed rhesus macaques, despite increased viral replication, pathology in the lungs was significantly lower in immunosuppressed animals ( ) . intriguingly some immunosuppressive agents used in treatment, including in ibd, have been found to interfere with viral replication ( ) . although the doses used were much higher in comparison to that in treatment of immune disorders such as ibd, thiopurines, when used for haematological malignancies, appeared to be specific inhibitors of sars cov- virus ( ) . ( , , , ) . in a monocentric cross-sectional study of patients, of whom (( %) were on calcineurin inhibitors alone, the immunosuppression with calcineurin inhibitors did not increase the risk of hospitalisation or mortality ( ) . cyclosporine was a c c e p t e d m a n u s c r i p t successfully used in a pregnant patient with acute severe ulcerative colitis with covid- ( ) . these promising signals, and the underlying pharmacological basis, have prompted some to suggest consideration of calcineurin inhibitors for treatment of coronavirus infections ( , ) . in a study of rheumatoid arthritis patients on methotrexate with resolved hbv infection, the incidence of hbv reactivation was very low at . / person-years ( ) . the patients on concomitant methotrexate in ibd so far has not reported any additional risks from sars-cov- ( , , ) overall, immunosuppressive therapy does neither seem to have a major impact on infection with sars cov- , mers-cov and sars-cov- nor does it seem to lead to a severe disease course in many cases. however, it must be kept in mind that reported case numbers are exceedingly small overall and continued vigilance is needed. furthermore, anti tnf treatment may diminish the degree of protective immunity resulting from vaccination, but levels achieved appear adequate with other viruses ( ). paradoxically, it is plausible that the use of biologics may have a beneficial effect in reducing the inflammatory immune responses following covid- by reduction of cytokines, including tnf. tnfα has been implicated in the severe immune-based pulmonary injury caused by sars-cov-, suggesting that tnfα inhibitors could be a potential treatment for the acute respiratory disease syndrome caused by coronavirus ( ). coronavirus viral spike protein is able to induce a tnf-α-converting enzyme (tace)-dependent shedding of the ace ectodomain, and this process, which appears to be strictly coupled to tnfα, is essential for the penetration of the virus into the cell. this means tnf inhibitors might be effective in blocking viral entry and the detrimental effects of exuberant tnf-α ( ). anti tnf agents, as an option for therapeutic modulation, were proposed during the sars-cov- outbreak although no human studies were performed ( ). following this, in a study on piglets to assess the efficacy of an anti-tnfα) therapy for endotoxin respiratory diseases, the investigators observed that tnfα blockade was not associated with decrease in disease severity( ) subsequently, anti tnf for therapy of viral infections caused by respiratory syncytial virus or influenza virus was studied again in animal models and showed that tnf however, it is important to recognise that it is equally plausible that theoretical possibility of increased viral burden resulting from inability of immune response may result in increases severity of inflammation ( ) . hence some concerns have been raised by some authors in relation to broad immunosuppression in the presence of an overwhelming infective illness ( ) . nevertheless, there is no evidence indicating that tnfα blockade is harmful to patients in the context of severe infections including septic shock ( ) . a randomised controlled trial of anti tnf agents with septic shock in intensive care units showed no evidence of increased infections in the anti tnf treated patients ( ) . no patients on anti tnf agents have been recorded in any of the series reported during the sars-cov- or mers outbreaks, but there is an increasing amount of literature suggesting that tnfα blockade is not harmful to patients in the context of covid- ( , , ) . these opinions are supported by the case series of patients on anti tnfs for immune mediated inflammatory disorders such as arthritis and inflammatory bowel diseases ( , , , , ) . in a report of rheumatology patients on disease modifying drugs, half of whom were on anti-tnf agents at the height of the pandemic in northern italy, monti et al however, the hospitalisation rates, intensive care treatment and mortality appears to be higher in the patients who are on combination therapy with anti tnf and immunomodulators ( %, % and % respectively), and therefore further data is required. vedolizumab selectively inhibits the interaction of α β with mucosal adhesion molecule - preventing the entry of t lymphocytes across the endothelium to the inflamed gastrointestinal mucosa ( ) . the receptors are present in gi tract, nasopharyngeal mucosa, and biliary epithelium. the gut-selective mode of action of vedolizumab should theoretically be associated with a lower risk of infections. no increase in viral infections including nasopharyngitis was noted in vedolizumab treated patients in a meta-analysis ( ) . higher, but statistically insignificant, rates of enteric infections occurred in vedolizumab-exposed patients ( . / pys; % ci: . - . ) to placebo ( . pys; % ci: . - . ) in this metaanalysis ( ) . no reactivation of hepatitis b or c was noted in patients with history of hepatitis b or c in a post marketing surveillance study of the licencing trials ( ) . in siv- of these patients required hospitalisation ( , , ) . the ig-ibd study included patients on vedolizumab, five of whom needed admission but reported no association with risk for covid- pneumonia ( ) . the secure ibd registry (accessed on th may ) has included data from patients on vedolizumab with a % hospitalisation rate; ( % in icu) and % mortality ( ). therapies targeting jaks may interfere with normal anti-viral response including inhibition of ifn-γ activity ( ) , and may potentially increase the risk of infection and/or reactivation of several viral infectious diseases including a dose dependent risk for vzv observed for tofacitinib ( ). in some studies the use of higher dose of tofacitinib along with corticosteroids was associated with serious infections ( ) . jak inhibitors also have anti-viral potential since they lower the pro-inflammatory response mediated by viruses and block many pro-inflammatory cytokines involved in cytokine storm such as il -, il , il- and tnf ( ). of relevance would be the il- or il _r blockade with jaks or specific anti il- r antibody tocilizumab ( ) . baricitinib, currently not used in ibd but approved for rheumatoid arthritis, blocks viral endocytosis and assembly of virus particles into pneumocytes, and has shown promising results in clinical trials in sars-cov- ( , ) . this potential beneficial effect is not seen with tofacitinib. fedratinib, a selective a c c e p t e d m a n u s c r i p t jak inhibitor which inhibits th mediated immune hyperstimulation, is also proposed for treatment of severe covid- infection ( ) . the new york series on imids had patients on tofacitinib, one among them needing hospitalisation but not ventilation ( ) . two patients on tofacitinib with covid- have been reported in rheumatology literature, both without severe outcomes ( ) . in a case report from washington ( ), a young patient on tofacitinib continued the treatment uninterrupted following diagnosis of covid- and had complete recovery without the need for hospitalisation. seventeen patients on vedolizumab have been reported so far to the secure ibd registry ( ), five of whom needed hospitalisation, with one mortality (accessed on th may ). hypothetically, blocking il and il which are involved in cytokine storm may have a beneficial effect in ameliorating the cytokine storm in covid- ( , ) . ustekinumab currently there is no data to indicate that therapies used in ibd will result in more severe outcomes in patients. whether drug-induced immunosuppression will prevent the cytokine storm in patients infected with covid- will require further investigation. until we have more data, a risk versus benefit grid based approach ( table ) covid- , sars and mers: are they closely related? who guidelines for the global surveillance of severe acute respiratory syndrome (sars) the global burden of premature mortality due to the middle 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interleukin- receptor (il- r) antagonist tocilizumab my be the key to reduce the mortality baricitinib as potential treatment for -ncov acute respiratory disease baricitinib as potential treatment for -ncov acute respiratory disease th responses in cytokine storm of covid- : an emerging target of jak inhibitor fedratinib case report of a sars-cov - infection in a patient with ulcerative colitis on tofacitnib il- , il- and il- in ibd: immunobiology and therapeutic targeting covid- and immunomodulation in ibd a c c e p t e d m a n u s c r i p t .cullen g, bader c, korzenik a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord- - og authors: danese, silvio; cecconi, maurizio; spinelli, antonino title: management of ibd during the covid- outbreak: resetting clinical priorities date: - - journal: nat rev gastroenterol hepatol doi: . /s - - - sha: doc_id: cord_uid: og the coronavirus disease (covid- ) worldwide outbreak has led to a dramatic challenge for all healthcare systems, including inflammatory bowel disease (ibd) centres. here, we describe the fast changes and clinical issues that ibd specialists could face during this sars-cov- infection pandemic, highlighting the potential rearrangements of care and resetting of clinical priorities. since december when the novel coronavirus ( -ncov as it was then termed, now named severe acute respiratory syndrome coronavirus (sars-cov- ) by the who) outbreak had been described in wuhan, hubei, china, the situation has dramatically evolved . the pandemic, as declared by the who, has led to > , cases worldwide reported as of march , in all continents, excluding antarctica, spreading on a logarithmic scale in europe. italy is currently the second most affected country after china and, as of march , europe was declared the centre of the pandemic . because of the very high transmission capacity, the who declared the outbreak of coronavirus disease (covid- ) caused by sars-cov- infection a public health emergency of international concern. as the need for hospitalization is very high among symptomatic cases (~ %), with an increased need to have access to intensive care units and mortality in the order of % globally , , european hospitals have started to intensively reduce elective activities, including surgery, to prepare for the high numbers of admissions. in addition, action by governments to contain the outbreak and slow the spread of covid- has restricted regions and nations (the entire country of italy, for example) by reducing their mobility within countries and across borders. but what are the implications of covid- for patients with inflammatory bowel disease (ibd)? with > , patients with crohn's disease and ulcerative colitis, our ibd center in milan, italy, has been flooded by requests from the patients themselves inquiring about the risk of infection in patients with ibd, and asking what precautions to take, particularly regarding, but not limited to, their immunosuppressive treatment. the covid- outbreak is a fast and evolving situation, and information on the incidence and/or risk of infection in patients with ibd is not yet available. however, it is important to counsel patients to inform them that > % of reported cases of covid- have been mild in published studies, and the proportion of fatal cases might be an overestimate as many asymptomatic cases are not identified . in our opinion, the best advice for patients with ibd is to try to minimize the risk of infection by following good hand hygiene (frequent washing with soap and water), covering the mouth and nose with a tissue or your sleeve (not hands) when coughing or sneezing, avoiding close contact with anyone with influenza-like and/or upper respiratory symptoms, and staying home or isolated if possible. in addition to these measures, emerging reports state that patients might have viral rna present in their faeces and live virus has been isolated from faecal samples [ ] [ ] [ ] . thus, caution should be taken when using public toilets given the implications for the potential route of faecal-oral transmission. an increasing number of patients with ibd treated with immunomodulators or biologic agents in our centre have asked whether a pause in their immunosuppressive therapy would be justified during the covid- outbreak. at the moment, there are no formal evidence-based recommendations from clinical societies or governments for patients on immunosuppression, such as those with ibd. however, a study in a tertiary care population of , patients with ibd followed for > , patient-years described an exhaustive characterization and validation (hospitalization reports) of all serious viral infections (for example, all that required hospitalization) including varicella zoster virus (vzv), herpes simplex virus (hsv), cytomegalovirus (cmv) and epstein-barr virus (ebv) but not sars-cov- (ref. the coronavirus disease (covid- ) worldwide outbreak has led to a dramatic challenge for all healthcare systems, including inflammatory bowel disease (ibd) centres. here, we describe the fast changes and clinical issues that ibd specialists could face during this sars-cov- infection pandemic, highlighting the potential rearrangements of care and resetting of clinical priorities. to anti-tnf agents, > , patient-years of exposure to immunosuppressants, and > , patient-years of follow-up in those > years. the researchers identified cases of serious viral infections related to ebv, cmv, vzv and hsv infection. the two independent drivers of the risk were clinically active ibd and exposure to thiopurines. no cases of severe seasonal flu and no deaths by seasonal flu were observed. although many of our patients have been vaccinated against seasonal flu, as recommended by european crohn's and colitis organisation (ecco) guidelines, vaccination strategy adoption is very low and the protective effect of the vaccine is moderate, particularly in patients with an immunosuppression status. thus, at the moment, it does not seem appropriate to recommend the pause of immunosuppressive treatment in patients with ibd, as stated by international organization of ibd ( fig. ), crohn's & colitis uk and crohn's & colitis foundation in their guidance for patients. moreover, although thiopurines have been associated with risk of serious viral infection in ibd , the ioibd recommends they continue to be taken as these agents take months to leave the body and so stopping these medications will not help in the short term. in addition, a survey from the european federation of crohn's and colitis association and ecco is ongoing to explore the need for education about covid- . additional recommendations for patients with ibd from the ioibd: • medicines such as mesalamine are safe • if possible, withdraw from steroid use • biologic agents used to treat ibd (e.g. anti-tnf agents, ustekinumab and vedolizumab) are generally safe; there are no recommendations to stop taking these medications and the effects of these drugs are present for many months • thiopurines and tofacitinib tend to inhibit the immune response to viral infections, but stopping these agents in the short-term will not help • get the influenza vaccination • stay at home and minimize social contact c | strategies to enable maintenance of our biologic agent clinic during the covid- outbreak in italy include checkpoints at hospital entrances for symptom screening and use of surgical masks for clinical staff and patients. a basic protective measures for the public according to the who. b recommendations by ioibd correct as of mar . c experience at the ibd center, humanitas university , milan, italy. as priorities and resources increasingly shift towards the covid- pandemic, will it be possible to maintain the high standard and quality of care for patients with ibd? multiple layers of complexity are faced during this challenging scenario. the interruption of any elective or routine follow-up clinic has caused anxiety among patients and clinicians. however, patients should be reminded that this interruption is temporary, and a shift towards virtual clinics can help patients and healthcare providers to avoid any potential loss of follow-up in case of clinical issues. this approach has also been initiated by ibd specialists in china during the covid- outbreak . the running of biologic agent clinics and drug administration during travel restrictions in tandem with recommendations by health authorities to stay at home have been of major concern. so far, ibd biologic agent clinics have been maintained in our centre following rigorous actions to avoid infection outbreaks ( fig. ), such as: checkpoints at the hospital entrance to screen and ask if there has been any cough or fever in the previous weeks; verification of patient contact information with people with similar symptoms such as cough and fever; respecting - m distance between chairs where patients sit for their infusions and during clinics; use of surgical masks for clinical staff and patients, and latex gloves for clinical staff. a topic of discussion is whether wearing surgical masks is a requirement, which is still a matter of debate. the who does not recommend wearing masks in the community as evidence is lacking . however, facing such a novel situation with limited options, the use of masks could lead to benefit. indeed, for influenza it has long been recommended that affected patients should wear masks to limit droplet spread and, therefore, the very same action could be used for immunosuppressed patients to reduce the risk of spread of infection. we have chosen to use surgical masks for both the clinical staff and the patients, but a global shortage of disposable surgical masks is creating challenges and this issue continues. as timely surgery is the other mainstay of ibd care, it is of deep concern that stopping scheduled surgery completely for several weeks for patients with ibd (allowing only oncological cases to undergo surgery) will soon result in increased numbers of emergency presentations and more complications from treatment delay. in the region of milan, italy, where elective surgery for every benign indication (including ibd) has been substantially slowed or stopped over the past weeks, with the prospect of further weeks of delays or cancellations, notable concerns have been raised for possible disease progression and poor outcomes of ibd surgery once performed. the covid- pandemic will continue to spread worldwide with increasing burden on our health-care systems and care of all patients with disease, not just those affected by covid- . to face these challenging circumstances, as clinicians we need to support political decision-making to rapidly adapt priorities, to reset temporarily the standards of quality of care and to help to communicate relevant information to patients. clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china clinical features of patients infected with novel coronavirus in wuhan coronavirus covid- global cases persistence and clearance of viral rna in novel coronavirus disease rehabilitation patients characteristics of pediatric sars-cov- infection and potential evidence for persistent fecal viral shedding detection of sars-cov- in different types of clinical specimens increased incidence of systemic serious viral infections in patients with inflammatory bowel disease associates with active disease and use of thiopurines. united eur update on covid for patients with crohn's disease and ulcerative colitis implications of covid- for patients with pre-existing digestive diseases advice on the use of masks in the community, during home care and in health care settings in the context of the novel coronavirus ( -ncov) outbreak: interim guidance has served as a consultant or speaker for ethicon, frankenman, oasis, pfizer, takeda and sofar. m.c. has served as a consultant or speaker for cheetah medical crohn's & colitis foundation international organization for the study of inflammatory bowel disease key: cord- -y b vml authors: suárez-fariñas, mayte; tokuyama, minami; wei, gabrielle; huang, ruiqi; livanos, alexandra; jha, divya; levescot, anais; irizar, haritz; kosoy, roman; cording, sascha; wang, wenhui; losic, bojan; ungaro, ryan; di’narzo, antonio; martinez-delgado, gustavo; suprun, maria; corley, michael j.; stojmirovic, aleksandar; houten, sander m.; peters, lauren; curran, mark; brodmerkel, carrie; perrigoue, jacqueline; friedman, joshua r.; hao, ke; schadt, eric e.; zhu, jun; ko, huaibin m.; cho, judy; dubinsky, marla c.; sands, bruce e.; ndhlovu, lishomwa; cerf-bensusan, nadine; kasarskis, andrew; colombel, jean frederic; harpaz, noam; argmann, carmen; mehandru, saurabh title: intestinal inflammation modulates the expression of ace and tmprss and potentially overlaps with the pathogenesis of sars-cov- related disease date: - - journal: gastroenterology doi: . /j.gastro. . . sha: doc_id: cord_uid: y b vml background and aims the presence of gastrointestinal symptoms and high levels of viral rna in the stool suggest active severe acute respiratory syndrome coronavirus (sars-cov- ) replication within enterocytes. methods here, in multiple, large cohorts of patients with inflammatory bowel disease (ibd), we have studied the intersections between coronavirus disease (covid- ), intestinal inflammation and ibd treatment. results a striking expression of ace on the small bowel enterocyte brush border supports intestinal infectivity by sars-cov- . commonly used ibd medications, both biologic and non-biologic, do not significantly impact ace and tmprss receptor expression in the uninflamed intestines. additionally, we have defined molecular responses to covid- infection that are also enriched in ibd, pointing to shared molecular networks between covid- and ibd. conclusions these data generate a novel appreciation of the confluence of covid- - and ibd-associated inflammation and provide mechanistic insights supporting further investigation of specific ibd drugs in the treatment of covid- . severe acute respiratory syndrome coronavirus (sars-cov- ) and the ensuing coronavirus disease (covid- ) , have evolved into a global pandemic of unprecedented proportions . angiotensin converting enzyme- (ace ) is a carboxypeptidase that catalyzes the conversion of angiotensin i into angiotensin - , and angiotensin ii into angiotensin - [ ] [ ] [ ] [ ] . ace can also be cleaved by serine proteases such as transmembrane serine protease (tmprss) , tmprss d and tmprss . early events in the pathogenesis of sars-cov- infection include attachment of the receptor binding domain of the viral spike (s) protein to epithelial ace [ ] [ ] [ ] [ ] . the s protein is then cleaved by tmprss , which facilitates viral entry into the cytoplasm of the host cell . following infection with sars-cov, ace is downregulated in the lungs, resulting in unopposed renin-angiotensin-aldosterone system (raas) and contributing to disease severity , . inflammatory bowel diseases (ibd) encompassing crohn's disease (cd) and ulcerative colitis (uc) are chronic, inflammatory disorders of the gastrointestinal (gi) tract that are treated with conventional immunosuppressive drugs such as corticosteroids, biologic therapies and immunomodulatory drugs , . given that sars-cov- co-opts receptors expressed by intestinal epithelial cells, covid- has the potential to intersect with the pathogenesis of ibd and by extension, its treatment, at a number of points , . for example, ace expression may be potentially be altered during gut inflammation or by ibd medications. further, immunomodulatory drugs used in ibd therapeutics , could potentially be used in covid- patients to manage the "cytokine storm" associated with severe disease. therefore, in this study we systematically examined potential areas of intersection between the uninflamed and inflamed gi tract and covid- disease. the results of this study may improve our molecular understanding of how covid- intersects ibd and may provide a rationale for further investigation of drugs used in ibd therapeutics for use in patients with covid- . specimens were obtained via clinical endoscopy during routine care (table s , s ). tissue was formalin fixed and paraffin embedded by the clinical pathology core at our institution. primary antibodies used included ace (abcam-ab , : ), epcam (abcam-ab , prediluted) and mouse anti- ii. the risk cohort : ace and tmprss in treatment-free pediatric cd (< years of age) patients was studied using rna-seq expression profiles from gse , which includes ileal biopsies from endoscopically defined inflamed samples (n= ), non-inflamed (n= ) and non-ibd controls (n= ). i) the gse series which includes expression profiles from the gut of anti-tnfα refractory cd patients and the blood from patients enrolled in a phase b crossover trial (certifi trial) with ustekinumab (detailed in supplementary methods). ii)the gse series includes gene expression profiles (affymetrix human gene . st arrays) from colonic biopsies from moderate-to-severe uc patients enrolled in two vedolizumab efficacy trials (gemini-i and gemini lts) (detailed in supplementary methods). gse series also included non-ibd colonic biopsies and colonic biopsies from uc patients before and - weeks after first infliximab treatment. response was defined as endoscopic mucosal healing. bgrns can capture fundamental properties of complex systems in states that give rise to complex (diseased) phenotypes . bgrns were generated from intestinal biopsy rna sequence data (msccr, using intestinal expression qtl information (eqtls) as priors). the bgrns were region-(ileum or colon/rectum) and disease-(cd, uc, and control) specific and included both inflamed and uninflamed biopsies and were constructed using rimbanet software and visualized using cytoscape . . we also used two publicly available bgrns from the risk and the certifi cohort (supplementary methods). ace and tmprss subnetworks: gene-centric subnetworks were generated by selecting either ace or tmprss from various bgrns and expanding out three to five layers (undirected) to obtain the nearest j o u r n a l p r e -p r o o f ace or tmprss neighbors. the connected subnetworks obtained were generally between - genes in total. we curated rna-seq based ibd and covid- response signatures by identifying differentially expressed genes (degs, supplementary methods). genes differentially expressed in blood , lung nhbe/a or human small intestinal organoids (hsio) following sars-cov- infection; ibd inflammation; or response to medications were separately projected onto various bgrns allowing for or nearest neighbors depending on the signature sizes. the most connected subnetworks were then extracted to generate model-specific sars-cov- infection-; ibd inflammation-; or drug-responseassociated subnetworks (supplementary methods). gene subnetworks were tested for functional enrichment using a fisher's exact test with benjamini-hochberg (bh) multiple test correction on a collection of genesets. the collection of genesets included i) reactome pathways sourced from enrichr , i) gene sets from smillie et al , ii) huang et al , iii) various macrophage perturbations (e.g. cytokines) , iv) ace co-expressed genes and v) reported ibd gwas genes (see supplementary methods). pathway and geneset enrichment, as well as intersection between networks were tested using a fisher's exact test and p-values were adjusted for multiple hypothesis with bh correction. key driver analysis (kda) identifies key or "master" driver genes for a given gene set in a given bgrn (supplementary methods). genesets for kda included those associated with nhbe-covid- infection or ibd inflammation. key driver genes (kdgs) were summarized by frequency across the networks. covid- response gene expression (from whole blood or epithelial models) was evaluated in the context of ibd related inflammation using gene set variation analysis (gsva). for each covid- response signature, a sample-wise enrichment score was quantified from each transcriptomic profile in the msccr and certifi cohorts using gsva. covid- response gsva scores were then modeled to test the association with patient-derived phenotypic information. healthy gut segments express ace and tmprss proteins j o u r n a l p r e -p r o o f to define the localization and distribution of ace , immunofluorescence (if) microscopy was performed on histologically normal gi tissue in adults ( males, females) and children ( males, females) ( figure and table s ). ace expression was observed on the small intestinal surface epithelium in all subjects in a continuous distribution with the exception of occasional breaks representing the mucin from goblet cells (figure a-b) . in all examined small intestinal segments, ace could be detected on the crypt epithelium, though to a lesser extent than on the surface epithelium. tmprss expression was less abundant in the small bowel, and when detectable, was exclusively found on crypt epithelium ( figure s a -b). there was no observable age or sex dependence of ace or tmprss protein expression in the small bowel. in the colon, ace expression was patchy and could not be identified in every subject, in contrast to the small bowel (figure c) . this inconsistency across different donors could not be readily associated with age, sex, ace inhibitor treatment, nor the colonic segment being examined. in contrast, tmprss expression was more robust in the colon and was readily detectable on both surface and crypt epithelia ( figure s c) . thus, ace in the healthy gut is higher in the small bowel than the large bowel and inversely, expresses more tmprss protein in the colon compared to the small bowel. next, we examined ace and tmprss mrna levels in the intestine of non-ibd controls and ibd patients with active and inactive disease enrolled in msccr ( table s ) . as the number of samples for the colon non-rectum locations was low (figure s a /b) and no discernable differences were observed, colon non-rectum biopsies were grouped together to increase statistical power. consistent with the protein data, ace gene expression was higher in the uninflamed ileum compared to the uninflamed colon or rectum. with inflammation, ileal ace mrna expression was significantly decreased compared to either uninflamed biopsies from ibd patients or non-ibd controls. in contrast, in the rectum ace mrna expression was increased with inflammation when compared to the uninflamed ibd patients or non-ibd controls (figure a, upper panels) . there were no significant differences by disease location or between patients with uc versus cd ( figure s ) . we further validated these results using the pediatric ibd risk cohort where ace mrna was significantly decreased in the ileum of patients with active ibd as well (figure b) . the expression of tmprss in the msccr cohort was moderately higher in the colon compared to ileum. in both ileum and colon biopsies tmprss expression was found significantly increased in the inflamed relative to non-inflamed samples, although the effect sizes were small (figure a (table s ) , we could not appreciate differences in ace expression in the ileum, which possibly stemmed from the elevated physiological expression of ace . in the colon, patchy epithelial ace expression from control non-ibd controls increased in ibd patients with inflammation and this increase was mostly evident on the crypt epithelium ( figure s a ). in the ileum, low intensity tmprss expression on the crypt epithelium was also comparable in inflamed or uninflamed mucosal segments from ibd patients and in non-inflamed mucosal segments from non-ibd controls. in the colon, tmprss expression appeared comparable in ibd patients and non-ibd controls. in the rectum, tmprss expression was enhanced by inflammation ( figure s a ). age and gender but not smoking increases ace mrna in the colon ace mrna was higher with age in the uninflamed rectum samples. however, these effects were essentially nullified in the presence of inflammation. a positive association with age in inflamed cd ileum and a negative association in inflamed uc rectum biopsies was observed. ace mrna in the uninflamed rectum was significantly lower in males versus females but no gender associations in tmprss mrna levels were found (figure s b-c) . the expression of ace and tmprss was similar when comparing active smokers to non-smokers, either between healthy controls or ibd patients (data not shown) and no significant interactions with inflammation status, region or other covariates were found. thus, age and gender, but not smoking modulates ace but not tmprss mrna expression in the ibd colon. we further evaluated the impact of non-biologic and biologic medication use (self-reported) on the expression of ace and tmprss mrna (table s ) in the ileum of ibd (cd) patients, corticosteroid, thiopurine or -aminosalicylate use had no impact on ace mrna expression in either inflamed or uninflamed biopsies (figure a-c) . in the rectum, however, a significant decrease in ace mrna expression was observed with corticosteroid use in inflamed biopsies. a similar decrease of ace mrna was noticed in thiopurine-treated non-inflamed samples from the rectum. the use of corticosteroids, thiopurine or -aminosalicylate did not significantly affect tmprss mrna expression in ileum samples. however, each of these three medications significantly decreased tmprss mrna expression in inflamed rectum or colon samples. thus, corticosteroid, thiopurine or -aminosalicylate attenuated ace and tmprss mrna expression in inflamed colon and rectum. we also defined the effect of current anti-tnf therapy (either adalimumab or infliximab) use on ace and tmprss (figure d ) expression. in the ileum, patients taking anti-tnf biologics did not show significantly different ace or tmprss mrna expression compared to those not on anti-tnf medication. in the large intestine, anti-tnf users showed increased ace and tmprss expression, particularly in the inflamed rectum. since the use cross-sectional cohort to study treatment effect has its limitations, we utilized published datasets from clinical trial cohorts, where longitudinal patient sampling was performed and information on endoscopic responses to treatment was available. the results from a patient cohort treated with infliximab or vedolizumab on the gemini lts trial are summarized (figure a and s ). compared to baseline ace expression, post-infliximab (week ) colonic ace gene expression was significantly lower and this decrease was observed predominantly in endoscopic responders ( figure s ). in contrast, post-vedolizumab (week ) ace gene expression did not significantly change, although a nominally significant decrease was observed in endoscopic responders (figure a and s ) . finally, neither vedolizumab nor infliximab modified tmprss expression. to study the impact of il- /il- -targeting ustekinumab on ace and tmprss mrna levels in ibd, we used the certifi trial cohort , . we first confirmed that, as in our msccr cohort, ace gene expression was higher in uninflamed ileum compared to the colon regions ( figure s a ). next, we observed that ileal ace gene expression was significantly decreased with inflammation as was observed in the ileal samples from both msccr and risk cohorts ( figure s a ). in colonic and rectal samples, a trend to increase ace expression in inflamed biopsies as compared with non-inflamed was observed ( figure s a ). to identify potential functions associated with ace and tmprss in the gut, we studied these genes in the context of bayesian gene regulatory networks (bgrn). these probabilistic graphical models consider all available trait data (gene expression and genotype) simultaneously in order to derive gene:gene casual relationships amongst thousands of intermediate molecular traits . the nearest neighbors of ace were extracted including genes within either to path lengths in each bgrn network ( networks total) keeping the subnetwork sizes relatively similar (~ - genes, figure a , d, table s ). immediate neighbors of ace in the ileum cd network included slc a (a known interacting partner of ace ), and other slc transporters but also other viralassociated receptor proteins, like dpp and ezr . a summary of the recurring genes across subnetworks is shown (figure b, e) . functional enrichment of ace -associated subnetwork was interrogated in the ileum (figure c) or colon (figure f ) using the reactome database. identified metabolic pathways included slc-mediated transport; xenobiotic metabolism; vitamins and cofactors; and hexose transport. additional ileumassociated pathways included amino acid and oligopeptide slc transporters, while colon-associated networks included interferon and immune signaling. to support our network approaches we verified a significant overlap was observed between the colonic ace -associated subnetworks and genes reported to be correlated with ace expression in colonocytes ( figure s a ). in addition, ace -subnetworks were significantly enriched in expression profiles associated with epithelial cell types, including enterocytes and absorptive cells , (figure s b c). colonic ace -associated subnetworks also co-enriched in immune cell types as well as macrophage gene signatures following various cytokine perturbations including ifnγ/β ( figure s d) . in summary our ace -subnetworks are a novel source of insight into the regulation and function of ace . to address the function of tmrpss , the subnetworks associated with tmprss were extracted from the bgrns (figure s a ) allowing three or four layers to obtain similar subnetworks sizes (~ - genes). tmprss was not found in the bgrn from the ileum of controls, likely due to a low expression variance, which is a filter used before bgrn network construction (table s ) (figure s c) . consistent with these functions, we observed enrichment of the tmprss subnetworks in genesets associated with enterocytes, goblet and secretory cells ( figure s a-b) . covid- is a multisystem disorder where innate and adaptive immune cells as well as non-immune cells likely play a role in disease pathogenesis. therefore, apart from alterations in the receptor expression, we investigated additional areas of overlap between covid- responsive pathways and pathways associated with ibd. as the first step, we examined a host molecular response signature generated following sars-cov- infection of a primary human lung epithelium (nhbe) cell and a transformed lung alveolar cell line (a ) . using gsva, we generated a per-sample score summarizing expression of either up or down-regulated covid- -responsive genes, and evaluated differences in these scores according to intestinal region, disease and inflammation status (figure a and s a-b) . as compared to uninflamed regions or non-ibd control subjects, an observation confirmed in the certifi cd cohort (figure s c-d) . we directly compared the genes associated with response to lung cell sars-cov infection and various ibd-centric genesets generated in our msccr cohort or ibd gwas genes. we observed that genes: up-regulated with inflammation, or positively associated with macroscopic or microscopic measures of disease, or associated with the risk of ibd, were significantly enriched with genes up-regulated by sars-cov infection of lung epithelial cells ( figure s e ). next, we examined for the congruence of covid- related peripheral blood gene responses and active ibd inflammation. we observed that genes up-regulated in the blood of covid- infected patients have significantly higher expression in the blood of ibd patients as compared to healthy control blood (figure b and figure s a ) as well as in patients with active ibd versus quiescent ibd (figure b and figure s b ). to further interrogate the molecular congruence between sars-cov- infection and ibd inflammatory responses, we generated nhbe-covid- associated subnetworks (nhbe-covid_subnet) and ibd inflammation associated subnetworks (ibd_inf_subnet) from three ibd bgrns (table s ) . we then determined the overlap between them, with the rationale that common gene membership implies similar molecular pathobiologies. we observed a significant overlap between ibd-inf and nhbe-covid_subnets across all three independent networks tested (figure c ). j o u r n a l p r e -p r o o f interestingly, the common genes ( in ileum cd, in colon uc and in colon cd networks) were significantly enriched in candidate ibd gwas genes (table s ) . to determine the underlying shared patho-biological mechanisms we evaluated enrichment of the three intersecting subnetworks against reactome pathways. interestingly, although subnetworks were generated by projecting a covid- response signature generated in an epithelial-based model the majority of the pathways and cell type enrichments were immune oriented with the most striking related to innate immune signaling via interferon and interleukin- (figure d, figure s , table s ). this is consistent with the fact that while sars-cov- infection initiates within the epithelium, covid- is a multisystem disorder where innate and adaptive immune cells as well as non-immune cells likely play a role in disease pathogenesis. our covid- associated gut subnetwork findings are consistent with a large body of recent data that describe a dramatic up-regulation of pro-inflammatory cytokines in patients with covid- including induction of ifn-stimulated genes . we confirmed these observations using the recent dataset where sars-cov- was shown to infect hsios (figure s a-b) . direct overlap of genesets from the sars-cov- infected hsios and various ibd-centric genesets showed significant gene enrichments, similar to those observed between the lung-covid- model and ibd (figure s c) . finally, subnetworks generated in the ileum cd using hsio-covid- responsive genesets (table s ) overlapped with ibd-inf associated subnetworks. the intersecting genes were similar to those found between ibd-inf and nhbe-covid -associated ileum cd subnetworks, containing many interferon-stimulated genes (figure s d-e) . next, as a data reduction approach, we evaluated each of the nhbe-covid- and ibd-inflammation molecular response subnetworks for key driver genes (kdgs). we then summarized the kdgs across the three bgrns and determined those shared by ibd inflammation and nhbe-covid- molecular responses ( figure s , table s ). the shared kdgs identified were interferon-stimulated genes such as irf , gbp / , parp / and several of these were replicated in bgrns from the risk and certifi cohort including cxcl , gbp , parp amongst others (data not shown). we also compared the intersections between covid- associated genes and those associated with three different murine models of intestinal injury or inflammation. we observed a significant overlap finally, we generated colonic or ileal gene subnetworks associated with response to infliximab or ustekinumab (table s ) and tested their enrichment in the tissue and ibd-type specific subnetworks and observed significant overlap between many of the genes associated with ibd therapy and either ibdinflammation (used as positive control) or covid-infection (figure c) indicative of commonality between covid- and response to ibd treatment. we also evaluated changes in the activity of the blood covid- response signature in the blood of certifi cd patients treated with ustekinumab and observed that covid- up-regulated genes significantly decreased after weeks of ustekinumab treatment (figure f) . altogether, through analyses of multiple genesets, we observed a significant overlap between covid- response genes and genes associated with ibd response. the objective of this study was to systematically determine the molecular intersections between covid- -associated inflammation, ibd and immunomodulatory drugs. our data provides mrna-and proteinlevel evidence of the regional distribution of ace and tmprss across different parts of the gi tract and the impact of the commonly used ibd medications on ace and tmprss expression in the inflamed and uninflamed intestines. additionally, our data highlight an overlap between covidresponsive pathways and pathways associated with ibd inflammation. these findings generate the possibility that some of the current and emerging therapies in ibd may be of benefit in patients with covid- . in exploring the intersections between covid- and ibd, we initially considered a) impact of inflammation on ace expression and tmprss expression in the intestines; b) impact of ibd medications on ace and tmprss expression. ace has a less appreciated raas-independent role in the intestine by promoting amino acid absorption. consistent with this function we observed high levels of ace expression in the small bowel brush border [ ] [ ] [ ] supportive of the role of ace in mucosal homeostasis . remarkably, the analyses of gene:regulatory networks empirically derived from the terminal ileum, showed that ace was co-regulated with slc a , an amino acid transporter that physically interacts with ace . the colon-derived ace subnetwork would also suggest, a potential metabolic role for ace in the colon including solute carrier dependent processes. while the physiological function of tmprss remains largely elusive, it has been linked to epithelial sodium channel (enac) regulation we have observed a reduction in ace expression in the inflamed ileum. since ace appears to be a brush-border enzyme, its reduction with inflammation in the ileum is consistent with loss of expression of other brush border enzymes during enteritis . that said, even during inflammation, the expression of ace remains significant in the small intestines. in contrast, inflammation associated with ibd enhances the expression of ace and tmprss in the rectum. overall, modulation of ace and tmprss by ibd-associated inflammation is complex and appears to be region-specific in the intestines. akin to the impact of inflammation on intestinal ace and tmprss expression, the effect of ibd medications was complex. corticosteroids, thiopurines or -aminosalicylates did not significantly affect tmprss mrna expression in the ileum. however, each of these three medications significantly decreased tmprss mrna expression in inflamed rectum or colon samples. additionally, the impact of ibd medications like tnf inhibitors may vary by the stage of treatment. one may speculate that in early stages of therapy, anti-tnf drugs, similar to anti-il- / agents, may increase the expression of ace in the intestines and potentially have a detrimental effect. however, these medications could have a beneficial effect in the long run due to their ability to reduce inflammation. importantly, while sars-cov- infection initiates with the viral attachment to ace and its cleavage by tmprss within the epithelial surfaces - , covid- is a multisystem disorder where innate and adaptive immune cells as well as non-immune cells likely play a role in disease pathogenesis. therefore, apart from alterations in the receptor expression, we investigated additional areas of overlap between covid- responsive pathways and pathways associated with ibd. a number of such intersections appeared. il , cxcl / / , pdpn, s a /a which were upregulated following sars-cov- infection of primary human lung epithelium (nhbe) were also significantly upregulated in the inflamed intestines of patients in our ibd cohort (msccr). similarly, a peripheral blood gene signature from covid- patients which included the upregulated genes, clec d, s a /a and fcar, were also significantly upregulated in the blood of msccr ibd patients (as compared to controls) and in patients with active ibd (as compared to patients with quiescent disease). additionally, a number of sars-cov- -associated genes were upregulated in murine models of intestinal injury with dss (dapp , pdpn, il rn, duox , il b , s a , cxcl , cxcl ) or tnbs (marcksl , ifitim , ifitim , c , agr , reg ) or adoptive t cell transfer colitis model (tap , marcksl , slp , parp , ifitim , mmp , il b, s a ) . these genes relate to a number of ibd-relevant pathways including those associated with inflammatory cytokine signaling (including il- , j o u r n a l p r e -p r o o f il- , ifn-g), chemokine signaling, but also with interferon-associated pathways, regulation of complement cascade, g protein coupled receptor (gpcr) signaling as well as collagen degradation. having observed significant molecular intersections between covid- -and ibd-associated pathways, we next examined the impact of biologic medications used in ibd therapeutics where pre-and post-treatment transcriptomic data was available. interestingly, our network analyses had identified a number of shared covid- and ibd-associated 'key driver genes', including cxcl , gbp , socs , parp and parp . importantly, we observed that these kdgs which were up-regulated with covid- and ibd inflammation, were all down-regulated following infliximab treatment. thus, the impact of ibd medications on attenuating some of the key inflammatory genes and pathways would be independent of their complex effects on the expression of ace and tmprss on enterocytes. given the unregulated inflammatory responses in patients with severe covid- , it has been argued that targeted use of anti-inflammatory medications be considered as a therapeutic option . this approach has been met with variable success. while the use of dexamethasone has provided a striking mortality benefit , the results of trials using anti-il- have been equivocal (nct ) and nct . an anti-tnf trial is currently underway in the uk (nct ). our data suggest that anti-il- / therapy could also be considered in the therapeutic armamentarium in patients with covid- . reassuringly, real-time data from a registry of ibd-covid patients did not report significant adverse outcomes associated with the use of biologic medications including anti-tnf and anti-il- / inhibitors. to the contrary, the risk of severe covid- was found to be reduced in ibd patients on anti-tnf inhibitor medication . this is consistent with the observation that individuals treated with cytokine inhibitors had lower rates of sars-cov- seroprevalence compared to controls. in summary, through detailed analyses of intestinal tissues in health and ibd, we conclude that high expression of ace and tmprss potentially supports local, gi-associated replication of sars-cov- . further, a number of overlapping inflammatory pathways between covid- and ibd are noted. these data support the use of specific anti-inflammatory agents in the treatment of patients with covid- . table s and s . under the model, we estimated the change in ace or tmprss gene expression between the medicated and non-medicated group according to disease subtype (cd, uc) region (ileum, colon, rectum) and tissue type (inflamed, non-inflamed). samples sizes are in table s . (table s for full results). (table s and s ). the three ileum-associated cd networks are shown for example. venn diagrams of the overlap in subnetwork genes including the fold enrichment (fe) and a p-value of the enrichment test are shown. using the bgrns drug response subnetworks were generated (see supplementary methods) for infliximab and ustekinumab response genes. the bar graph summarizes the fe for the overlaps between the drug response and either the i) covid- subnetworks; ii) ibd inflammation subnetworks or iii) the intersecting nodes between covid- and ibd-inf subnetworks. a full table of network nodes and enrichment results can be found in table s and s (d) the sets of genes in the intersecting subnetworks from c were interrogated for enrichment in the reactome database. heatmap depicting the fe in pathways (bh adj p< . and minimum -fold enrichment, table s ). anti-tnf inf n/a n/a n/a n/a certifi risk n/a n/a rectum ace tmprss inf inf non.i n/a n/a n/a n/a n/a n/a tissue location and in ammation state bend cct p cd chrna clcn egfr-as ensg espn fads gna haao hnrnpa p jdp lrrc a ms a muc nbpf nsmf nucks oit plb rhou serpina slc a slc a slc a slc a r tcn tmem tmem b acsl acy adgrg anpep ca cndp dgat entpd mep b mgam plekhs sat sec b slc a slc a tkfc tmem d. e. f. adj p value * = . - . , **= . - . , ***> . ace associated subnetworks ace associated subnetworks the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- covid- ) situation report - : world health organization aceh/ace is a novel mammalian metallocarboxypeptidase and a homologue of angiotensin-converting enzyme insensitive to ace inhibitors a novel angiotensin-converting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase ace -angiotensin-( - )-mas axis and oxidative stress in cardiovascular disease sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor the origin, transmission and clinical therapies on coronavirus disease (covid- ) outbreak -an update on the status evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission angiotensin receptor blockers as tentative sars-cov- therapeutics sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury differential downregulation of ace by the spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus nl crohn's disease ulcerative colitis management of ibd during the covid- outbreak: resetting clinical priorities are patients with inflammatory bowel disease at increased risk for covid- infection? journal of crohn's and colitis pediatric crohn disease patients exhibit specific ileal transcriptome and microbiome signature a functional genomics predictive network model identifies regulators of inflammatory bowel disease effect of vedolizumab (anti-α β -integrin) therapy on histological healing and mucosal gene expression in patients with uc cytoscape: a software environment for integrated models of biomolecular interaction networks transcriptomic similarities and differences in host response between sars-cov- and other viral infections imbalanced host response to sars-cov- drives development of covid- sars-cov- productively infects human gut enterocytes enrichr: a comprehensive gene set enrichment analysis web server update intra-and inter-cellular rewiring of the human colon during ulcerative colitis mucosal profiling of pediatric-onset colitis and ibd reveals common pathogenics and therapeutic pathways transcriptome-based network analysis reveals a spectrum model of human macrophage activation ace expression by colonic epithelial cells is associated with viral infection, immunity and energy metabolism ustekinumab induction and maintenance therapy in refractory crohn's disease an integrative genomics approach to infer causal associations between gene expression and disease tissue-specific amino acid transporter partners ace and collectrin differentially interact with hartnup mutations ezrin interacts with the sars coronavirus spike protein and restrains infection at the entry stage op a molecular measure of inflammation in ibd patients based on transcriptional profiles from intestinal biopsies clinical features of patients infected with novel coronavirus in wuhan conserved transcriptomic profile between mouse and human colitis allows unsupervised patient stratification temporal colonic gene expression profiling in the recurrent colitis model identifies early and chronic inflammatory processes temporal genome expression profile analysis during tcell-mediated colitis: identification of novel targets and pathways tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis ace links amino acid malnutrition to microbial ecology and intestinal inflammation distribution of brush-border membrane peptidases along the rat intestine a protein complex in the brush-border membrane explains a hartnup disorder allele regulation of the epithelial na+ channel and airway surface liquid volume by serine proteases tmprss , a serine protease expressed in the prostate on the apical surface of luminal epithelial cells and released into semen in prostasomes, is misregulated in prostate cancer cells endothelial jam-a promotes reovirus viremia and bloodstream dissemination the ubiquitin e ligase trim promotes aggregation and activation of the signaling adaptor mavs through lys -linked polyubiquitination irtks negatively regulates antiviral immunity through pcbp sumoylation-mediated mavs degradation early biochemical responses of the small intestine of coeliac patients to wheat gluten trials of anti-tumour necrosis factor therapy for covid- are urgently needed dexamethasone in hospitalized patients with covid- -preliminary report secure-ibd database public data update patients with immune-mediated inflammatory diseases receiving cytokine inhibitors have low prevalence of sars-cov- seroconversion key: cord- -srzysoqy authors: segal, jonathan p.; mullish, benjamin h.; quraishi, mohammed n.; iqbal, tariq; marchesi, julian r.; sokol, harry title: mechanisms underpinning the efficacy of faecal microbiota transplantation in treating gastrointestinal disease date: - - journal: therap adv gastroenterol doi: . / sha: doc_id: cord_uid: srzysoqy faecal microbiota transplantation (fmt) is currently a recommended therapy for recurrent/refractory clostridioides difficile infection (cdi). the success of fmt for cdi has led to interest in its therapeutic potential in many other disorders. the mechanisms that underpin the efficacy of fmt are not fully understood. importantly, fmt remains a crucial treatment in managing cdi and understanding the mechanisms that underpin its success will be critical to improve its clinical efficacy, safety and usability. furthermore, a deeper understanding of this may allow us to expose fmt’s full potential as a therapeutic tool for other disease states. this review will explore the current understanding of the mechanisms underlying the efficacy of fmt across a variety of diseases. faecal microbiota transplantation (fmt) is currently a recommended therapy for recurrent/ refractory clostridioides difficile infection (cdi). [ ] [ ] [ ] [ ] it is also being explored in the research setting for many other indications. however, there are a number of associated concerns regarding its use, including the unpleasant prospect of the procedure, the potential need for invasive administration, the small, but recognised risk of transmission of infection, and the complex regulation associated with its use. the covid- pandemic and potential risk of viral transmission through donor stool samples has brought its limitations to the fore. as such, from a therapeutic perspective, understanding the mechanisms that underpin the efficacy of fmt may enable us to refine fmt from its current relatively crude state to a more refined 'microbiome therapeutic', which is no longer fmt, but could have a greater overall safety profile. this review will explore the current understanding of the mechanisms that underpin the efficacy of fmt across a variety of diseases. there has been a wealth of evidence demonstrating that fmt for cdi is effective for recurrent and refractory cdi, and the treatment has therefore been adopted in national and international guidelines. [ ] [ ] [ ] a meta-analysis of all these studies highlights clinical resolution in % ( % ci - %) of cases. the success of fmt for cdi has led to interest in its therapeutic potential in many other disorders, , but a report on these is beyond the scope of this review. in cdi, the suppression of the native gut microbiome, often by antibiotic treatment, enables c. difficile spores to germinate into vegetative cells, which produce enterotoxins that cause inflammation and result in debilitating diarrhoeal symptoms. the key rationale for using fmt as treatment for cdi is that this therapy restores the gut microbial communities. indeed, the 'healthy commensals' reintroduced through fmt will compete for the ecological niches and prevent colonisation by pathogens, a well-described phenomenon known as 'colonisation resistance'. the role of the gut microbiota as a factor in the pathogenesis of many conditions including inflammatory bowel disease (ibd), metabolic syndrome and subgroups of patients with irritable bowel syndrome (ibs) is accepted. however, the relative importance of the gut microbiota in the overall pathogenesis is different from one disease to another and we cannot yet quantify it for many diseases. for example, it has been noted that in cdi, changes in the composition of the gut microbiota represents the predominant factor in cdi pathogenesis and, in ibd, it plays a very important role. for many other conditions, its role might be more limited compared with other factors. furthermore, other than for cdi, mechanistic studies are largely lacking, and it remains overall unclear whether these microbiota changes play a role significant enough to be efficiently targeted by fmt or other microbiome-based intervention. more recent data have shown that the efficacy of fmt in the treatment of recurrent cdi (rcdi) may not be explained by purely restoration of gut bacteria per se, but also by a number of additional factors. for instance, in one pilot study, researchers prepared a sterile faecal filtrate by passing fmt slurry through progressively narrower pore filters, culminating in a . µm pore filter. the administration of the sterile faecal filtrate via a nasojejunal tube was effective in treating five patients with rcdi (> months), comparable with that degree of efficacy seen after administration of conventional fmt. the authors concluded that, rather than fmt directly requiring live, intact bacteria for its efficacy, it was instead likely that one or more soluble factors associated with bacteria within the filtrate potentially mediated its mechanism of action. within the following sections, the potential contributions of such factors are discussed. bacteriophages are viruses that target and replicate within bacteria or archea. importantly, phage exposure can alter both the virulence and biofilm of its host. from fmt/cdi studies, it has been shown that abundance of the order of bacteriophages named caudovirales reduced significantly in stool after fmt, with fmt success more likely if donors had a higher fraction of caudovirales within their stool virome. following fmt for rcdi, a recipient's core virome quickly resembled that of a donor and remained stable over at least the next -month period and even up to months. , in terms of other diseases, there are controversial data regarding phages, but successful fmt for ibd was associated with low eukaryotic viral richness in recipients before fmt. further supporting the role of the virome was a recent mouse study that transferred lean faecal virome into mice fed with high-fat diet. the virome transfer led to reduced weight gain and normalised blood-glucose relative-control mice. the authors concluded that the faecal virome exerts it effects via changes in the gut microbiota. importantly, eukaryotic viruses can be found in food and hence diet could be a confounding factor. however, it is likely that the gut virome plays a significant part by its interaction with the other components of the gut microbiota, but from a mechanistic perspective there are limited data to explain how bacteriophages and the virome contribute to a successful fmt and at present, we are limited to associative studies, and hence studies that infer causation are needed. it is likely that bacteriophages can alter their bacterial hosts indirectly by reprogramming their metabolism, to include transfer of phage genes that encode for antibiotic resistance and alterations in pathogen virulence. it is therefore likely that the enteric virome may contribute to some of the mechanisms that underpin the success of fmt, but this requires further exploration. from a fungal perspective, it has been suggested that patients with cdi who responded to fmt experienced colonisation with particular donorderived fungal taxa (in particular, members of saccharomyces and aspergillus genera), whereas non-response was associated with a dominant presence of candida within donor stool. individuals not responding to fmt and/or patients treated for rcdi with antimicrobials alone retained overgrowth of candida. in a mouse model of cdi, the presence of candida albicans was associated with reduced efficacy of fmt, while use of antifungal therapy helped restore efficacy. utilising internal transcribed spacer (its ) sequencing, it was demonstrated that the fungal microbiota is skewed in ibd, with an increased basidiomycota/ascomycota ratio, a decreased proportion of saccharomyces cerevisiae and an increased proportion of c. albicans compared with healthy controls. in samples from a large randomised controlled study utilising fmt for ulcerative colitis (uc) it was found that high candida abundance pre-fmt was associated with a clinical response, whereas decreased candida abundance post-fmt was indicative of ameliorated disease severity. the authors suggested that high candida abundance in the recipient might promote the engraftment of donor's bacteria by freeing ecological niches, and that fmt may reduce candida which culminates in the success of fmt. these potential mechanisms need further exploration while acknowledging caveats associated with fungal infections in the presence of immunosuppression. importantly, trans-kingdom-fungi-bacteria interactions have good evidence in many ecosystems and are beginning to be further understood in the gut. while the perturbation of gut fungal profiles and their influence upon fmt outcomes are of interest, their significance as potentially contributing to the efficacy of fmt remains unclear. in view of the established relationship between antimicrobial treatment and overgrowth of candida within the gut, any changes in gut mycobiota profiles may only possibly be proxies of gut bacterial alterations. as such, the specific contribution of bacteriophages and fungi to the efficacy of fmt remains undefined. metabonomics is defined as 'the quantitative measurement over time of the metabolic responses of an individual or population to drug treatment or other intervention'; this differs from metabolomics, that explore the metabolic responses present in the whole cell or tissue. metabonomics, therefore, explore responses of an individual or community, whereas metabolomics explore responses in a cell, or bacterial population. metabonomics utilise integrated-systems biology to provide a way of investigating the metabolic status of an organism or ecosystem by studying 'real' metabolic endpoints. the contribution of gut microbiota-derived metabolites, or 'cometabolites' produced through the interaction between the microbiota and host, has also been a key area of interest in the study of mechanisms of fmt. short-chain fatty acids. one particular group of metabolites that have been well studied in this field include short-chain fatty acids (scfas), which are the products of bacterial fermentation of partially digestible and non-digestible dietary carbohydrates and amino acids. mice treated with broad-spectrum antibiotics experienced a marked reduction in levels in scfas in stool, and higher scfa levels correlated with protection from c. difficile growth, suggesting an interaction between antibiotics, scfas, and cdi risk. more recent work used a bioreactor/chemostat model of cdi to demonstrate that cessation of broad-spectrum antibiotics was associated with spontaneous recovery of the microbial synthetic recovery of most scfas, but not that of valerate, the five-carbon scfa. in vitro, valerate caused a dose-dependent inhibition of the growth of a range of c. difficile ribotypes, without any adverse effects against any commensal bacteria. furthermore, in a mouse model of cdi, oral gavage of glycerol trivalerate was demonstrated to cause a rapid reduction in c. difficile colony-forming units detectable within stool. further experiments demonstrated that successful fmt for rcdi in humans was associated with the rapid, sustained restoration of stool valerate levels. beyond the dominant scfas of acetate, butyrate and propionate, these data support a specific role of valerate recovery in the success of fmt for rcdi. , furthermore, scfas seem to be critical in driving intestinal homeostasis through immunometabolic pathways in ibd. scfas, specifically butyrate, have been shown to promote regulatory t-cell response in murine models of ibd. gut microbiota analysis of fmt-treated mice showed significant increases of commensals, including members of lactobacillaceae and streptococcus along with of the scfa-producing taxa erysipelotrichaceae and ruminococcaceae. administration of fmt is associated with enrichment of specific clostridium clusters that include the scfa-producing families ruminococcaceae and lachnospiraceae and genus roseburia in clinical studies. taken together, these findings suggest that restoration of gut microbial scfa producers through fmt may drive regulatory immunological responses and homeostatic balance in ibd. bile acids. a further group of metabolites of particular interest in the field of fmt/cdi is the bile acids. initial experiments in vitro over years ago demonstrated the differential effects of different classes of bile acids upon c. difficile. specifically, primary bile acids have a 'pro-c. difficile' effect, primarily through the promotion of spore germination; in particular, the conjugated bile acid taurocholic acid (tca) strongly promotes c. difficile germination in vitro in the presence of glycine as co-germinant. , conversely, the secondary bile acids (including deoxycholic and lithocholic acid) have a net 'anti-c. difficile' effect, particularly through the inhibition of vegetative growth and toxin activity of the bacterium. , the transition from primary to secondary bile acids within the gut occurs through the activity of enzymes produced by the gut microbiota (in particular, the enzymes bile salt hydrolase (bsh) and -α-dehydroxylase). rodent studies supported the concept that restoration of bacterial bile-metabolising capacity to the gut microbiota was protective against cdi, prompting interest into whether this could also be a mechanism of efficacy of fmt. in this context, a range of in vitro, rodent and human studies have collectively demonstrated that while the pre-fmt stool bile-acid milieu is enriched with primary bile acids (and particularly tca), the post-fmt stool bile-acid pool is much more comparable with that of healthy donors, with high levels of secondary bile acids. , , , more recent work has directly demonstrated that successful fmt in those with rcdi results in maintained restoration of microbial bsh functionality to the gut microbiota, and that restoration of bsh in a mouse model of cdi is sufficient to significantly reduce c. difficile counts within stool. further research in this area has demonstrated that successful fmt for cdi is associated with an increase in circulating fibroblast growth factor (fgf)- and reduction in fgf- , consistent with upregulation of the bile-acid receptor farnesoid x receptor (fxr)-fgf pathway. an additional surprising finding of interest has been the recent demonstration that bacteria with -α-dehydroxylase bile-metabolising activity (including clostridium scindens) are also able to produce tryptophanderived antibiotics which inhibit the cell division of c. difficile. while evaluation of the effect of fmt for rcdi upon scfas and bile-acid metabolism has focused on their direct effects upon the life cycle of c. difficile, it is possible that this may also have other beneficial effects. for instance, fmtmediated changes in bile-acid-fxr interactions may directly impact upon the colitis caused by c. difficile; administration of an fxr agonist in a mouse model of colitis resulted in significantly reduced colonic inflammation and a more intact intestinal barrier, while microbially mediated production of particular secondary bile acids exhibit anti-inflammatory effects on intestinal epithelial cells and have been recently recognised as promoting generation of peripheral regulatory t cells. scfas have also been demonstrated as able to regulate the size and function of the colonic regulatory t-cell population, which was directly shown to be a protective mechanism against the development of colitis in mice. other metabolites. a further related area of interest relates to the ability of c. difficile to 'scavenge' for metabolites within the antibiotic-treated gut as energy sources to facilitate growth. in particular, after antibiotic treatment, the loss of bacteria that compete with c. difficile for metabolites including the amino acid proline, the organic acid succinate, the monosaccharide sialic acid (derived from intestinal mucus) and dietary trehlose allows c. difficile to scavenge these metabolites unopposed, and exploit them for its growth and division. as such, it may be hypothesised that a further mechanism by which fmt functions is by restoring microbial competition within the gut, and therefore minimising an ecological niche that c. difficile deploys to derive energy sources. metabonomics have also been applied to explore mechanisms underlying the efficacy of fmt in treating uc. an experimental model in rodents found that fmt given from dextran sulfate sodium-induced uc rats to healthy rats induced uc-like changes. it was also found that fmt from healthy rats to colitic rats induced remission. when exploring the metabonomic changes associated with this remission, it was observed that urinary hippuric acid was significantly reduced in the uc group compared with normal rats. specifically, it was noted that there were increases in c : acylcarnitine, hydroxyphenylpropionylglycine, and riboflavin. in a second experiment, researchers transferred the microbiota from those rats with uc to untouched rats. it was noted that hippuric acid decreased in the normal rats but was restored to normal levels at day and , and further found that the changes induced by fmt correlated with the genera oscillospira and dehalobacterium and the families bacillaceae and exiguobacteraceae. importantly it has been shown that hippuric acid is reduced in cd and uc due to gut microbial metabolism; this therefore suggests that fmt can alter the gut microbiome to change the metabolic drivers of disease states. further supporting this concept was a study on pigs, where it was noted that fmt resulted in significant increases of the typical microbiotaderived tryptophan catabolite indole- -acetic acid in the colonic lumen, suggesting that tryptophan metabolites may be important actors in the efficacy of fmt. in a human study, exploring fmt for children with uc, responders to fmt highlighted that bacilli and betaproteobacteria were positively correlated with metabolites from the 'disease-associated' cluster (such as creatinine and norvaline), and clostridia were positively correlated with metabolites from the 'healthy' cluster (such as xanthine and -hexadecanol). there has been one randomised controlled trial (rct) in uc that measured metabolites following fmt using metabonomic and shotgun metagenomics. they noted that specific bacterial functional pathways were associated with a positive outcome, including: benzoate degradation, glycerophospholipid metabolism, secondary bile-acid biosynthesis, guanosine pentatetra-phosphate biosynthesis, pyruvate fermentation to acetate and lactate, biosynthesis of ansamycins, and starch degradation. furthermore, it was found that these pathways were correlated with the abundance of eubact erium, ruminoccus, lachno spiraceae, roseburia, dorea and coprococcus taxa. linking metabolic function to specific bacteria is likely to provide key mechanistic insights into the active components in fmt and potentially help refine fmt. fmt metabolites and autophagy. autophagy is a crucial housekeeping process in cellular function that removes and recycles dysfunctional components such as misfolded proteins or damaged organelles. this process is particularly active and important for the function of proliferating cells such as intestinal epithelial cells. it has been noted that fmt could trigger intestinal mucosal autophagy and alleviate gut-barrier injury caused by specific bacteria such as escherichia coli. specifically, it was noted that metabolites, such as lactic acid and succinic acid, were enhanced and upregulated in piglets, following fmt. these upregulations were then responsible for changes in metabolic pathways such as linoleic acid metabolism, which culminated in a decrease in intestinal permeability and enhancement of mucins and mucosal expression of tight junction proteins in the recipient. it is therefore possible that fmt alters autophagy through its influence on the gut microbiomes metabolic pathways. through a complex and bidirectional relationship, the gut microbiome plays a critical role in shaping the gut mucosal immune response. our initial insight of how fmt impacted the immune system was from cdi-fmt studies. in a dextran sodium sulfate (dss)-induced colitis mouse model, it was noted that response to fmt was associated with activation of a variety of immunemediated pathways which ultimately lead to interleukin (il- ) production by innate and adaptive immune cells. these included cd + t cells, invariant natural killer t (inkt) cells and antigen-presenting cells. furthermore, it was demonstrated that fmt reduces the ability of dendritic cells, monocytes and macrophages to present major histocompatibility complex class-ii-dependent bacterial antigens to colonic t cells. it has also been shown that patients with recurrent cdi who responded to fmt had a reduction in complexity serum n-glycosylation profiles. glycans are associated with epigenetic modification that affects multiple immunological pathways and enable cross talk between gut bacteria/ pathogens and host epithelial cells. the relevance of this molecular mechanism in relation to response to fmt deserves further exploration. a breakdown in the innate and adaptive immune mechanisms appears to be fundamental in the development of chronic immune-mediated diseases such as ibd. there is now increasing evidence to suggest that the gut microbial perturbations observed in these diseases contribute to (or possibly even trigger) this homeostatic immunological imbalance. therapeutic advances in gastroenterology journals.sagepub.com/home/tag transfer of gut microbiota from patients with ibd into germ-free mice has been shown to significantly increase the numbers of pro-inflammatory intestinal t-helper (th ) cells and while reducing regulatory rorγt+ t-regulatory-cell (treg) populations when compared with gut microbiota from healthy individuals. moreover, microbiota from patients with ibd exacerbate colitis in an immunological mouse model of ibd with correlations observed between proportions of th and rorγt+ treg cells and patient inflammatory status. the majority of mechanistic work incorporated into the five rcts in ibd , - focused on shifts in gut bacterial and metabolomic profiles, with only one exploring immunological effects of fmt on disease response. this study did not find any significant change in proportions of γδ t cells, nk cells and t-cell subsets in colonic lamina propria immune cells. they did, however, observe a slight increase in peripheral blood mononuclear gut-homing cd t-cell populations following fmt when adjusted for clinical disease-activity scores (p = . ). it was unclear if responders to fmt had specific shifts in immune subsets compared with non-responders. our group (quraishi and iqbal) recently evaluated the host mechanistic response to fmt in patients with active uc as part of the pilot phase of the stop-colitis trial. , in the patients enrolled into this mechanistic arm, a clinical response was seen in eight patients following fmt. the responders had a significant reduction in mucosal th cells along with a significant increase in regulatory t cells, effector-memory tregs and gut-homing tregs. furthermore, we observed a significant increase in il- -producing cd cells and reduction in il- -producing cd -cell and cd -cell populations in responders, following fmt. colonic mucosal transcriptome analysis demonstrated that clinical response to fmt was associated with significant downregulation of host antimicrobial defence response, antimicrobial peptides and pro-inflammatory immune pathways. there was a significant upregulation of butyrate and propionate metabolic pathways in fmt responders. a study in two patients with immune-checkpoint inhibitor colitis observed that immunological response after fmt was associated with an increase in foxp + cd cells along with a substantial reduction in the colonic mucosal cd + t-cell population. there was a concomitant expansion in the population of bifidobacterium species, clostridia and blautia. treatment of mice with bifidobacterium has been shown to ameliorate dss-induced colitis following immunecheckpoint blockade. this protective effect was, however, abrogated in treg-depleted mice. collectively, these findings indicate an emerging role of fmt and specific agents in the gut microbiota in mitigating inflammation via induction or modulation of treg function. furthermore, in a rodent study that inoculated -week old neonatal mice with faeces from clostridium-associated mice, it was demonstrated that there was a significant increase in clostridium clusters iv and xiva in the treated mice accompanied by a significantly higher number of colonic foxp + tregs, highlighting the potential interactions between the microbiome and the local/systemic immunity. in a follow-up study exploring this concept, researchers inoculated germ-free mice with either treated or untreated chloroform human stool and noted a significant increase in the percentage of foxp + tregs among cd + t cells in the colons of germ mice inoculated with untreated human faeces compared with germ-free mice. when applied to those with ibd, a study that used colonic lamina propria lymphocytes (lpls) and peripheral blood lymphocytes (pbls) from healthy individuals and those with colon cancer and ibd, demonstrated that dp α t cells exhibited a highly skewed repertoire toward the recognition of faecalibacterium prausnitzii, which is decreased in patients with ibd. they further demonstrated that the frequencies of dp α pbl and colonic lpl were lower in patients with ibd than in healthy donors and in the healthy mucosa of patients with colon cancer, respectively. these data together suggest that clostridium species are key regulators of inflammation through their influence on the gut immune system. a further study which stimulated cells known to respond to f. prausnitzii measured their production of il- and their downstream cell activity. they demonstrated that the proportion of circulating ccr +/ cxcr + dp α t cells was significantly reduced (p < . ) within the total population of cd + t cells from patients with ibd compared with patients with infectious colitis or controls. summarising these findings suggests that components of the gut microbiome are key regulators of immune function and significantly impact the mechanisms that underpin gut homeostasis, and therefore, fmt may work by promotion of some of the gut homeostatic immune functions and downregulating the pro-inflammatory immune responses. when considering specific metabolites, it has been demonstrated that scfas, specifically butyrate, have been shown to induce tregs and promote anti-inflammatory il- production in mice. , it is likely that introduction or enrichment of specific gut microbial species via fmt attenuates inflammation by promoting treg proliferation in the colonic mucosa through products of bacterial metabolism including scfas, tryptophan and polysaccharide. [ ] [ ] [ ] when exploring tryptophan specifically, it has been demonstrated that the transfer of microbiota from card mice into wild-type germ-free mice increases their susceptibility to colitis. the mechanism that appears to underpin this is the card susceptibility gene alters the gut metabolism of tryptophan into aryl hydrocarbon receptor ligands, leading to inflammation. importantly, this phenomenon was ameliorated by inoculation of mice with lactobacillus strains capable of metabolizing tryptophan, suggesting a key link between genetics and microbiota. small-bowel microbiota importantly, the majority of published studies focus on the colonic microbiota, as assessed by stool (or, in some cases, by mucosal biopsies). the small bowel also harbours a complex microbial community, albeit with less diversity and abundance (≈ - microbial cells/g) than the colonic microbiota (≈ cells/g)). , its influence on the mechanisms and effect of fmt is currently poorly understood. importantly, fmt is known to have comparable efficacy in cdi when infused into the upper gastrointestinal tract. specifically, when considering ibs, small-bowel microbiota alterations have been associated with symptoms, and hence future studies will need to explore the role of the small-bowel microbiota in efficacy of fmt. data from non-cdi fmt studies such as in ibd have demonstrated that some recipients of fmt have an exceptional response while others do not. , it is therefore possible that there are factors associated with both the recipient and the donor that may underpin the success of fmt. when considering donor factors, it is likely that particular components are driving the therapeutic effect from fmt and hence analysing the donor stool remains an important element in understanding the mechanisms underpinning the efficacy of fmt. studies have speculated about what makes a 'super donor'. the origins of a putative 'super donor' effect were from an fmt-uc study in where 'donor b' induced significant more remission than other donors. this therapeutic effect was associated with significant increases for the family lachnospiraceae and the genus ruminococcus in 'donor b' microbiota. such evidence lead researchers to conclude that a donor's microbiota diversity may have an influential effect on the success of fmt in ibd. , furthermore, specific taxa have been associated with disease response such as, clostridium clusters iv and xiva , and ruminococcaceae and lachnospiraceae families. specific bacteriaproducing scfas such as butyrate are also suggested to be important in the efficacy of fmt. furthermore, as previously stated, a meta-analysis exploring the role of fmt for ibs demonstrated that fmt had no effect in ibs, following this, however, an rct using a single 'super donor' showed a high success at reducing ibs symptoms, suggesting that the stool donor may have significant effects on the efficacy of fmt. importantly, when considering cdi, most patients respond to fmt, which suggests that disease-specific factors may be driving the success rather than the donation itself. in view of this, studies exploring donor-specific factors associated with an unsuccessful response may provide valuable insight into mechanisms that underpin fmt success. another major consideration is that diet plays a large influence on the gut microbiota and hence is likely to affect the donation and fmt efficacy. uncovering the dietary aspects that may influence the efficacy of fmt will be an important consideration. lastly, it is plausible that specific constituents in an fmt will provide benefit for one person but not another. it is therefore possible that a 'one size fits all' fmt might be replaced by a more personalised fmt as our knowledge improves regarding mechanisms that underpin a successful donor. another important consideration is studies have not been powered to date to understand the donor characteristics associated with success. despite these findings, however, the mechanisms underpinning the 'super donor' phenomenon are yet to be detailed. an important consideration is to determine what part of the fmt engrafts into the host and may be a significant factor that underpins efficacy. currently, there is no robust definition of engraftment. a significant consideration is to understand if fmt promotes growth of suppressed host microbiome constituents or introduces new constituents into the host. specific strain tracking may help understand this and studies are attempting to further define this. , post-fmt era given this central importance of the restoration of the gut microbiota to the efficacy of fmt in the treatment of cdi, there have been a number of different approaches towards a more defined 'narrow spectrum' microbiota product of wellcharacterised bacteria as an alternative to fmt. proof-of-concept use of 'defined bacterial communities' as an alternative cdi treatment has been demonstrated in bioreactor and rodent models, , as well as human studies. for instance, as early as , bacteriotherapy was discussed in the treatment of cdi. more recently, in the 'repoopulate' study, different commensal bacterial species were cultured from the stool of healthy donors; these were used to synthesise a 'stool-substitution therapy', consisting of a mixture of purified bacterial cultures derived from these stool bacteria. after colonoscopic administration of this mixture to two patients with rcdi, both patients achieved a rapid and sustained remission. as an alternative approach, healthy donor stool was ethanol treated (to kill vegetative cells); the surviving spores were fractionated and capsulised, and delivered orally as a preparation named ser- . in a cohort of patients, achieved clinical remission from rcdi after one or two administrations of ser- . however, despite early promise, ser- produced negative results when administered in a phase ii clinical trial, with potential issues related to the differentiation of true cdi recurrence from post-cdi ibs, and the dosing of the treatment regimen. , this concept has been further expanded into other disease areas with a consortium of microorganisms being explored for treatment of mild-to-moderate uc in a phase ii study. live biotherapeutics refer to live microorganisms that are used to prevent or cure human disease. the concept relies on specific microbes causing a beneficial effect to the host. these can be isolated from the gut microbiota of healthy people or engineered microbiomes. as previously mentioned, these have been studied for diseases such as cdi and ibd but have shown promise in other disease areas. specifically, they have shown promise in the treatment of cancer, with one study highlighting that a commensal of healthy human-associated bacterial strains can induce interferon γ+ cd t cells that confer resistance to the intracellular pathogen listeria monocytogenes, and inhibit tumour growth in conjunction with immune-checkpoint inhibitors. in another study, human-derived clostridium strains (ve ) were able to reverse histological colitis in a mice model. there are many commercial companies aiming to find biotherapeutics for a whole range of diseases. as we learn more about the mechanisms that underpin the efficacy in fmt, it is likely that these will feature in more clinical trials. importantly, any engineered microbiota-based therapies will need to be examined in clinical trials to assess if they have clinical equipoise with, or are even superior to, fmt. phage therapy refers to the therapeutic use of viruses that infect bacteria, bacteriophages, to treat disease. phage therapy aims to specifically kill their respective bacterial host while preserving other microorganisms and human cells. this has been a growing area of interest in view of the rising incidence of antibiotic resistance. phage therapies in clinical practice are very much still in the research stage with concerns over regulation and safety. in an in vitro human model study, phage øcd showed significant reduction in c. difficile cell numbers and toxin production without major effects on other members of the microbiota. as previously demonstrated, the virome plays a significant role in the efficacy of fmt and hence further exploration into phage therapy may help us understand the mechanisms that underpin fmt efficacy (figure ). as highlighted in this review, much of our current understanding of mechanistic insights into the the authors declare that there is no conflict of interest. the authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: the division of digestive diseases at imperial college london receives financial support from the national institute of health research (nihr) imperial biomedical research centre based at imperial college healthcare nhs trust and imperial college london. bhm is the recipient of an nihr academic clinical lectureship. systematic review with 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difficile and toxin production in an in vitro human colon model system key: cord- -hn airxq authors: bayoumy, ahmed b.; van liere, elsa l. s. a.; simsek, melek; warner, ben; loganayagam, aathavan; sanderson, jeremy d.; anderson, simon; nolan, jonathan; de boer, nanne k.; mulder, chris j. j.; ansari, azhar title: efficacy, safety and drug survival of thioguanine as maintenance treatment for inflammatory bowel disease: a retrospective multi-centre study in the united kingdom date: - - journal: bmc gastroenterol doi: . /s - - - sha: doc_id: cord_uid: hn airxq background: thioguanine (tg) is a thiopurine which has been used for patients with inflammatory bowel disease (ibd), who have failed azathioprine (aza) or mercaptopurine (mp) due to adverse events or suboptimal response. its widespread use has been hampered due to concerns about nodular regenerative hyperplasia (nrh) of the liver. the aim of this study was to investigate the long-term efficacy and safety of low-dose tg therapy in ibd patients failing aza and mp. methods: a retrospective multicentre study was performed in ibd patients who failed prior treatment with conventional thiopurines with or without following immunomodulation (thiopurine-allopurinol, biologicals, methotrexate, tacrolimus) and were subsequently treated with tg as rescue monotherapy between and at three hospitals in the united kingdom. clinical response, adverse events, laboratory results, imaging and liver biopsies were retrospectively collected. results: a total of patients ( % female and % crohn’s disease) were included, with a median daily tg dose of mg (range: – mg), a median treatment duration of months (iqr – ) and a median follow-up of months (iqr – ). the clinical response rate at months was and % remained on tg until the end of follow-up. adverse events consisted primarily of elevated liver tests ( %), myelotoxicity ( %) and rash ( %). nrh was histologically diagnosed in two patients and two other patients ( %) developed non-cirrhotic portal hypertension. the median -tgn and tpmt levels were pmol/ × ( ) rbc (iqr – ) and mu/l (iqr . – ). conclusions: long-term follow-up suggests that tg can be an effective and well-tolerated therapy in more than half of difficult-to-treat and multi-therapy failing ibd patients. findings of this study indicate that tg can be used safely and the occurrence of hepatotoxicity was low. the incidence rate of nrh was within the background incidence. conclusions: long-term follow-up suggests that tg can be an effective and well-tolerated therapy in more than half of difficult-to-treat and multi-therapy failing ibd patients. findings of this study indicate that tg can be used safely and the occurrence of hepatotoxicity was low. the incidence rate of nrh was within the background incidence. keywords: thioguanine, thiopurines, ibd, crohn's disease, ulcerative colitis, drug repositioning, drug survival, the classic thiopurines mercaptopurine (mp), and its licenced and more widely used prodrug azathioprine (aza), are still first line immunosuppressives for treating inflammatory bowel diseases (ibd). in recent times, their role is increasingly being questioned because of their relative poor performance, mainly due to treatment and dosage limiting adverse events, when compared to biologics [ ] [ ] [ ] . nonetheless, a sizable subgroup of individuals experience a positive response ( - %) with meaningful clinical outcomes such as sustained steroid sparing/ free remission combined with a reduced need for hospitalisation, surgery and biologic agents [ ] [ ] [ ] [ ] [ ] [ ] [ ] . there is an emerging interest in small molecules, as they have advantages over biologicals (e.g. oral administration, lower costs, no antibody formation and therefore sustained efficacy) [ ] . the thiopurines are well-established classical small molecules and hence share these benefits. however, the conventional thiopurines (aza/mp) have been well described and accepted shortcomings include slow onset of action, relatively high rate of adverse events and subsequent frequent need for monitoring and/or dose optimisation. these time-consuming and often distressing problems have resulted in an increase of early use of biological therapy [ , ] . thioguanine (tg) was introduced in as treatment for leukaemia and about five decades later, clinicians started using it for treating ibd. modern cases series suggest that tg offers better outcomes in terms of efficacy ( - %) as rescue therapy, low incidence of adverse events and possibly a more rapid onset of action compared to conventional thiopurines [ ] [ ] [ ] . however, its widespread use has been hampered due to concerns about hepatotoxicity (nodular regenerative hyperplasia [nrh] ) but this is thought to be dose-dependent [ ] [ ] [ ] . other long-term risks of tg might be the development of lymphoma and skin malignancy, as has been shown with aza/mp and anti-tnf therapies [ , ] . having considered these potential risks, the benefits of tg remain important in the treatment of patients with ibd [ ] . for this reason, a few centres continue to use tg but only at a lowered dose ( - mg/day). reports of outcomes from these centres in the united kingdom (uk), the netherlands and australia confirm a good efficacy and lack of hepatotoxicity [ , , [ ] [ ] [ ] . moreover, in the netherlands, tg has been provisional licenced for ibd since a few years [ , ] . at the moment an estimated dutch ibd use tg as rescue treatment for ibd [ ] . for the reasons outlined, it is important to evaluate the long-term outcomes of a low dose tg approach. the aim of this study was to investigate the long-term efficacy and safety of low dose tg in a large ibd cohort failing conventional thiopurines, derived from three uk centres. a retrospective, multicentre cohort study was performed in three centres in the uk (st. thomas' hospital, queen elizabeth hospital and east surrey hospital). patients were identified by using local hospital pharmacy dispensing records dating from to , as tg is only dispensed through the hospital pharmacy. patients were included if they were diagnosed with crohn's disease (cd), ulcerative colitis (uc) or ibd-unclassified (ibd-u) according to clinical, endoscopic and/or histological criteria and if they were treated with tg, either as monotherapy or concomitant therapy. montreal classification were used to classify the ibd patients [ ] . patients without complete documented follow-up were excluded. patient and disease characteristics, drug history and clinical, biochemical, radiological and histopathological data were reviewed using the patients' medical records. laboratory data were taken at months prior to tg initiation, at months after initiation and at final follow-up. laboratory parameters included haemoglobin, white blood cell count (wbc), platelet count, aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase (alp), gamma glutamyl-transferase (ggt), bilirubin, albumin and -thioguanine nucleotides ( -tgns). most recent abdominal ultrasonography, magnetic resonance imaging (mri) and liver biopsies were assessed. safety of tg therapy was assessed by evaluating ) haematological parameters for myelotoxicity and nrh (through alkaline phosphatase) and ) radiological, histopathological and biochemical parameters for liver toxicity. histopathological liver biopsy reports were assessed and the pathologists' findings and conclusions were used to assess the occurrence of nrh. patients were categorized into 'signs of nrh' when minimal focal hyperplasia was present. emphasis has been put on signs or findings of non-cirrhotic portal hypertension (ncpht) (e.g. hepato-or splenomegaly, nodular abnormalities, ascites, varices). the occurrence of adverse events and reasons for withdrawal during the entire follow-up were recorded. adverse events were defined as laboratory abnormalities and signs or symptoms that occurred after initiation of tg, and were listed according to the common terminology criteria for adverse events (ctcae) (version . , released november , ) [ ] . a positive clinical response was defined as: no (re)initiation of corticosteroids, (re)initiation of biological therapy or ibd-related surgery. crp and esr levels were collected to determine biochemical response. tg use at final follow-up was evaluated to assess long-term beneficial effect. primary resistance was assigned when criteria for clinical effectiveness were not met within the first months of therapy. lastly, -tgn levels were collected to evaluate a possible correlation with clinical response. data was presented as numbers with percentages, medians with interquartile range (iqr) or means with standard deviations. depending on the kind of parameter, distribution, parametric or nonparametric tests including the mann-whitney u test, wilcoxon signedrank test, kruskal wallis, and the student t-test or chisquare test were used to test for differences within and between groups. a kaplan-meier plot was performed to assess the tg attrition rate. the survival curves were compared using the mantel-cox test. this study was reported according to the strengthening the reporting of observational studies in epidemiology statement [ ] . ibm spss statistics v. was used for the statistical analysis. a p-value less than . was accepted as statistically significant. according to the guidelines of the uk health research authority, as the data were collected as part of routine clinical care and were evaluated retrospectively, the study was considered a review of clinical practice and ethical approval was not required [ ] . this study was conducted in accordance with the declaration of helsinki [ ] . all data in this study was anonymised. in total, ibd patients treated with tg were identified. sixteen patients were excluded due to incomplete follow-up data, leading to a total number of patients whom were included in the study. of these, patients were female ( %), had cd ( %), uc ( %) and ibd-u ( %). perianal disease was diagnosed in patients ( %). median age at ibd diagnosis and initiation of tg was (iqr - ) and years (iqr - ), respectively. sixty-three patients ( %) had a history of bowel resection prior to tg therapy. patient and disease characteristics are shown in table . prior to tg initiation, aza was used in patients ( %), mp in patients ( %) and both aza and mp in patients ( %). seventy-six of patients ( %) had used co-therapy with allopurinol to optimise aza (aza-allo). the median daily dosages of aza and mp were mg (range: - mg) and mg (range: - ). the dosage used for aza-allo was mg for aza and mg for allopurinol. the most commonly reported reason for prior thiopurine failure was intolerance to thiopurines, which occurred in patients ( %). intolerances to thiopurines mostly recorded were nausea ( %), pancreatitis ( %) and arthralgia/myalgia ( %). resistance to thiopurines occurred in patients ( %). twelve of those resistant patients ( %) were primary non-responders. the rest of the patients were switched to tg for reasons such as patients' preference, suboptimal -tgn levels and non-compliance. furthermore, patients ( %) had a history of biological therapy (see table ). sixty-one patients ( %) had loss of response on biologicals, six patients ( %) suffered from rash, five ( %) had infusion site reactions, two ( %) suffered from tonsillar abscess and three patients ( %) had psoriatic skin abnormalities. the median daily dose for tg was mg; patients ( %) were treated with mg once daily and patients ( %) with mg twice daily. patients of east surrey hospital were commenced on mg twice daily and after months the lowest effective dose was achieved by reducing to mg/day. at st. thomas' hospital most patients were started on mg once daily although some received mg/day ( mg twice daily) with the desire to reduce to mg once daily if response allowed. at queen elizabeth hospital, all patients received mg once daily. the median treatment duration of tg was months (iqr - ) and the median follow-up time of months (iqr - ). thirty-three patients ( %) started tg along with biological therapy; adalimumab ( mg, two weekly) was started in patients, infliximab ( mg/kg, eight weekly) in patients, ustekinumab ( mg, eight weekly) in one patient and vedolizumab in one patient ( mg, eight weekly). the median duration of follow-up was months (iqr - ), in which out of patients ( %) were still using biologicals along with tg. the reasons to stop biologicals were due to loss of response and side-effects (i.e. infusion reactions). clinical response at and months of tg therapy was seen in % (n = ) and % (n = ) of patients, respectively. the proportion of patients who continued tg therapy is depicted in a kaplan-meier survival curve ( fig. ). of the entire cohort, ( %), ( %) and ( %) were still using tg at respectively and months after initiation, and at final follow-up. drug survival did not depend on age, gender, type of ibd, bowel resection history, short bowel syndrome or ibd behavior. no statistical significant difference was observed between using a dosage of or mg per day (fig. ) . thioguanine was discontinued in patients ( %) during follow-up. forty-three patients ( %) stopped tg due to intolerance, ( %) due to resistance, ( %) due to malignancy (melanoma, diffuse large b-cell lymphoma, recurrent breast cancer), ( %) due to pregnancy, ( %) due to non-compliance, ( %) due to complete remission and ( %) due to miscellaneous reasons. the median time till cessation of therapy due to intolerance or resistance was (iqr - ) and months (iqr - ), respectively. of the patients who failed prior thiopurine therapy due to intolerance, patients ( %) did not experience any intolerances while on tg. of these patients, patients ( %) were still using tg at final follow-up. furthermore, of the patients ( %) who were primary (n = ) or secondary (n = ) resistant to conventional thiopurines, patients ( %) had a positive response to tg at months. of the primary resistant patients, nine out of ( %) were responding to tg at months. seventy-one adverse events occurred in a total of % (n = ) of patients. overall, % of patients had to stop tg therapy due to intolerance. no deaths occurred in our cohort study related to tg therapy. adverse events to tg are shown in table . laboratory abnormalities occurred in % (elevated liver function tests or myelotoxicity) and signs/symptoms in % (mainly rash). pancreatitis occurred in of patients with a history of conventional thiopurine-induced pancreatitis. malignancies occurred in three patients ( %): two patients developed moderate graded (i.e. requiring minimal, local or non-invasive intervention; melanoma and recurrent breast cancer) and one patient severe graded malignancy (diffuse large b-cell lymphoma). the latter was diagnosed on surgical specimen following excision of age at diagnosis treatment duration of thioguanine (months, iqr) enterocutaneous fistula and redo of ileocolic anastomosis after years of tg treatment. this patient was previously treated with both anti-tnf therapy and methotrexate. non-cirrhotic portal hypertension (ncpht) occurred in two patients ( %). the first patient was between and years old and diagnosed with crohn's disease who started tg therapy in after poor response to both aza and low dose aza with allopurinol afterwards. tg was initiated at mg twice daily ( mg/day) and gradually reduced to mg on alternate days with occasional complete breaks in treatment. in january , the tg was stopped after the patient had been established on anti-tnf therapy for months combined with recurrent episodes of pancytopenia. anti-tnf therapy was ceased in july due to loss of response and a severe psoriatic reaction. in december , the patient was admitted with ascites and features of portal hypertension. investigations (within weeks) determined normal portal pressures and a liver biopsy was reported as normal. the further work up for chronic liver disease and coagulopathy was negative. a clinical diagnosis on ncpht was established and therapy was discontinued. symptoms improved with conservative measures such as spironolactone treatment. the second case of ncpht was between and years old, diagnosed with crohn's disease and had three ileocecal resections prior to using tg. tg was started after not tolerating aza and failure of both infliximab and adalimumab. azathioprine was discontinued because of mild fibrosis and steatosis observed in the liver biopsy performed due to abnormal liver enzymes. during tg therapy, no follow-up liver biopsy was performed. after using tg mg twice daily ( mg/day) for years, the patient presented with abdominal pain. gastroscopy revealed gastric fundal varices and a ct-scan showed widespread intra-abdominal non-haemorrhagic varices and small volume ascites. tg was discontinued and the patient was given atenolol for primary prevention of variceal bleeding. no varices bleeding occurred over time. laboratory parameters at initiation of therapy and months of treatment are depicted in table . among the entire cohort, alanine transaminase (alat) and alkaline phosphatase (ap) levels prior to tg initiation did not differ from levels at months. however, a statistically significant increase in bilirubin concentration ( to μmol/l) and a decrease in platelets ( to × /l) was observed (p < . ). the platelet level was lower in the response group compared to the non-response group but still within the normal range ( versus × /l, p < . ). regarding efficacy, a biochemical response at months was observed in the patients experiencing a positive clinical response as their crp level was statistically significantly lower compared to the nonresponders ( versus mg/l, p = . ). there was no statistical difference in erythrocyte sedimentation rate (esr) level between responders and nonresponders (p > . ). tpmt measurements were performed in patients ( %), the median tpmt level was mu/l (iqr . - ). in total, three patients ( %) had low tpmt activity (< mu/l) and patients ( %) had high tpmt dili drug-induced liver injury, dili grade is defined as alanine aminotransferase (alt) and aspartate aminotransferase (ast) > . and ≤ upper limit of normal (uln) and total bilirubin > . and ≤ uln, grade is defined as alt and ast > and ≤ uln and total bilirubin > and ≤ uln. bone marrow suppression: grade < = % reduction from normal cellularity for age, grade : > -< % reduction from normal cellularity for age, grade : > -< = % reduction cellularity from normal for age activity (> mu/l). all patients in the low activity group suffered from mild side-effects (e.g. myalgias, nausea and rash) on thiopurines. in the high tpmt activity group, % of patients discontinued tg, while in the normal tpmt activity group % of patients discontinued tg during follow-up (p = . ). however, if a cutoff level of mu/l (based on the rate of tg discontinuation) was used, % of patients above this cut-off level discontinued tg while % of patients below this level discontinued tg (p = . , chi-square test). eleven patients above this cut-off value stopped tg due to non-response, while no patients stopped due to nonresponse under this cut-off level (p = . , chi square test). in these patients, the mean tpmt level was . ± . mu/l. median steady-state -tgn concentrations during the first months of tg therapy were available in patients ( %). the median -tgn concentration was pmol/ × rbc (iqr - ). median -tgn metabolite levels were not significantly higher in clinical responders compared to non-responders ( [iqr - ] versus [iqr - ], p = . ). no significant differences were found between quartile -tgn levels (fig. , p = . ) . the response rate at months was % in patients with a -tgn cut-off level below pmol/ × rbc, whereas the response rate was % in patients with a cut-off above pmol/ × rbc. this difference was not statistically significant. imaging and/or biopsies of the liver were performed in case of suspicion of ncpht or as part of routine screening. an ultrasound was performed in patients ( %) at a median of months (iqr - ). thirtysix patients ( %) had no abnormalities, ( %) borderline splenomegaly, ( %) signs of steatosis and one ( %) other unrelated abnormalities. four patients demonstrated both steatosis and splenomegaly. eighty-three patients ( %) underwent an magnetic resonance imaging (mri) at a median follow-up of months (iqr - ), showing no abnormalities in patients ( %), borderline splenomegaly in seven ( %), steatosis in nine ( %), focal nodular hyperplasia in one ( %), hepatomegaly in ( %) and other abnormalities (all liver cysts) in seven ( %). liver biopsies were performed in patients ( %) during tg treatment, at a median follow-up of months (iqr . nrh was histologically diagnosed in two patients, signs of possible nrh were found in five patients and normal histology was found in the remaining patients. one nrh patient used mg once daily, the other nrh patient mg twice daily. all five patients with signs of nrh were started on mg/ day, which was reduced to mg on alternate days in one patient. the first nrh patient, also diagnosed with primary sclerosing cholangitis, suffered from varices grade i on gastroscopy for which this patient underwent band ligation. nrh was detected months after cessation of tg, which had been used for months. the second nrh patient was asymptomatic and developed nrh after months of tg therapy. both patients with nrh had a history of methotrexate therapy and sideeffects on thiopurines. all five patients with signs of nrh on liver biopsy were asymptomatic and did not develop clinical features of ncpht during follow-up. four of those five ( %) were able to continue tg until final follow-up. nine of the patients ( %) demonstrating abnormalities (spleno-or hepatomegaly) on ultrasound and/or mri underwent a liver biopsy. three patients of the remaining five patients who did not receive a liver biopsy were still using tg at final follow-up without signs of ncpht. two remaining patients who did not receive liver biopsy discontinued tg. one patient discontinued due to persistent thrombocytopenia and elevated liver function enzymes, and the other patient (known with cirrhotic liver disease) discontinued because of elevated liver enzymes and perisplenic and gastro-oesophageal table laboratory parameters at initiation of tg (t ) and months of treatment (t ), shown as mean (sd). levels of response and non-response group at t are compared, as are levels at t and t of the entire cohort ( ) ( ) ( ) ( ) ( ) ( ) alat alanine aminotransferase, ap alkaline phosphatase, crp c-reactive protein, esr erythrocyte sedimentation rate *: statistical significantly different (p < . ) varices. the latter had portal hypertension which developed due to postoperative portal vein thrombosis after a right hemicolectomy, which was performed years before tg initiation. this retrospective, multicentre study sought to look at the real-world outcomes of tg from three large uk ibd units (over ibd patients per unit). a complete data capture was achieved through a search of local tg databases (prescriptions and ibd databases). this yielded patients with a median follow up of years and a median dose of mg/day. thioguanine can be started at its therapeutic dose as it is well tolerated, resulting in a positive clinical response of % at months and % at months as demonstrated in our study. following this initial positive response, most patients continued to tolerate the treatment long-term with an overall efficacy at final follow-up of %. in our study, no correlation was found between -tgn levels and clinical response. meijer et al. published a systematic review on the efficacy of tg in ibd. they reported results of studies, comprising of patients who were treated with a median tg starting dose of mg/day after failing prior conventional thiopurine therapy. clinical improvement in terms of decreased disease activity score or the ability to cease/decrease corticosteroid use was observed in % of patients (n = ) [ ] . recently, simsek et al. published the largest tg cohort consisting of ibd patients, with a long-term follow-up of years. they found that tg was tolerated in % of patients, % was responding at months and % showed sustained clinical effectiveness. although % of patients developed adverse events ( % mild, % moderate and % severe), only % overall discontinued due to intolerance. these authors also provided a meta-analysis of ibd patients on at least months of tg treatment. they reported that patients achieved a positive clinical effect (pooled proportion: , % ci: - %) [ ] . the results described above are broadly in line with the outcomes of our cohort. in our study there were two patients with histological features of nrh in their liver biopsies. five patients had histological characteristics suspicious of nrh during liver biopsy, but none of them developed clinical signs of ncpht. only one patient, who did not respond to aza and aza-allo, discontinued tg during follow-up also due to poor-response. additionally, two other patients ( %) were diagnosed with ncpht in this cohort, without nrh-related characteristics in their liver biopsies. as compared to historical data from dubinsky et al. ( ) in which high rates of nrh were described: of liver biopsies demonstrated features of nrh ( %), our numbers are comparable to the background incidence of nrh ( %). the worrying high nrh rate in dubinsky's study is not completely understood, but may be associated with the used higher doses of tg (median mg/day, with an undocumented doses in the subgroup of patients who developed liver toxicity) and/or a selection bias as tg was used in patients with hepatotoxicity during aza/mp therapy [ ] . more recently, van asseldonk et al. assessed the occurrence rate of nrh during tg therapy in ibd patients. these patients had at least one liver biopsy, a mean daily tg dose of mg (sd mg) and a median treatment duration of months. in this group only seven patients ( %) were histologically diagnosed with nrh and none had complications [ ] . ward et al. evaluated patients treated with a mean tg dose of mg/day in a group of patients intolerant or resistant to fig. correlation between -tgn concentration and clinical response rate within months. no significant difference was found between the four quartiles (p = . , pearson chi square). at a cut-off level of pmol/ × rbc no significant difference was found below and above the cut-off level (p = . , t-test) conventional thiopurines or methotrexate. no nrh was observed in their study [ ] . pavlidis et al. investigated a similar sized cohort of ibd patients who received a median tg dose of mg twice daily. the findings included mild nrh in one patient who was procoagulant due to antiphospholipid syndrome ( . %), resulting in a portal vein thrombosis with complications. this group also had a patient with signs of nrh that resolved after dose reduction of tg to mg once daily [ ] . to determine the background incidence of nrh, de boer et al. did liver biopsies (during gastrointestinal surgery) in a thiopurine-naive ibd cohort and detected a nhr prevalence of % [ ] . furthermore, wanless et al, reported cases ( . %) of nrh among consecutive autopsies in the general population [ ] . these observations suggest that patients treated with low dose tg (not exceeding mg/day) do not have a considerable higher risk of developing nrh when compared to non-tg receiving patients. furthermore, it is important to note that the conventional thiopurines (aza/ mp) can cause nrh as well and were used in all our patients [ , ] . moreover, both of our nrh cases in our study were previously treated with methotrexate and one was also diagnosed with primary sclerosing cholangitis. regarding the two ncpht patients, one of them presented with signs of ncpht (with a normal liver biopsy) almost years after tg cessation. the other patient had already steatosis and fibrosis on liver biopsy while still on aza. it is therefore important to state that we cannot determine which of the nrh and ncpht patients of our study may have actually developed nrh and ncpht due to other causes. furthermore, van asseldonk et al. demonstrated that although vascular liver abnormalities of any degree are commonly observed ( %) in tg treated ibd patients, they rarely become progressive or are of any clinical relevance [ , ] . it was found by morris et al. that nrh survival is highly variable and mostly related to age and underlying disease [ ] . seven patients in this study withdrew tg due to pregnancy, as the current uk guidelines do not allow this treatment in pregnancy, as compared to aza/mp. theoretically, there should be no difference between tg and aza/mp. thiopurines are not associated with any congenital abnormalities in men or women [ , ] . although data about safety of tg during pregnancy is scare, there is no evidence demonstrating negative effects. there is one case series published on pregnancies in ibd patients who were treated with low dose tg ( mg/day). all patients conceived healthy new-borns, supporting the safe use of tg during pregnancy [ ] . to our knowledge, the present study is the second largest study evaluating the safety and efficacy of tg in ibd patients. patients from three different hospitals were included, which created the possibility of assessing slightly different low-dose tg treatment regimens. secondly, the patients of our study have a long follow-up period, allowing us to assess the long-term effects of tg treatment. as a result of the retrospective design of the study, we acknowledge a few limitations. firstly, it was difficult to compare the laboratory and imaging results between patients, as these were not regularly performed at fixed time intervals. secondly, splenomegaly, hepatomegaly and signs of nrh were not defined using a standardised definition, therefore it was complicated to objectify and compare these parameters. thirdly, it was not possible to assess mucosal response and clinical symptoms, as too limited endoscopic reports and disease activity indices (simple clinical colitis activity index or harvey-bradshaw index) were available. furthermore, faecal calprotectin was not routinely measured and therefore not evaluated by us. lastly, -tgn and tpmt levels were not available in the majority of our patients what led to calculating the correlation of -tgn level and clinical response using a relatively small number of patients. also, the -tgn data is likely to be affected by selection bias as metabolite levels are mostly performed in patients having an insufficient response to tg. a -tgn cut-off level above pmol/ × rbc seems to be associated with better clinical effectiveness [ ] . in our study, patients having -tgn levels above pmol/ × rbc had a response rate of %, while patients who had -tgn levels under this cut-off level had a response rate of %. this difference in response, although not statistically significant, might suggest that -tgn levels above pmol/ × rbc can be associated with better clinical effectiveness as demonstrated by simsek et al. [ ] . one of the causes of low -tgn levels might be the result of drug incompliance. so, -tgn levels can be used to assess drug compliance in ibd patients treated with tg. furthermore, out of patients who stopped tg due to non-response had tpmt levels above the cut-off level of mu/l. this is in line with results that were found in literature for aza treated ibd patients. ansari et al. [ ] found that high tpmt activity (> u/ml) was significantly associated with non-response in aza-treated ibd patients (or . , % ci . - . ). it seems that high tpmt activity might also be associated with non-response in tg-treated ibd patients. although, the numbers in this study are small, it might indicate that further optimization of tg dosing might be useful by measuring the tpmt level. the outcomes of this study are in a subset of difficultto-treat patients who all have failed prior thiopurine therapy. moreover, a substantial number of patients in our study were already unsuccessfully treated with biologicals and/or allopurinol co-therapy ( and % respectively). still, in this heavily treated group of patients, tg has shown to be an effective and welltolerated therapy. a prospective study is needed to establish the role of tg as a rescue therapy for ibd patients who fail conventional thiopurine or other immunomodulation therapy (biological therapy, azaallo, methotrexate, tacrolimus, tofacitinib), and as firstline maintenance therapy for ibd. long-term follow-up in this english cohort of difficult-to-treat ibd patients suggests that low-dose tg (≤ mg/day) can be effective and well-tolerated in more than half of patients who had failed prior thiopurine therapy and other immunomodulation or biological therapy. hepatotoxicity was not common and the incidence rate of nrh was similar to the background incidence of nrh in ibd. there were no differences between and mg/day in terms of efficacy and safety, but we recommend to use the lowest possible effective dose of mg/day or lower. a prospective trial is recommended to further establish the role of tg in ibd patients as first-line or rescue treatment. infliximab, azathioprine, or combination therapy for crohn's disease evolving considerations for thiopurine therapy for inflammatory bowel diseases-a clinical practice update: commentary is there still a role for thiopurines in crohn's disease? 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-experience in a london district general hospital continued development of drugs: the path of thioguanine unrealized potential of drug repositioning in europe during covid- and beyond: a physician's perspective finding hidden treasures in old drugs: the challenges and importance of licensing generics the montreal classification of inflammatory bowel disease: controversies, consensus, and implications department of health and human services. common terminology criteria for adverse events (ctcae) version the strengthening the reporting of observational studies in epidemiology (strobe) statement: guidelines for reporting observational studies revising the declaration of helsinki efficacy of thioguanine treatment in inflammatory bowel disease: a systematic review -thioguanine can cause serious liver injury in inflammatory bowel disease patients the prevalence of nodular regenerative hyperplasia in inflammatory bowel disease patients treated with thioguanine is not associated with clinically significant liver disease histopathology of liver biopsies from a thiopurine-naive inflammatory bowel disease cohort: prevalence of nodular regenerative hyperplasia micronodular transformation (nodular regenerative hyperplasia) of the liver: a report of cases among , autopsies and a new classification of benign hepatocellular nodules nodular regenerative hyperplasia in patients with inflammatory bowel disease treated with azathioprine review article: the association between nodular regenerative hyperplasia, inflammatory bowel disease and thiopurine therapy limited relevance and progression of histological alterations in the liver during thioguanine therapy in inflammatory bowel disease patients clinical course of nodular regenerative hyperplasia in thiopurine treated inflammatory bowel disease patients nodular regenerative hyperplasia of the liver: survival and associated features in a uk case series systematic review and meta-analysis on the effects of thiopurines on birth outcomes from female and male patients with inflammatory bowel disease the associations of thiopurines with male fertility and paternally exposed offspring: a systematic review and meta-analysis safety of tioguanine during pregnancy in inflammatory bowel disease thiopurine methyltransferase activity and the use of azathioprine in inflammatory bowel disease publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable.authors' contributions ab collected and analyzed the data, wrote and conceptualized the manuscript. el analyzed the data, wrote and conceptualized the manuscript. ms established the database, wrote and reviewed the manuscript. bw, al, js, sa, jn, nb, cm reviewed and commented on the manuscript. aa established the study, wrote and conceptualized the manuscript. all authors have read and approved the manuscript. no funding was obtained for the study. the datasets generated and/or analysed during the current study are not publicly available but are available from the corresponding author on reasonable request. according to the guidelines of the uk health research authority, as the data were collected as part of routine clinical care and were evaluated retrospectively, the study was considered a review of clinical practice and ethical approval was not required [ ] . this study was conducted in accordance with the declaration of helsinki [ ] . key: cord- - se mxq authors: botti, fiorenzo; mazza, stefano; costa, stefano; farina, elisa; baldari, ludovica; prati, matteo title: urgent surgery for inflammatory bowel diseases during the covid- outbreak in a high incidence area - experience from a high-volume centre date: - - journal: dig liver dis doi: . /j.dld. . . sha: doc_id: cord_uid: se mxq nan since december , a novel coronavirus named sars-cov- (severe acute respiratory syndrome coronavirus ) was identified as the causative agent of a respiratory syndrome named covid- . [ ] during the following months, the virus rapidly spread around the world, until the world health organization (who) defined covid- as a pandemic on march . lombardy region was severely hit by covid- since february , posing serious challenges to healthcare system due to an extremely high number of simultaneous cases. our hospital (fondazione irccs ca' granda ospedale maggiore policlinico) is a tertiary care university centre located in the centre of milan and it is a referral centre for the treatment of inflammatory bowel diseases (ibd). in order to deal with the difficult scenario due to the pandemic, our hospital, like many others in northern italy, was deeply reorganized to ensure an adequate care for the huge number of covid- patients. almost all intensive care units (icus) were dedicated to covid- , new icus were created, and specialist departments, both medical and surgical, were reduced and merged into covid-free wards. because of such a rearrangement and according to national and international indications, [ , ] only urgent surgical procedures were allowed, while elective surgeries were drastically reduced and limited to oncological cases. [ ] as a tertiary referral centre for the surgical management of ibd patients, it was important to clearly define which complicated ibd cases were to be operated, by taking into account several variables including disease severity and quality of life, presence of comorbidities, feasibility of an alternative procedure. according to the british society of gastroenterology (bsg) guidelines on the ibd management during the covid- pandemic, [ ] routine elective operations should be deferred, while emergency procedures (e.g. colectomy in acute severe ulcerative colitis [uc] or intestinal resection in septic complications of penetrating crohn's disease [cd]) should continue. complex surgical ibd cases should also be deferred when possible and its timing must be regularly reviewed at multidisciplinary meetings, trying to optimize medical treatment (e.g. using partial or exclusive enteral nutrition regimens or draining abscesses in combination with antibiotic therapy) and to avoid surgery or change the timing from emergency to urgent or semi-urgent. in the case of subacute obstructive symptoms, guidelines suggest the possibility to avoid or delay surgery by using partial or exclusive enteral nutrition regimens. [ ] we hereby report our experience on surgical management of ibd patients as a high-volume centre located in a high-incidence region for covid- . from the beginning of march until may , ibd patients ( cd and uc, mean age yrs, m/f / ) were admitted to our hospital for an ibd-related urgent condition. all cases were handled by a multidisciplinary team including surgeons, gastroenterologists, and radiologists. one of them (an -year-old man) was treated conservatively after being discharged weeks earlier from a non-referral centre, and then admitted to our surgical department for severe malnutrition and the presence of a pelvic abscess caused by cd. he was treated with total parenteral nutrition and antibiotic therapy with regression of the abscess, successful reintroduction of oral nutrition, and was discharged after days of hospitalization. the remaining patients underwent surgical treatment; their characteristics are summarized in table . three of them ( cd and uc) were already followed-up by our ibd unit and an indication for surgery was already posed. during the lockdown period, they were monitored with regular contacts by phone or e-mail, until symptoms and clinical condition worsened, and the surgical approach could not be postponed further; at that point they were hospitalized electively. three other patients ( cd and uc) were admitted from the emergency department because of an acute complicated presentation of ibd; the ibd was previously unknown in one case and already followed-up at our centre in cases. the remaining patient, with cd, was referred to our unit from a non-tertiary hospital due to a rapid worsening of his ibd-related clinical condition. for all patients the timing of surgery was defined on the basis of how long surgery could be delayed without causing harm to the patient: less than hours (emergency), to days (urgency), or to days (semi or delayed urgency). [ ] surgical indications and timing were as follows: -a -year-old male uc patient underwent emergency surgery the same day of presentation. in january he received proctocolectomy with ileo pouch-anal anastomosis and protective loop ileostomy for severe uc, followed by the closure of the ileostomy in december . two months after the recanalization, a perforation of the pouch developed, that evolved into purulent peritonitis with septic shock: a new loop ileostomy was performed during the emergency intervention. -two patients underwent surgery with urgency timing. a -year-old man was operated for evolution into toxic megacolon after failure of medical therapy (including rescue therapy with infliximab) for severe uc; a laparoscopic total colectomy with terminal ileostomy and closure of the rectum was performed. a -year-old women developed an acute intestinal obstruction due to a long ( cm) cd-related stenosing ileal segment, which was resolved with a laparo-assisted cm-long ileal resection and a cm-long ileo-ileal anastomosis with atypical strictureplasty. both these patients were operated after days of hospitalization. -four patients were operated with a delayed urgency timing. in a -year-old man with previously unknown ibd, an open right hemicolectomy was performed for severe inflammatory mass with deep abscess in the right flank, suspected for cd (at radiological end endoscopic examinations) and unresponsive to medical therapy. it was later confirmed as cd at histology. two patients underwent surgery because of worsening of a pre-existing sub-occlusive state: in a -year-old man with longstanding uc, a laparoscopic total colectomy with terminal ileostomy was performed due to a colonic stenosis; a -year-old man with cd underwent a laparo-assisted ileocaecal resection for a stenosis of the ileocaecal valve. the remaining -year-old women, affected by uc, was treated with a laparoscopic total colectomy for rapid clinical worsening of a moderate-severe steroiddependent disease; a terminal ileostomy was made. all the patients operated in the urgent and delayed urgent surgical setting were screened for covid- before surgery. two nasopharyngeal swabs for sars-cov- were performed hours apart, along with a chest x-ray followed by a chest ct scan were appropriate. [ ] none of these patients tested positive. they were operated in dedicated covid- -free surgical rooms and the surgical technique was the same as the one that would have been chosen in the pre-covid era. regarding the case of emergency surgery, it was not possible to wait for the result of sars-cov- swabs. thus, the surgery took place in a dedicated room for covid- patients, maintained at negative pressure and with a high frequency air exchange; furthermore, an open approach was preferred, instead of laparoscopic or laparo-assisted, according to the current international guidelines. [ , ] for all surgical patients, the choice to perform the protective ileostomy was not affected by the covid- status. five out of the surgical patients had a regular post-operative course and were discharged within days after surgery. one patient experienced a delayed canalization after a laparotomic intervention, that prolonged the hospitalization and was discharged on day . the patient who received the laparo-assisted ileocaecal anastomosis developed a major surgical complication (anastomotic leak with peritonitis and septic shock) which required an urgent reintervention. covid- did not develop in any patient after hospital discharge over a -weeks follow-up period. all patients were followed-up for at least months after surgery, during which no late surgical complications or early disease recurrence were observed. all physicians involved in the ibd management underwent regular sars-cov- swabs without any positive results. the challenging scenario related to the covid- outbreak has contributed to highlight the importance, for an ibd-referral centre, of having and maintaining an efficient integrated network with peripheral centres; this in fact allowed patients from neighbouring hospitals to be treated in a referral centre like ours, despite the difficulties related to the epidemic. the importance of multidisciplinary management of ibd patients must also be stressed. multidisciplinary team is composed by surgeons, gastroenterologists, and radiologists with large experience in the management of ibd, and seems to be a key element for a successful treatment without overloading the hospital facilities in such difficult times. in our experience, this has led to avoid surgery whenever possible, or to define the optimal timing for surgery once needed. moreover, urgency and delayed urgency timings allowed us to bring patients to surgery with a known negative covid- status, with important implications in terms of logistical simplicity (i.e. using of covid-free surgical rooms); it also allowed us to overcome the covid- -related concerns about the use of laparoscopy and the risk of a primary anastomosis execution. furthermore, the choice of the minimally invasive surgical technique was not affected. our case series finally showed that the rate of surgical complications has been similar to the pre-covid era, as reported by other experiences. [ ] overall, our experience can serve as an important example for other ibd referral centres, especially in the case of very challenging settings like the covid- outbreak. a new coronavirus associated with human respiratory disease in china american college of surgeons coronavirus pandemic and colorectal surgery: practical advice based on the italian experience unchanged surgical management of patients with cholangiocarcinoma during the covid- pandemic. dig liver dis british society of gastroenterology guidance for management of inflammatory bowel disease during the covid- pandemic surgery scheduling in a crisis covid- outbreak in northern italy: viewpoint of the milan area surgical community sages and eaes recommendations regarding surgical response to covid- crisis factors associated with surgical mortality and complications among patients with and without coronavirus disease all authors have no conflict of interests or funding information to declare. key: cord- -icteg y authors: namai, fu; shigemori, suguru; ogita, tasuku; sato, takashi; shimosato, takeshi title: microbial therapeutics for acute colitis based on genetically modified lactococcus lactis hypersecreting il- ra in mice date: - - journal: exp mol med doi: . /s - - - sha: doc_id: cord_uid: icteg y the increased incidence of inflammatory bowel disease (ibd) in western and rapidly westernizing developing countries poses a global pandemic threat. the development of affordable drugs for treating ibd worldwide is thus a priority. genetically modified lactic acid bacteria (gmlab) as microbial therapeutics are inexpensive protein producers suitable for use as carriers of protein to the intestinal mucosa. here, we successfully constructed gmlab hypersecreting interleukin receptor antagonist (il- ra). oral administration of these gmlab suppressed body weight reduction and exacerbation of the disease activity index score in mice with acute colitis and decreased the number of cd (+) il- a(+) cells in the mesenteric lymph nodes. these data suggest that the gmlab deliver il- ra to the colon, where it inhibits il- signaling. we thus developed a novel ibd therapeutic that blocks il- signaling using a gmlab protein delivery system. this system could be an inexpensive oral microbial therapeutic. the increasing incidence of inflammatory bowel disease (ibd) is a problem worldwide, particularly in developing countries, thus raising the possibility of a serious global pandemic . considering the economic burden associated with ibd, the development of less expensive and easierto-use therapeutics is an important issue. il- signaling plays an important role in ibd immunity and inflammation. in ibd patients with crohn's disease and ulcerative colitis, mucosal mononuclear cells express il- β, and a positive correlation between severity and il- β expression has been demonstrated , . il- signaling is regulated by endogenous il- receptor antagonist (il- ra), but an imbalance between il- and il- ra activity occurs in the gastrointestinal tract of ibd patients . in a study using a murine il- ra knockout model, deletion of il- ra induced spontaneous ibd-like symptoms , . therefore, inhibition of il- signaling and correction of the agonistantagonist imbalance in the inflamed portions of the intestinal tract are targets for the treatment of ibd. to date, several animal studies targeting il- signaling using il- ra or an anti-il- antibody have been conducted and demonstrated an anti-inflammatory effect [ ] [ ] [ ] . however, in clinical trials, serious side effects were reported in . % of patients receiving subcutaneous administration of anakinra, a recombinant human il- ra . therefore, the delivery of il- ra directly to the intestinal tract is desirable, as this approach might reduce the side effects and produce a beneficial anti-inflammatory effect . in this context, genetically modified lactic acid bacteria (gmlab) delivery of il- ra, the production of which does not require protein purification or sophisticated techniques for culture, could be an attractive strategy . gmlab are classified as microbial therapeutics and nextgeneration probiotics, and new opportunities for drug delivery using gmlab are expected , . indeed, compared with the oral administration of protein alone, an equivalent effect can be demonstrated with a smaller amount of protein when it is transported by gmlab , . here, we successfully constructed a microbial therapeutic based on genetically modified lactococcus (l.) lactis hypersecreting recombinant mouse il- ra (rmil- ra) with the goal of facilitating a cost-effective treatment for ibd. plasmid, bacteria, and growth conditions a protein secretion vector plasmid for l. lactis, pnz # :sec , was used for recombinant gene expression. the sequence encoding mil- ra optimized for l. lactis mg was synthesized, and the sequence was cloned into ptakn- . l. lactis nz was purchased from mobitec (goettingen, germany) as the host and cultured at °c in m broth with . % glucose (gm ). gm-l. lactis nz was cultured in gm cm, which is gm medium with chloramphenicol ( μg/ml). both ends of the mil- ra gene encoded by ptakn- were cleaved using the restriction enzymes hindiii and kpni (takara bio, inc., tokyo, japan). the resulting dna fragment was ligated to pnz # :sec digested with hindiii and kpni. the constructed plasmid (pnz # :sec-il ra) was subjected to sequence analysis (eurofins genomics) to examine consistency with the desired sequence. pnz # :sec-il ra was introduced into l. lactis nz to generate the gmlab (designated nz-il ra). simultaneously, the original plasmid lacking the mil- ra gene was also introduced into l. lactis nz to generate the vector control gmlab (designated nz-vc). precultured gmlab were inoculated into gm cm at a final concentration of % and incubated for - . h (until the od reached . ). nisin was then added (final concentration . ng/ml) and incubated for h. the culture was separated into cells and supernatant by centrifugation ( × g, °c, and min), and protein samples were prepared. bacterial pellets were washed with tris-buffered saline (tbs) and suspended in tbs + protease inhibitor cocktail (tbs + pic). glass beads ( . mmφ) were added to the solution, and the cells were crushed using a bead crusher. the resulting liquid of each sample was obtained by centrifugation ( , × g, °c, and min). a total of µl of trichloroacetic acid was added to µl of supernatant sample and incubated for h on ice to precipitate the protein. the precipitate was collected and washed twice with µl of acetone. after centrifugation, the acetone was removed by incubation at °c, and the precipitate was dissolved in µl of . m naoh. samples were separated by sodium dodecy sulfate polyacrylamide gel electrophoresis (sds-page), and the resolved proteins were stained with coomassie brilliant blue (cbb) or transferred onto amersham hybond p pvdf membranes (ge healthcare, buckinghamshire, uk). the membranes were blocked for h using skim milk and then incubated overnight with purified an anti-his tag antibody (biolegend, san diego, ca, usa), followed by reaction for h with an anti-mouse igg (whole molecule) peroxidase-conjugated antibody (sigma-aldrich, st. louis, mo, usa). labeling was detected using imagequant las (ge healthcare). rmil- ra contained in the cell pellet lysate and supernatant was quantified by enzyme-linked immunosorbent assay (elisa). the soluble fraction from the crushed cell pellet and supernatant collected from nz-il- ra incubated for h was diluted using tbs + pic and subjected to elisa (mouse il- ra/il- f elisa, r&d systems, minneapolis, mn, usa). nz-il ra was cultured ( l), and rmil- ra expression was induced as described above. the bacteria were pelleted by centrifugation ( × g, °c, and min). supernatant-binding buffer ( mm na po h o, mm nacl, and mm imidazole in the supernatant) was prepared and filtered ( . µm). after equilibrating a his-trap hp -ml column with binding buffer, the filtrate was loaded, and the column was washed with binding buffer ( column volumes [cvs]). the proteins adsorbed to the column were eluted with at least a -cv gradient of - mm imidazole at ml/min using an akta pure system (ge healthcare). the obtained samples (supernatant, flow-through, wash, and eluate: e- to e- ) were subjected to sds-page and western blotting. the resulting fraction (e- ) containing rmil- ra was then dialyzed against phosphate-buffered saline (pbs) ( mm disodium hydrogen phosphate, . mm potassium chloride, mm sodium chloride, and . mm potassium dihydrogen phosphate [ph . ]). the concentration of rmil- ra in the eluted fraction was determined by elisa (mouse il- ra/il- f , r&d systems). mouse ascites lymphoma lymphoblast el .nob- cells that highly express il- r were purchased from sigma-aldrich and maintained in complete rpmi medium, which is rpmi (sigma-aldrich) containing fetal calf serum ( %, sigma-aldrich), streptomycin ( mg/ml), penicillin ( u/ml), hepes ( mm), nonessential amino acids, sodium pyruvate ( . mm), and -mercaptoethanol ( . %). el .nob- cells were maintained at °c with % co and passaged every days. assay of rmil- ra activity el .nob- cells ( × / µl) were plated in -well plates and incubated for h. the preincubated el .nob- cells were stimulated with il- β/il- f ( pg/ µl: r&d systems) and various concentrations ( . - ng/ µl) of purified rmil- ra, commercially available mil- ra (r&d systems), or commercially available human il- ra (hil- ra) (r&d systems) for h. after incubation, the supernatants of the el .nob- cells were collected, and the concentration of il- was assessed using the mouse il- elisa (r&d systems). intestinal delivery of rmil-ra by oral administration of nz-il ra c bl/ mice (female, weeks old) were purchased from japan slc (shizuoka, japan) and maintained for weeks under controlled light and temperature conditions. the mice were provided a standard diet and autoclaved water ad libitum. the animal protocol was approved by the animal experiment committee of shinshu university (no. ). mice were separated into two groups (nonadministration group and nz-il ra administration group, n = - ). nz-il ra was cultured in a volume of ml, and gene expression was induced as described above. nz-il ra was orally administered at . × cfu/ µl every min for doses. thirty minutes after the last oral administration, the mice were euthanized, and the serum and contents of the cecum and colon were collected. the cecal and colonic contents were suspended in pbs ( mg/ml). the suspension was streaked onto a gm cm agar plate and incubated for days. eight of the resulting colonies were subjected to polymerase chain reaction (pcr) using the universal primers f ( ′-agagtttgatcctggctcag- ′) and r ( ′-ggctaccttgttacgactt- ′) to amplify the s rrna region, and the resulting dna fragments were sequenced and identified using blast. the supernatant of the cecum and colon content suspensions was obtained by centrifugation ( , × g, °c, and min). the mil- ra concentration in the prepared sample was measured using the mouse il- ra/il- f elisa (r&d systems). c bl/ mice (female, weeks old) were purchased and maintained for weeks as described above. after prehousing, mice were separated into two groups: nz-vc (n = , dss and nz-vc treatment) and nz-il ra (n = , dss and nz-il ra treatment). to induce acute colitis, mice were provided water supplemented with % dss (mw = - kda; mp biomedicals, llc, solon, oh, usa) from day to day . on day , the contents of the water bottle were switched to water alone until day . body weight and disease activity index (dai) scores were recorded daily . for oral administration, gmlab were cultured in a volume of ml, and gene expression was induced as described above. gmlab were suspended in pbs at . × cfu/ml, and mice were immediately administered µl of the gmlab suspension ( . × cfu) intragastrically for consecutive days (days - ). on day , the mice were euthanized, the colon, mesenteric lymph nodes (mlns), and cecal contents were immediately collected, and the colon length was measured. distal colonic tissues were frozen in optimal cutting temperature compound and sliced using a leica cm s cryostat. the resulting sections were stained with he. tissue sections were observed using a bz-x microscope (keyence, osaka, japan). the total mucosal area was measured using the hybrid cell count system in the bz-x viewer (keyence). quantitative pcr (qpcr) was performed as described previously , . primers for five genes (encoding ifn-γ, tnf-α, il- β, il- , and il- ) were obtained from takara bio, inc. alexa fluor ® anti-mouse cd (cat# ; biolegend) and percp/cy . anti-mouse il- a antibodies (cat# ; biolegend) were used. mln cells were cultured in complete rpmi medium containing brefeldin a, ionomycin, and phorbol myristate -acetate for h. after fixation, cells were incubated with an alexa fluor ® anti-mouse cd antibody for min at °c for surface staining. after permeabilization, cells were reacted with a percp/cy . anti-mouse il- a antibody for min at °c for intercellular staining. the percentage of cd + il- a + cells was determined by flow cytometry. the acquired data were analyzed using flowjo software (llc, ashland, or, usa). next-generation sequencing samples of day cecal contents were prepared as previously described . sequencing quality control and trimming of the f and r forward and reverse primer sequences were conducted using dada . sequencing data were analyzed using the qiime pipeline, including clustering, chimera checking, and α-diversity and β-diversity analyses . the taxonomy assignment of each representative sequence was performed according to the tutorial on the qiime website (https://qiime .org) using the greengenes database (greengenes _ % operational taxonomic units [otus] ). graphpad prism software (version , graphpad, san diego, ca, usa) was employed for statistical analysis, and significance was accepted at p < . . for in vitro rmil- ra bioactive assays, one-way anova and tukey-kramer test were performed. for in vivo and related experiments, the rout method was used to identify outliers, and the data were then analyzed using unpaired t tests. lactococcus lactis remarkably secretes rmil- ra following nisin stimulation pnz # :sec-il ra (fig. b) was constructed by inserting sequences encoding mil- ra into the multicloning site of pnz # :sec (fig. a, c) . the plasmid was introduced into l. lactis nz to generate vector control gmlab (nz-vc) or gmlab that secrete rmil- ra (designated nz-il ra). upon nisin stimulation, a band corresponding to pre-rmil- ra was detected from the nz-il ra cell pellet by western blotting ( . kda, nz-il ra (+), fig. d ). in contrast, no bands from nz-vc and nz-il ra without nisin were detected. in the supernatant of nz-il ra stimulated with nisin, a band corresponding to secreted rmil- ra was detected ( . kda, nz-il ra(+), fig. d ), but this band was not detected in the supernatant of nz-vc and nz-il ranisin(−). these results clearly indicate that nz-il ra secretes rmil- ra in a nisin stimulation-dependent manner. the yield of recombinant protein was as follows: cell pellet . ± . µg/mg and supernatant . ± . mg/l, immense quantities of rmil- ra. we successfully obtained gmlab that hypersecrete rmil- ra in a nisin stimulation-dependent manner. purification and bioactivity of rmil- ra rmil- ra was purified from the nz-il ra supernatant, and a chromatogram of the absorbance at nm (a ) is shown in fig. a . based on the absorbance value, the purified solution was separated into five fractions (e- to e- ). the purity was assessed by sds-page followed by staining with cbb (fig. c) or western blotting (fig. b) . bands corresponding to secreted rmil- ra ( . kda) were confirmed in e- to e- , confirming that rmil- ra was highly purified, particularly in fraction e- . therefore, in future experiments, e- was used as purified rmil- ra. elisa indicated that e- contained µg of rmil- ra. the ability of purified rmil- ra to inhibit il- signaling was examined using el .nob- cells . il- secretion was observed in el .nob- cells stimulated with il- β but not in unstimulated cells (fig. ) . il- β-stimulated il- secretion was significantly reduced with increasing concentrations of purified rmil- ra and commercial mil- ra and hil- ra (fig. ) . the effect of suppressing il- production was stronger in the order of mil- ra, hil- ra, and purified rmil- ra (fig. ) . nz-il ra was orally administered every min for doses. thirty minutes after the last administration, serum and the contents of the cecum and colon were collected (fig. a) . in a plating assay, the growth of colonies was confirmed in the nz-il ra group, and the s rrna region of randomly selected colonies exhibited homology with l. lactis ( %) (fig. b) . in contrast, no colonies were observed in the nonadministration group (fig. b) . the serum mil- ra level was dramatically increased with serial nz-il- ra administration (fig. c) . furthermore, fig. construction of an il- ra gene expression vector and analysis of rmil- ra expression. a vector map of the lactococcal secretion vector pnz # :sec. p, nisin a-regulated promoter; sp sequence of the signal peptide from the usp protein, his-tag dna sequence encoding the × histidine tag, fxa dna sequence encoding the factor xa recognition site, mcs multiple cloning site, t terminator, rep replication gene, cat chloramphenicol acetyltransferase gene; b vector map of pnz # :sec-il ra. mil- ra, the mg -optimized dna sequence encoding mil- ra. c scheme of the procedure for constructing the il- ra gene expression vector. d nz-vc or nz-il ra was cultured in the presence (+) or absence (−) of nisin ( . ng/ml) for h to induce recombinant protein expression. in the cell fraction, bands corresponding to the sizes of the sp and rmil- ra complex (gray arrowhead: . kda) and secretory rmil- ra (black arrowhead: . kda) were detected. a secreted rmil- ra (black arrowhead: . kda) band was also detected in the supernatant fraction. the mil- ra level in the cecal and colonic contents increased with nz-il ra administration compared with that in the nonadministration group (fig. d, e) . mice with dss-induced colitis were orally administered nz-il ra or nz-vc, and the experiment was performed according to the schedule shown in fig. a . colitis severity was determined by measuring body weight and determining the dai score daily . on the last day (day ), the mice were euthanized, the colon was collected, and its length was measured. in mice allowed to freely drink water containing % dss, body weight decreased significantly from days to , and the dai score increased, confirming that dssinduced acute colitis (fig. b) . after day , no increase in body weight was observed in the nz-vc oral administration group. in contrast, a significant recovery of body weight was observed in the nz-il ra oral administration group (fig. b) . the dai score also decreased significantly in mice orally administered nz-il ra (fig. c) . nz-il ra oral administration also significantly improved the colitisassociated shortening of the colon, as measured on the last day of the experiment (fig. d) . he staining of colon sections removed on the last day revealed a decrease in immune cell infiltration in the nz-il ra administration group and a significant decrease in the total mucosal tissue area (fig. e-g) . in the ibd model, tnf-α and ifn-γ are overexpressed, and the production of il- a by th cells is greatly affected by il- signaling [ ] [ ] [ ] [ ] . the colon and mlns were collected and analyzed to investigate the antiinflammatory effects. the proportion of cd + il- a + cells in mlns was determined using flow cytometry. the nz-il ra group exhibited a significant decrease in the proportion of cd + il- a + cells vs. the nz-vc group (fig. a-c) . total rna was extracted from the colon and mlns, and the expression of tnf-α, ifn-γ, and il- a was measured by qpcr (fig. d-i) . excluding tnf-α expression in mlns, there was a trend toward decreased inflammatory cytokines. significantly decreased expression of tnf-α in the colon and ifn-γ in the mlns was observed in the nz-il ra group (fig. e, g) . considering that ibd occurs in the gastrointestinal tract, studies examining effects on the microbiota are critical. therefore, the cecal microbiota in mice with dssinduced acute colitis was analyzed using a nextgeneration sequencing approach. the cecal microbiota compositions in the nz-il ra and nz-vc groups are shown in fig. a . there was no significant change in αdiversity (observed otus) following the oral administration of nz-il ra compared with that in the nz-vc = ) . b, c change in body weight and dai score, respectively, of mice with dss-induced colitis. data are the mean ± se (n = ). the p values of day results are shown. d colon length on day . data are the mean ± se (n = ), and each dot in the plot represents one mouse. e, f representative images of colon tissue stained with he ( × magnification). e nz-vc, f nz-il ra. g total mucosal area on day . data are the mean ± se (n = ), and each dot in the plot represents one mouse. group (fig. b) . similarly, there was no significant change in β-diversity (bray-curtis) following the oral administration of nz-il ra (fig. c ). gmlab not only produce proteins at low cost but also deliver the proteins produced directly to the intestinal mucosa . focusing on suppression of ibd by controlling the il- signaling involved in the exacerbation of inflammation, we constructed a gmlab that secretes rmil- ra and investigated its efficacy using an acute colitis model. as the expression cassette is controlled by the p nisa promoter, the addition of nisin, a type of bacteriocin, initiates the transcription of genes downstream of the promoter . western blotting indicated that nisinstimulated nz-il ra to markedly secrete rmil- ra. interestingly, we found that the amount of secreted il- ra was extremely high ( mg/l) compared to that of other interleukins for which secretion was found to be controlled by the nice system in previous studies ( table ). the expression efficiency of nz-il ra was also remarkable as an endotoxin-free bacterial factory for il- ra. although it is unclear why the expression level was so high, these results emphasize the importance of ensuring compatibility between the expression system and target protein in applications of microbial therapeutics. we also investigated whether rmil- ra produced by nz-il ra exhibits biological activity in inhibiting il- signaling. el .nob- cells highly express il- r and are known to produce il- upon stimulation with il- β . production of il- by el .nob- cells stimulated with il- β was suppressed by increasing the concentration of rmil- ra purified from nz-il ra supernatant, commercially available mil- ra, or hil- ra. these results indicate that rmil- ra produced by gmlab can play an effective role as an endogenous antagonist of il- . inhibition of il- signaling increased in the order of mil- ra, hil- ra, and purified rmil- ra, suggesting that the affinity of gmlab-secreted rmil- ra for il- r declined due to a change in the three-dimensional structure due to cleavage of the signal peptide and/or introduction of the his-tag. next, we investigated the delivery of rmil- ra to the intestinal tract by oral administration of nz-il ra. the results suggested that nz-il ra reached the colon alive and secreted rmil- ra in situ because l. lactis colonies were confirmed in a plating assay, and the mil- ra concentration increased in the contents of the cecum and colon. furthermore, oral administration of nz-il ra resulted in a dramatic increase in the serum mil- ra level, which was below the detection limit in the nonadministration group. these results suggested that highly secreted rmil- ra translocated into the blood and that nz-il ra oral administration could be a useful approach for suppressing il- signaling. delivery of il- ra directly to the intestinal tract not only reduces the prevalence and severity of side effects seen with systemic administration but also produces a beneficial anti-inflammatory effect . indeed, in clinical trials involving the subcutaneous administration of anakinra (a recombinant hil- ra), serious side effects were reported in . % of patients. by contrast, gmlab expressing recombinant target protein functions as an inexpensive tool for protein delivery directly to the fig. effects of nz-il ra on the cecal microbiota. a the cecal microbiota composition in the nz-il ra and nz-vc groups (n = ). the v -v region of s rrna from cecal contents was amplified and sequenced. the resulting sequences were analyzed using the qiime pipeline and classified using the greengenes database. b α-diversity (the rarefaction curve of observed otus) in the nz-il ra and nz-vc groups (n = ). c β-diversity (the bray-curtis index) in each group (n = ). intestinal tract without the requirement for purification or sophisticated techniques for culture . therefore, we investigated the anti-inflammatory effect of nz-l ra using an acute colitis model. oral administration of nz-il ra from day to day promoted weight gain from day and decreased the dai score. furthermore, the results of he staining of tissue sections indicated a reduction in the thickening of the mucosal tissue area of the colon caused by inflammation, suggesting that orally administered nz-il ra alleviates acute colitis. in dssinduced colitis, inflammatory cytokines, such as tnf-α and ifn-γ, are overexpressed and known to be involved in the worsening of colitis symptoms . in the nz-il ra group, inflammatory cytokine mrna expression on day was decreased, suggesting that nz-il ra suppressed the excessive inflammatory response. notably, th cell proliferation increases in ibd, as characterized by cd and il- a production, and this expansion of th cells is enhanced by il- signaling , , . in this study, we measured the proportion of cd + il- a + cells in mlns. compared with those in nz-vc, cd + il- a + cells decreased in the nz-il ra administration group. this result suggests that administration of nz-il ra suppresses il- signaling in vivo and suppresses the increased expansion of th cells. in addition to testing the intestinal delivery of rmil- ra by nz-il ra oral administration performed in this study, shigemori et al. demonstrated that gmlab transport target proteins to the mucosal tissue of the colon and that oral administration of nz-vc does not improve dss-induced colitis. these results suggest that rmil- ra delivery to the colon by oral administration of nz-il ra alleviates symptoms by inhibiting il- signaling. however, given that l. lactis does not colonize the intestinal tract and that -fold more il- ra than il- is required to inhibit signaling , , preadministration or posttreatment with nz-il ra may not be effective. breakdown of the inflammatory immune response balance is known to be a cause of ibd, but changes in the gut microbiota are also an important factor . we therefore collected cecal contents on the last day of in vivo studies and analyzed the cecal microbiota based on s rrna gene sequencing. for diversity analysis, we determined the indices of α-diversity (observed otus) and β-diversity (the bray-curtis index). however, there was no significant difference with respect to αor β-diversity. these results suggested that nz-il ra improves the symptoms of acute colitis by primarily affecting host il- signaling rather than through effects on the microbiota. our metagenome analysis, however, did not exclude a possible alteration of minor but important microbial species. the anti-inflammatory effect of nz-il ra via the microbiota should be further investigated. the increasing number of patients with ibd in rapidly westernizing developing countries may cause an unprecedented pandemic . combatting this problem using existing ibd drugs would not only be very expensive but also require additional expensive equipment and technology for production and administration. the use of nz-il ra, by contrast, does not require protein purification or difficult culture techniques, and the ability to administer nz-il ra orally makes it an attractive nextgeneration agent for treating ibd. however, the use of genetically modified organisms involves ethical issues, and adequate monitoring is necessary to control for release into the environment and potential side effects in the body. although clinical trials examining gmlab have demonstrated the effectiveness of biological containment strategies and safety in patients, further research is needed to evaluate the effectiveness of gmlab in treating diseases such as ibd [ ] [ ] [ ] . in conclusion, we successfully constructed gmlab that hypersecrete bioactive mil- ra. oral administration of nz-il ra to acute colitis mice alleviated colitis symptoms and suppressed excessive immune reactions in the intestinal tract and mlns. these results suggest that rmil- ra reaches the colon via nz-il ra and inhibits il- signaling. nz-il ra could be used as an inexpensive and effective tool for the treatment of colitis by targeting il- signaling in the colon. we hope that this study will advance the application of microbial therapeutics. worldwide incidence and prevalence of inflammatory bowel disease in the st century: a systematic review of population-based studies enhanced production of interleukin -beta by mononuclear cells isolated from mucosa with active ulcerative colitis of crohn's disease imbalance between interleukin- agonists and antagonists: relationship to severity of inflammatory bowel disease mucosal imbalance of il- and il- receptor antagonist in inflammatory bowel disease. a novel mechanism of chronic intestinal inflammation interleukin is a key driver of inflammatory bowel disease-demonstration in a murine il- ra knockout model alginate/chitosan microcapsules for in-situ 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of autoimmune diseases il- beta promotes th differentiation by inducing alternative splicing of foxp excess il- signaling enhances the development of th cells by downregulating tgf-beta-induced foxp expression years of the nisin-controlled gene expression system (nice) in lactococcus lactis survival of lactococci during passage through mouse digestive tract biological properties of recombinant human monocyte-derived interleukin receptor antagonist gut microbiota and ibd: causation or correlation? ag , a mouth rinse formulation of lactococcus lactis secreting human trefoil factor , provides a safe and efficacious therapeutic tool for treating oral mucositis a phase i trial with transgenic bacteria expressing interleukin- in crohn's disease a phase a randomized placebo-controlled double-blind multi-center dose escalation study to evaluate the safety, tolerability, pharmacodynamics and efficacy of ag in patients with moderately active ulcerative colitis nisincontrolled extracellular production of interleukin- in lactococcus lactis strains, without the requirement for a signal peptide sequence secretion of biologically active porcine interleukin- by lactococcus lactis serine protease inhibitors protect better than il- and tgf-beta anti-inflammatory cytokines against mouse colitis when delivered by recombinant lactococci enhanced secretion of biologically active murine interleukin- by lactococcus lactis intranasal immunization with recombinant lactococcus lactis secreting murine interleukin- enhances antigen-specific th cytokine production expression of biologically active murine interleukin- in lactococcus lactis secretion of biologically active human interleukin (il- ) by lactococcus lactis preventative delivery of il- by lactococcus lactis ameliorates dss-induced colitis in mice we thank chikako miyazaki and masami tsukagoshi of the institute for biomedical sciences, shinshu university, for technical support. we also thank the research center for support of advanced science, shinshu university, for use of their facilities. funding: this study was supported by the kato memorial bioscience foundation, award #h b.author contributions f.n. and s.s. performed the experiments and analyzed the data; t. sato, t. shimosato, and t.o. contributed the reagents, materials, and/or analytical tools; t. shimosato designed the research project; and f.n. and t. shimosato wrote the paper. the authors declare that they have no conflict of interest.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - bobmkvu authors: kaplan, gilaad g.; windsor, joseph w. title: the four epidemiological stages in the global evolution of inflammatory bowel disease date: - - journal: nat rev gastroenterol hepatol doi: . /s - - -x sha: doc_id: cord_uid: bobmkvu inflammatory bowel disease (ibd) is a global disease; its evolution can be stratified into four epidemiological stages: emergence, acceleration in incidence, compounding prevalence and prevalence equilibrium. in , developing countries are in the emergence stage, newly industrialized countries are in the acceleration in incidence stage, and western regions are in the compounding prevalence stage. western regions will eventually transition to the prevalence equilibrium stage, in which the accelerating prevalence levels off as the ibd population ages and possibly as a result of an unexpected rise in mortality during the covid- pandemic. mitigating the global burden of ibd will require concerted efforts in disease prevention and health-care delivery innovations that respond to changing demographics of the global ibd population. in this perspective, we summarize the global epidemiology of ibd and use these data to stratify disease evolution into four epidemiological stages. the journey from a few sporadic cases of inflammatory bowel disease (ibd) to millions of people with ibd is a -year path beginning with the industrial revolution . the distribution of ibd was initially thought to be bound by ethnicity and geography -predominantly affecting people of western european descent . by the end of the twentieth century, these boundaries had been shattered with the disease recorded on every populated continent of the globe , . whereas the incidence of ibd in the western world increased in the twentieth century, the incidence began to increase in newly industrialized countries at the turn of the twenty-first century . in , omran proposed the epidemiologic transition theory to explain human population growth via predominant drivers of mortality . the pre-industrial society was labelled the age of pestilence and famine, in which populations were limited by epidemic outbreaks, deprivation and wars. the age of receding pandemics reflects a reduction in mortality crises and a greater burden of endemic diseases (for example, malaria and tuberculosis). the final the next is a result of human influence: the factors that contribute to these transitions include industrialization, urbanization and westernization of society . by combining epidemiological principles with temporal prevalence trends, a fourth stage can be postulated: prevalence equilibrium, the countervailing force between the increasing mortality of an ageing ibd population and the incidence of ibd (fig. ) . prevalence equilibrium represents a future epidemiological stage that has yet to materialize but provides the framework for predicting the future global burden of ibd. understanding the changing epidemiological patterns is critical to provide clues to the pathogenesis of disease and to support health-care systems in addressing the rising global burden. in this perspective, we provide evidence of the epidemiological stages of ibd using real-world data from different regions across the globe. we show how postulating a fourth stage in ibd evolution provides a theoretical framework for predicting the future burden of disease. a glossary of relevant terms and new terminology introduced herein is detailed in box , and a video abstract and interactive map are available online (see related links). stage one: emergence of ibd in many ways, the evolution of ibd parallels the evolution of biological species; thus, we take a cue from the father of evolution, charles darwin, who had a surprising connection to the emergence of ibd himself. on december , darwin embarked on a -year journey from england to the coast of south america, across southern australia, around the horn of africa and back to england. only months of this journey was on water; the rest of the time, darwin explored foreign lands and made observations that formed the basis of the theory of evolution. in , his seminal book on the origin of species by means of natural selection was published, which revolutionized our understanding of biology . throughout his adult life, darwin was plagued -even while writing his most famous book -with undiagnosed, chronic, gastrointestinal symptoms (for example, abdominal pain, vomiting, ulcerations in the mouth, eczema and peripheral transition proposed by omran was the age of degenerative and human-influenced diseases that arises in industrialized and urbanized societies . mortality is dominated by non-communicable diseases, the occurrence of which is influenced by environmental pressures such as nutrition and lifestyle behaviours . this final stage is a proposed anthropological explanation for the rise in chronic inflammatory conditions . in a editorial, kedia and ahuja postulated that ibd is an example of omran's final stage . the evolution of ibd occurs within omran's degenerative and human-influenced diseases age and has occurred across three distinct epidemiological stages ( fig. ) : emergence, acceleration in incidence and compounding prevalence. emergence is represented by sporadic case reports of ibd . acceleration in incidence is a tipping point whereby incidence steadily increases over several decades, although prevalence remains low . compounding prevalence reflects a steady increase in the population living with ibd, despite stabilization or even a decline in incidence . the transition from one stage in the evolution of ibd to neuropathy) . initially, it was believed that he had contracted chagas disease in south america, but this has been challenged on the basis that he experienced coexisting eye and skin inflammation suggestive of an underlying diagnosis of crohn's disease -a diagnosis that was unheard of during darwin's lifetime ( - ) . the first stage in the evolution of ibd -emergence -occurred in north america and western europe following the advent of the industrial revolution. james watt modified the steam engine in , which triggered the industrial revolution that led to fundamental societal shifts involving manufacturing, urbanization, agriculture and transportation. in this milieu, ibd emerged in the western world, starting as a handful of case studies published at the end of the eighteenth century to taking root as a new affliction of health . matthew baillie, in his publication morbid anatomy of some of the most important parts of the human body, reported among the earliest cases consistent with a diagnosis of ibd . in , john abercrombie published a book describing digestive diseases that included patients who, retrospectively, seem to have had ibd as evidenced by ileocaecal disease . however, the first published case that specifically denotes a unique, non-infectious, gastrointestinal disease was in by british physician samuel wilks -the same year that darwin published his book while possibly affected by the yet-unrecognized crohn's disease . on the heels of wilks's classification, several case reports were published in europe and the usa, including a case series from william hale white in that introduced the term ulcerative colitis into the medical lexicon . with the formal naming of ulcerative colitis, the western world became rooted in the first stage of ibd evolution . by the early s, awareness of ulcerative colitis was mounting with the transition from sporadic case reports to regularly recognizable diagnoses. in , the royal society of medicine assembled a symposium in london, england, to address the surge in the number of cases of ulcerative colitis. the physicians at the symposium reviewed the documented cases of ulcerative colitis that had been collected from seven london hospitals (guy's, london, st mary's, st thomas' , st bartholomew's, st george's and westminster) over the preceding years ( - ) . in the first collaborative effort to investigate the disease, the physicians documented that two or three patients with ulcerative colitis were admitted per annum at each hospital . awareness of ulcerative colitis expanded during the early twentieth century; additionally, an inflammatory disorder outside the colon was beginning to be recognized. wilks's original case report in (later understood to be a case of crohn's disease) described ileal inflammation . t. kennedy dalziel reported a surgical case series in that described chronic inflammation of the terminal ileum . finally, the landmark paper by crohn et al. on regional ileitis fundamentally entrenched two distinct gastrointestinal chronic inflammatory diseases under the paradigm of ibd and established crohn's disease as clinically distinct from ulcerative colitis . by the s, ulcerative colitis and crohn's disease were established in the western world, which was then in the process of shifting to the second epidemiological stage in the evolution of ibd. by contrast, recognition of ibd in the first half of the twentieth century in developing countries of the time lagged behind that in the western world, though chopra and ray described cases of ulcerative colitis in kolkata, india, in (ref. fig. | four epidemiological stages of ibd evolution. the changing pattern in the incidence (orange) and prevalence (blue) of inflammatory bowel disease (ibd) across the four the stages of ibd evolution. developing countries are in the emergence stage, during which sporadic cases of ibd are documented. newly industrialized countries are in the acceleration in incidence stage, during which incidence rises and prevalence is relatively low. countries of the western world are in the stage of compounding prevalence, during which incidence is stable, but prevalence is rising steeply. the slope of the prevalence increase will level off with the transition to the prevalence equilibrium stage, which represents the opposing force between an ageing ibd population and the incidence of ibd. www.nature.com/nrgastro was reported in (ref. ) -a century after darwin's publication of the theory of evolution and wilks's first published case report in england . in , crutzen and stoermer postulated that the effect of human activity on the earth's ecosystem had shifted the planet's geological timescale : they named this period the anthropocene . the anthropocene began with the industrial revolution, coinciding with the emergence of ibd in the western world. during the anthropocene, human population growth has increased tremendously. temporal trend analyses detected an inflexion point after world war ii, confirming an exponential increase in the global population not observed during any preceding period in human history . the global human population doubled, from three billion in to six billion in (refs , ). the dramatic rise in the population since was termed the 'great acceleration' , . the great acceleration is attributed to multiple factors including technological advances, improvements in health care and hygiene, energy utilization, transportation, agriculture and diet, urbanization and the interconnectivity of global economies and societies . in parallel to the great acceleration in human population, the western world transitioned to the second epidemiological stage in the evolution of ibd: the (great) acceleration in incidence. the components of the second epidemiological stage include exponentially increasing incidence in conjunction with low prevalence. in the western world, numerous population-based epidemiological studies show that the acceleration in incidence of ibd spanned the second half of the twentieth century . the majority of population-based studies evaluating inception cohorts from the s to the s showed steadily increasing incidence for both ulcerative colitis and crohn's disease . meticulous longitudinal studies clearly documented this steady increase in incidence of ibd , . for example, a study of the incidence of crohn's disease in cardiff, wales, is the longest continuous epidemiological analysis of the incidence of crohn's disease that dates back to the original description of regional ileitis in (ref. ). initially, the incidence was low and flat ( . and . per , in and , respectively), but showed a spike towards the s ( . per , in , . per , in , . per , in and . per , in ). thereafter, the incidence stabilized ( . per , in and . per , in ) a systematic review of population-based studies investigating the incidence of ibd from to revealed a shifting paradigm of stabilizing incidence in the western world . in many regions in the western world, the rapidly increasing incidence of ibd levelled off in the last decades of the twentieth century and, in some regions, reduced at the turn of the twenty-first century. for example, the most recent data from canada suggest that incidence is stable, but varies according to region: incidence is stable in alberta, decreasing in manitoba, nova scotia and quebec, and variable in ontario by age group (increasing in children and adults aged - years, but stable among other age groups) , . since , more than three-quarters of studies reported stable or decreasing incidence of ibd in the western world . most of these studies report incidence across all ages, though the decline in incidence of ibd might be driven by adult-onset ibd. by contrast, some studies have found that ibd incidence in children , including ibd of very early onset , has increased in the western world over the past two decades. furthermore, • ceiling incidence range a : the range of maximal reported incidence values as defined by the th to th percentiles of the amalgamated incidence data reported in a region. • coalescing incidence range a : the range of reported incidence values as defined by the th to th percentiles (the interquartile range) of the amalgamated incidence data reported in a region. • developing countries: countries/regions categorized as least developed by the united nations ( definitions) . • epidemiologic transition theory: a theory of epidemiological epochs defined by population growth rates and predominant drivers of mortality. • ewar-ibd a : the elderly to working age ratio (ewar); the ratio of the prevalence of inflammatory bowel disease (ibd) among older people to the prevalence among the working-age population, where the older population is defined as those aged > years and the working-age population is defined as those aged - years. • newly industrialized countries: countries/territories categorized as economies in transition or developing economies by the united nations ( definitions) . • prevalence doubling period a : the time taken for the prevalence in a defined region to double. • stage : emergence a : the first stage of ibd evolution, in which sporadic incident cases begin to emerge in a population. • stage : acceleration in incidence a : the second stage of ibd evolution, in which a dramatic increase in the number of incident cases is observed, but overall prevalence remains low. • stage : compounding prevalence a : the third stage of ibd evolution, in which incidence rates stabilize or even decline, but the slope of prevalence continues to accelerate owing to decades of high incidence with low mortality. • stage : prevalence equilibrium a : the theoretical fourth stage of ibd evolution, in which the slope of prevalence begins to level off owing to stable incidence and an ageing prevalent population with higher mortality. • true rise of incidence a : an increase in the incidence of disease as a result of exposure to environmental determinants. • unmasking of incidence a : an increase in the incidence of disease due to economic factors that improve awareness and detection of persons contracting the disease. • western world: countries/territories from western europe and regions that were predominantly colonized by europeans (that is, north america and australia) that share cultural, lifestyle, dietary and socioeconomic influences in their society (developed economies as classified by the united nations ( definitions)) . a indicates a new term used in this perspective. nature reviews | gastroenterology & hepatology incidence continues to increase in certain countries including new zealand and some countries/regions in europe, such as spain and denmark . even among the countries/regions with increasing incidence, the collective data indicate that this might be particular to western nations catching up to a common incidence. for example, in southern spain, the incidence of ibd increased from . per , in - to . per , in - , which is still lower than most reported incidence values in the western world, for which the mean incidence for the period - was . times higher ( . per , ) . future studies will demonstrate whether incidence will eventually level off in most countries/regions in the western world; such stabilization in incidence will denote the end of the second epidemiological stage. demarcating the end of the second epidemiological stage is fundamentally important because it enables the calculation of the coalescing incidence range and the ceiling incidence range. the coalescing incidence range is the range of incidence values reported in most regions within a unit of time. ng et al. performed a systematic review of incidence values between and , which provides our current understanding of the coalescing incidence range and ceiling incidence range for the western world . figure displays the coalescing incidence range as defined by the interquartile range. since (that is, the end of the second stage within the western world), the coalescing incidence ranges of crohn's disease and ulcerative colitis were - per , and - per , , respectively ( - per , collectively) in the western world (fig. ). as the year following diagnosis of ibd is often the most costly and resource intensive, the coalescing incidence range is relevant for health administrators and policy makers attempting to define the burden of ibd in patients with newly diagnosed disease as compared with the burden in the prevalent population as a whole. additionally, the systematic review data of ng et al. provide the ceiling incidence range for the western world, which is the range of maximal reported incidence values as defined by the th to th percentiles. for crohn's disease, the ceiling incidence range is in the range . - . per , (refs , , ), and for ulcerative colitis, it is in the range . - . per , (refs , , ). the ceiling incidence range needs to be interpreted cautiously because outliers can represent unique clinical scenarios or methodological biases that skew the incidence range upwards. for example, nova scotia, canada, has been reported to have among the highest incidences of ibd in the world at per , , which is nearly double the values reported for other provinces in canada . the high incidence of ibd in nova scotia might be methodological in origin because incidence calculations relied on administrative databases using non-validated coding algorithms, which are subject to misclassification biases that might have inflated the incidence . by contrast, the danish territory of the faroe islands had the highest worldwide average annual incidence of ibd at . per , in , with a predominance of ulcerative colitis . the investigators articulated an elegant argument that the increased incidence of ibd in the faroe islands is not simply an artefact of improved awareness, diagnosis and data capture . instead, the exceedingly high incidence of ulcerative colitis was explained by a genetic shift in a stable, geographically enclosed society in conjunction with unique environmental exposures within the population . as most countries in the western world transitioned into the third stage in the evolution of ibd at the end of the twentieth century, newly industrialized countries in asia and latin america entered the second stage: the acceleration in incidence. this stage is associated with rapidly rising incidence and low prevalence. for example, the annual incidence of ibd in bahrain increased from an average of per , to per , during the period - , but the total prevalent population remained under persons at per , ( . %) (ref. ). also, a four-decade analysis of the incidence of ibd in malaysia showed a slow increase from the s ( . per , ), to the s ( . per , ) and to the s ( . per , ), and then more than doubling to . per , in the past decade ( - ); however, the prevalence of ibd in malaysia remained low at per , ( . %) in (ref. ). moreover, a systematic review of clinical and epidemiological studies on ibd from latin america documented the emergence of ibd in the twentieth century with a rapidly rising incidence during the twenty-first century . similarly, prevalence remains low in most regions in latin america. for example, in the state of são paulo, brazil, the incidence of ibd was . per , during the period - (ref. ) and peaked at . per , during the period - , but the prevalence remained low at . per , ( ) ( ) ( ) ( ) ( ) . tremendous heterogeneity between the reported incidence rates exists among newly industrialized countries as well as among regions within these countries. for example, in the inception cohort ( - ) followed in the asia-pacific crohn's and colitis epidemiology study (access) , , the incidence of ibd ( - ) was a low . per , in the philippines as compared with . per , in hong kong . furthermore, within nine separate regions in mainland china, the incidence varied from . per , in xi'an to . per , in guangzhou . in part, the explanation for this heterogeneity is the challenge of conducting high-quality population-based chronic disease surveillance research in newly industrialized countries, due to the differences between regional populations (for example, the degree of westernization of diets and lifestyles) that make it difficult to generalize findings , , . however, the data available suggest that the rising incidence of ibd and the heterogeneity of reported values in newly industrialized countries at the turn of the twenty-first century can be explained by two fundamental factors: first, the unmasking of incidence in which economic advances improve awareness of ibd and access to health care, which reveals undiagnosed ibd; and, second, the true rise of incidence due to westernization of society that genuinely increases the number of people being diagnosed with ibd. the unmasking of incidence is essentially a diagnostic bias: more new cases of ibd are diagnosed as the ability to detect ibd improves. this factor is influenced by economic factors, leading to increased awareness, improved diagnosis, greater access to health care and improved disease surveillance . collectively, these factors reveal the incidence of ibd and, over time, the reported incidence steadily increases. an analogy to the unmasking of incidence is the ratio of the number of ulcerative colitis diagnoses to the number of crohn's disease diagnoses in a region. historically, ibd typically emerges in a population as cases of ulcerative colitis, eventually followed by cases of crohn's disease. over time, crohn's disease becomes more commonly diagnosed as the incidence approximates and, in some cases, surpasses that of ulcerative colitis . for example, in malaysia the ulcerative colitis to crohn's disease ratio was : in the s and fell to . : from to (ref. ). the diagnosis of ibd is skewed towards ulcerative colitis in lower income regions with limited technology (for example, access only to sigmoidoscopy); however, as economies improve, so too does health-care infrastructure (for example, access to specialists and colonoscopy), which improves the ability of physicians to accurately diagnose crohn's disease . moreover, newly industrialized countries in asia and latin america are experiencing a rising incidence of ibd that is independent of the unmasking phenomenon. the true rise in incidence is fundamentally an epidemiological transition state whereby individuals carrying genetic susceptibility mutations are exposed to environmental factors associated with the westernization of society that might alter their intestinal microbiota, particularly early in life, which could trigger ibd later in life . several reviews have contrasted the environmental determinants of ibd in the western world with those of newly industrialized countries , , . overall, a pattern is observed such that the highest incidence of ibd has been reported in regions with the highest population density . for example, over the past years, china experienced a massive population shift from rural communities to megacities, each with a population of more than ten million . inhabitants in these urban environments have been exposed to westernization of their society including diets containing higher levels of fat and refined sugars and changes in lifestyle behaviours, such as an increase in smoking, less breastfeeding, greater exposure to antibiotics and improved hygiene and sanitation . all these factors have introduced a pressure that is driving the true incidence of ibd upwards. newly industrialized countries in asia and latin america are entrenched in the second stage of ibd evolution. preliminary data suggest that, over the next decade, the incidence in these regions is likely to approximate that in the western world. for example, some regions in brazil, china and india already have published incidence values that are close to the coalescing incidence range (for example, - per , for ulcerative colitis) in the western world , , , . most importantly, with decades of rising incidence, these countries will witness an exponential growth in prevalence until they eventually transition into the third epidemiological stage in the evolution of ibd: compounding prevalence. in , a seminal paper by freeman and hutchison was published in the american journal of epidemiology, demonstrating that, in a stable population, prevalence is the product of the incidence and the average duration of disease . this concept was initially applied to acute, self-limited conditions (that is, infectious diseases). the formula also estimates the prevalence of chronic diseases in an ageing population in which disease duration is relatively short owing to high mortality (for example, chronic obstructive pulmonary disease, dementia) , or owing to recovery within a specified time period (for example, within a -year window, patients with certain cancers either die or are classified as survivors) , . consequently, health-care providers and administrators can integrate information on incidence and disease duration to predict the burden of acute and chronic diseases. however, this formula cannot explain prevalence patterns observed in populations with ibd. although ibd can occur in individuals of any age, it is most commonly diagnosed in adolescents and young adults (ages - years) . without a cure, and owing to relatively low mortality, patients with ibd diagnosed at a young age can live a long life . consequently, estimating average disease duration over the course of a lifetime is not feasible in ibd; that is, the prevalence of ibd cannot be estimated by multiplying incidence by average disease duration. fewer than % of the prevalent population of patients with ibd living in the western world were over the age of years in (ref. ). with a life expectancy of ~ years in the western world, most patients living with ibd in are alive today in . in other words, most patients with ibd cared for in a gastroenterology clinic in are also being cared for now, in . even if the incidence of ibd in the western world stabilizes, or declines, new diagnoses of ibd continue to increase the prevalent population of patients with ibd . moreover, immigration from low-incidence countries/ regions (for example, developing countries) to the western world will lead to a further increase in prevalence as the first-generation offspring of immigrants have a similar risk of acquiring ibd as individuals in western populations . in a chronic, incurable disease of the young, as long as incidence surpasses mortality (that is, there are more patients with newly diagnosed ibd than patients with ibd dying) then prevalence will steadily rise -a phenomenon called 'compounding prevalence' (fig. ) . during the second half of the twentieth century, incidence rapidly rose in the western world, but prevalence remained relatively low (that is, second epidemiological stage). after each decade of escalating incidence, the prevalence accumulated; this aspect is analogous to compound interest in which interest (that is, incidence) earned over time is added to the principal fund (that is, prevalence) and is more than withdrawals (that is, mortality). in the third epidemiological stage, the 'tug-of-war' between incidence and mortality greatly favours incidence, and so prevalence increases. the prevalence doubling period -the time taken for the prevalence in a defined region to doubleconceptualizes the magnitude of rising prevalence. in the western world, the prevalence doubling period is currently likely to be - years; for example, in olmsted county, mn, usa, the prevalence doubling period was approximately years from per , in to per , in (refs , , ). in , the prevalence of ibd was per , in olmsted county, and therefore almost doubled again in the years from (ref. ). population-based epidemiological studies from canada and scotland published in documented the compounding prevalence of ibd: during the twenty-first century, prevalence rose by % and % per year in canada and scotland, respectively , . in , the calculated prevalence of ibd in canada and scotland was . % and . % , respectively, which was similar to the . % reported in olmsted county in (ref. ). forecasting models indicate that the prevalence doubling point of % would occur over years in canada ( to ) and years in scotland ( to ) . owing to natural population growth over time, including both new births and projected immigration patterns, the doubling of prevalence from . % to . % in some countries/regions, represents a more than % rise in the number of individuals with ibd. for example, in canada, . % prevalence of ibd in represented , individuals with ibd, whereas , individuals are predicted to be living with ibd in -a % increase from . the consistency of reported and forecast data between north america and europe highlights an impending challenge for health-care providers, administrators and systems. table shows the population of individuals with ibd in selected regions in the western world with currently high ibd prevalence assuming a growth in prevalence from . % in , to . % in and to % in . accounting for natural population growth and assuming a % prevalence in , the number of people living with ibd in the western world over the next decade could surpass ten million -the most people with ibd ever recorded (table ) . perhaps more concerning than the growth to a % prevalence over the next decade is whether the western world will experience another doubling of prevalence by , bringing the ibd population to %. fortunately, it is unlikely that another doubling of prevalence is in store for regions in most of europe, north america or oceania; it is far more likely that these regions will transition to the fourth epidemiological stage (prevalence equilibrium) before . individuals born between and are known as the 'baby boomer generation' from the sharp increase in births stemming from the economic prosperity following the end of world war ii . in , this generation will be aged - years, meaning that nearly one in five people living in the western world will be over the age of years . economists highlight the potential fiscal challenge associated with the shifting age demographic as the proportion of older individuals increases relative to those aged - years (considered here to represent the working-age population) , . moreover, life expectancy has increased with advances in public health and innovations in medicine . consequently, health-care systems face an expanding demographic burden as baby boomers age and life expectancy increases . the ibd population in the western world will undergo similar shifts in population demographics that will ultimately led to transition in the epidemiological stages from compounding prevalence to the fourth epidemiological stage -prevalence equilibrium. prevalence equilibrium is a hypothetical state in which the incidence of ibd approximates the mortality, causing prevalence to stabilize and, in some regions, decline (fig. ) . the ibd population has aged over time: there are more older individuals with ibd in than in . for example, the prevalence of ibd among older individuals in ontario, canada, increased by . % per year between and (ref. ). thus, the likelihood of the ibd cohort sur viving to was higher than it is for the ibd cohort surviving to . the ageing ibd popu lation will have reduced life expectancy from age-related comorbidities such as cardiovascular disease and cancer, as well as a small increased risk of death of patients with ibd as compared with age-matched individuals without ibd as controls , . consequently, the prevalence of ibd is unlikely to double to %. although the prevalence equilibrium stage is conjectural, several signs are indicative of its arrival: decline in the slope of prevalence over time; prolongation of the prevalence doubling period; and an elevated ratio of the prevalence of ibd among older individuals to the prevalence among the working-age population (elderly to working-age ratio; ewar-ibd). a shift to the fourth epidemiological stage will be preceded by a change in the slope of prevalence over time. a steep slope -positive or negative -is representative of rapid acceleration or deceleration in prevalence, whereas minor fluctuations in a mostly flat slope indicate relative stability (fig. ) . as the ibd population ages, their mortality increases and, in conjunction with stable (and even more so with declining) incidence, the steep positive slope indicative of compounding prevalence will eventually become more moderate until the point where prevalence levels off, and potentially declines thereafter. as regions begin to transition between compounding prevalence to prevalence equilibrium, the prevalence doubling period will increase in length. in the western world, the prevalence doubling period is currently - years , , . although prevalence is anticipated to increase over the next decade, as regions transition to the fourth stage, the steep slope of prevalence will decline, leading to a lengthening of prevalence incidence true rise in incidence (environmental) unmasking of incidence (economic) fig. | the interplay and determinants of ibd incidence, prevalence and mortality. the figure shows the unmasking of incidence by economic factors that improve the detection of undiagnosed inflammatory bowel disease (ibd) (for example, access to colonoscopy) and the true rise in incidence due to westernization of society (for example, smoking) that influence the extent of incidence (water from the tap), which adds to the prevalent population (accumulation of water in the sink). the prevalence level is then affected by the rate of mortality, which is influenced by ageing of the ibd population with complications from longstanding disease, age-related comorbidities and covid- . www.nature.com/nrgastro the prevalence doubling time. this second indicator of the prevalence equilibrium will be seen in future studies that will document prevalence not rising by an anticipated % within a period of - . years. by contrast, newly industrialized countries that are undergoing rapid increases in incidence currently show very short prevalence doubling periods, which extend as the regions get nearer to stage . for example, in taiwan the ewar-ibd represents the relationship between the prevalence of ibd in the population over the age of years and the prevalence in those aged - years. the ewar-ibd is conceptually analogous to the old-age dependency ratio that is used in population demography to estimate the number of individuals older than years for every individuals aged - years . older individuals with ibd are the fastest growing population with ibd in canada . in canada, the total (all ages) prevalence of ibd rose from per , in to per , in to per , in (ref. ). during this period the ewar-ibd in canada increased from . ( ) to . ( ) to . ( ) . an ewar-ibd of . in means that individuals with ibd were older than years for every individuals with ibd aged - years in canada. once the ewar-ibd exceeds . (that is, one individual with ibd older than years for every two individuals with ibd aged - years), then we hypothesize that a region might begin to transition to prevalence equilibrium. so, are regions in the western world at the threshold of entering the fourth epidemiological stage in ? no. for example, the incidence of ibd in alberta, canada, which has among the highest reported incidence of ibd in the world , fell by . % per year from . per , in to . per , in ; however, based on mortality of individuals with ibd in , the incidence of ibd has to fall below per , for prevalence to stabilize . consequently, individuals with newly diagnosed ibd will continue to add to the prevalent population, which will continue to drive compounding prevalence over the next decade, with prevalence forecast to be % by (ref. ). the gap between incidence and mortality is so wide that decades will probably pass before countries in the western world enter prevalence equilibrium. global ibd epidemiology: to every region in the world is currently in one of the first three epidemiological stages of ibd evolution and, with time, will transition through all four stages (fig. ) . developing countries are currently in the first stage of evolution (emergence) and can expect to transition to the second stage if their economies advance and their societies westernize. most newly industrialized countries are entrenched in the second stage (acceleration in incidence), but if these regions follow the epidemiological patterns of the western world, they are likely to transition into the third stage over the next three decades. the western world is in the third epidemiological stage (compounding prevalence), and is anticipated to cross towards prevalence equilibrium within the next years. the clinical challenges are different for each of the epidemiological stages of ibd evolution. understanding the transition from the current epidemiological stage (in ) to the next stage is paramount to prepare for the evolving burden of ibd over the next years in each of these different regions. developing countries have a low per capita income, with their economies predominantly driven by agriculture, and are at the earliest stages of industrialization. health-care infrastructure in these countries is limited, and disease surveillance can be rudimentary. consequently, observational data on the incidence and prevalence of ibd in these regions are lacking. for example, high-quality population-based epidemiological data from developing countries in africa are essentially non-existent. however, ibd can, and does, manifest among individuals from developing countries. for example, children of immigrants from sub-saharan most importantly, developing countries should be aware that, if their economies advance and their societies show greater westernization, the incidence of ibd will rise throughout the twenty-first century. lessons from newly industrialized countries in asia and latin america that transitioned to the second stage at the turn of the twenty-first century can prepare developing countries before they move beyond the emergence stage. the international ibd community should invest in clinical care and epidemiological surveillance systems of ibd in developing countries -both to support the anticipated rising incidence as seen in the developing world and for the unique opportunity to institute research aimed at exploring the social and environmental determinants of ibd. newly industrialized countries in asia and latin america that are embedded in the second epidemiological stage are currently (in ) experiencing a juxtaposition of rapidly rising incidence with low prevalence. if newly industrialized countries follow the same epidemiological patterns as the western world, then, over the next years, these countries should approximate the coalescing incidence range for ibd in the western world: - per , . moreover, after several decades of accelerated incidence, prevalence will begin to climb rapidly as these regions enter the compounding prevalence stage, similar to the experience in the western world at the turn of the twenty-first century. for example, japan and south korea experienced rapid industrialization after world war ii and transitioned into the second stage earlier than other countries in asia , . japan and south korea serve as examples of the anticipated escalation in prevalence that will manifest throughout asia over the next years. a nationwide study in japan demonstrated a prevalence of ibd of per , in (ref. . regions in the first stage (emergence) are highlighted in green, regions in the second stage (acceleration in incidence) are highlighted in yellow and regions in the third stage (compounding prevalence) are highlighted in orange. regions with black borders are regions for which population-based incidence or prevalence studies are available. regions with grey borders lack data on incidence or prevalence, highlighting current gaps in knowledge of ibd epidemiology , . www.nature.com/nrgastro years -as newly industrialized countries transition from the second to the third stage -these countries can draw from the lessons learned from the western world over the past years. newly industrialized countries will soon need to contend with the adoption and implementation of clinical care paradigms established by the western world, but modified for local needs. owing to the tremendous heterogeneity of socioeconomic status between newly industrialized countries, and within disparate regions within any given region, the major challenge will be the equitable, affordable and accessible distribution of care for patients with ibd. consequently, health-care systems will need to prepare personnel, resources and medical infrastructure in readiness for the global increase in the number of cases of ibd. compounding prevalence in the western world, in conjunction with an ageing ibd population, will change the face of gastroenterology clinics in the near future. over the next decade, gastroenterologists involved in adult care will contend with an escalation in the number of patients living with ibd while simultaneously managing an ageing population that will have a mixture of complications of longstanding ibd and age-related comorbidities. older individuals with ibd will continue to make up a higher proportion of the adult patients attending gastroenterology clinics (that is, with ewar-ibd exceeding . ) and octogenarians with ibd will become more common. addressing these future clinical pressures will require innovation to health-care delivery in gastroenterology clinics. potential innovations in delivery of care are multifaceted, but include advances in multidisciplinary care models , personalized medicine , telehealth , data mining and electronic medical records , and artificial intelligence used to improve monitoring and treatment of ibd , . in parallel with innovating health-care delivery, the ibd community should focus on efforts to shorten the transition time from compounding prevalence to prevalence equilibrium. expediting this transition will facilitate health-care system stability as the number of cases will be relatively stable year to year, enabling easier and/or better resource planning by health-care administrators and policy-makers. moreover, prevalence equilibrium might result in a decline in prevalence in certain jurisdictions, which would reduce the overall burden of ibd to society. a concerted focus on the prevention of ibd could drive the decline in ibd incidence. new research on the gut microbiome and environmental determinants that focus on ibd disease prevention are ultimately needed to decelerate the slope of the increasing prevalence of ibd across the world. among western countries/regions where incidence is reported to be decreasing, the causes for the declining incidence should be explored and proven environmental modification strategies should be adopted in other regions . finally, unexpected events could entirely change the evolution of the global epidemiology of ibd. a fundamental discovery that leads to a cure for ibd would dramatically decrease its incidence. alternatively, the ongoing covid- global pandemic has the potential to dramatically affect mortality among the prevalent ibd population . surveillance epidemiology of coronavirus under research exclusion (secure-ibd) is an international registry reporting on patients with ibd who have tested positive for covid- . an interactive updated online map of the secure-ibd registry can be found in related links. the primary risk factor for a severe complication of covid- -defined as ventilator use, intensive care unit admission or death -in patients with ibd is advanced age. among people over the age of years who were reported in secure-ibd, % experienced a severe complication of covid- , with older age (adjusted or . , % ci . - . ) and two or more comorbidities (adjusted or . , % ci . - . ) associated with poor outcomes. consequently, older people are the most vulnerable population with ibd during the pandemic . this tragic, unprecedented pandemic could contribute to an earlier transition to prevalence equilibrium. ibd serves as a case study on the evolution of modern diseases. this perspective provides a novel framework for describing the changing epidemiological patterns of ibd globally: from the industrial revolution, to the current state in , and projected out to (box ). additionally, this article synthesizes terminology and introduces new definitions that explain the evolution of ibd across epidemiological transition periods (box ). the four epidemiological stages of ibd are emergence, acceleration in incidence, compounding prevalence and prevalence equilibrium. the global burden of ibd can be mitigated by shortening the transition from compounding prevalence to prevalence equilibrium. to accomplish this goal, the ibd community should focus on disease prevention to drive down the incidence of ibd. in the meantime, developing, newly industrialized regions and countries of the western world all need to innovate their current health-care delivery to address the evolving demographics of their ibd populations as they transition across epidemiological stages. • the evolution of inflammatory bowel disease (ibd) occurs across four epidemiological stages: emergence, acceleration in incidence, compounding prevalence and prevalence equilibrium. • in , newly industrialized countries/regions in asia and latin america are in the acceleration in incidence stage, which is associated with rapidly rising incidence and low prevalence. • acceleration in incidence is driven by the unmasking of incidence, in which economic advance improves detection of ibd, and the true rise of incidence occurs owing to westernization of society. • the western world is in the compounding prevalence stage: stable incidence (coalescing incidence range of - per , persons) and rapidly increasing prevalence, which will be as high as % by in many regions. • the western world will transition to prevalence equilibrium stage (stabilization of prevalence) owing to the advancing age of the ibd population as denoted by the ratio of the prevalence among older individuals (aged > years) to the prevalence among the working-age population (aged - years). • shortening the transition to prevalence equilibrium via measures for ibd prevention and innovating health-care delivery to address changing demographics of the ibd population might help stem the global ibd burden. the global burden of ibd: from to globalisation of inflammatory bowel disease: perspectives from the evolution of inflammatory bowel disease in the uk and china increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review worldwide incidence and prevalence of inflammatory bowel disease in the st century: a systematic review of population-based studies the epidemiologic 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associations between genetic factors and response to treatment of inflammatory bowel diseases performance of a deep learning model vs human reviewers in grading endoscopic disease severity of patients with ulcerative colitis development and validation of machine learning models in prediction of remission in patients with moderate to severe crohn disease a new coronavirus associated with human respiratory disease in china corticosteroids, but not tnf antagonists, are associated with adverse covid- outcomes in patients with inflammatory bowel diseases: results from an international registry health nutrition and population statistics united nations world economic situation and prospects the authors thank f. underwood and s. coward for contributing to the article through literature searches, providing advice and graphic design. the authors have received funding from the canadian institutes for health research operating grant reference number pjt . the authors contributed equally to all aspects of the article. the authors declare no competing interests. nature reviews gastroenterology & hepatology thanks v. ahuja, j. burisch and m. kappelman for their contribution to the peer review of this work. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. global ibd visualization of epidemiology studies: https:// wpsites.ucalgary.ca/gilkaplan/ioibd-gives/ kaplan lab interactive maps: https://wpsites.ucalgary.ca/ gilkaplan/ secure-ibd interactive map: https://wpsites.ucalgary.ca/ gilkaplan/secure-ibd/ video abstract: https://youtu.be/ cp tacc xm key: cord- - my t authors: turner, dan; huang, ying; martín-de-carpi, javier; aloi, marina; focht, gili; kang, ben; zhou, ying; sanchez, cesar; kappelman, michael d.; uhlig, holm h.; pujol-muncunill, gemma; ledder, oren; lionetti, paolo; dias, jorge amil; ruemmele, frank m.; russell, richard k. title: corona virus disease and paediatric inflammatory bowel diseases: global experience and provisional guidance (march ) from the paediatric ibd porto group of european society of paediatric gastroenterology, hepatology, and nutrition date: - - journal: j pediatr gastroenterol nutr doi: . /mpg. sha: doc_id: cord_uid: my t introduction: with the current coronavirus disease (covid- ) pandemic, concerns have been raised about the risk to children with inflammatory bowel diseases (ibd). we aimed to collate global experience and provide provisional guidance for managing paediatric ibd (pibd) in the era of covid- . methods: an electronic reporting system of children with ibd infected with sars-cov- has been circulated among pibd centres affiliated with the porto and interest-group of espghan. a survey has been completed by major pibd centres in china and south-korea to explore management during the pandemic. a third survey collected current practice of pibd treatment. finally, guidance points for practice have been formulated and voted upon by pibd authors and porto group members. results: eight pibd children had covid- globally, all with mild infection without needing hospitalization despite treatment with immunomodulators and/or biologics. no cases have been reported in china and south korea but biologic treatment has been delayed in children, of whom ( %) had exacerbation of their ibd. among the porto group members, face-to-face appointments were often replaced by remote consultations but almost all did not change current ibd treatment. ten guidance points for clinicians caring for pibd patients in epidemic areas have been endorsed with consensus rate of % to %. conclusions: preliminary data for pibd patients during covid- outbreak are reassuring. standard ibd treatments including biologics should continue at present through the pandemic, especially in children who generally have more severe ibd course on one hand, and milder sars-cov- infection on the other. supplemental digital content: an infographic accompanying this article can be found at. (jpgn ; : - ) a global pandemic of coronavirus disease is now apparent. sars-cov- infection resulting in coronavirus disease causes significant pulmonary disease in adults leading to intensive care and sometimes death. the impact in adults including on mortality is greater in patients with chronic diseases. the first paediatric inflammatory bowel disease cases to have sars-cov- infection have been described. coronavirus disease in children, including paediatric inflammatory bowel disease, appears to be mild. standard paediatric inflammatory bowel disease treatments should continue since delay in infusions in endemic areas has been associated with disease flare. c oronavirus disease is caused by the zoonotic coronavirus sars-cov- and started in china in december ( ) . by march , it had been declared by who as a global pandemic. it is predominantly spread by airborne droplets but there is also viral shedding in stool, giving the potential for faecal-oral transmission ( , ) . in adults, covid- predominantly presents with cough and fever resulting in a proportion of patients developing acute respiratory distress syndrome (ards) ( ) . sars-cov- infection may also cause gastrointestinal symptoms ( , ) . the disease course of covid- in children is predominantly benign with mild or even no symptoms, and almost no reported mortality ( ) ( ) ( ) . the severe pulmonary involvement of the virus may be caused also by hyperinflammation and a secondary hemophagocytic-lymphohistiocytosis (hlh)-like presentation ( , ) . sars-cov- enters cells via the angiotensin-converting enzyme- (ace- ) receptor that is abundantly expressed in cells of the lungs, oral cavity, and the gastrointestinal tract. lymphopenia and elevated c-reactive protein (crp), ferritin and lactate dehydrogenase levels are associated with a more severe course ( ) . increased levels of cytokines and chemokines, such as interleukin- (il- ) , have been associated with increased disease severity in adults ( ) and children ( ) . it seems that the most severe presentations of covid- result from hyperinflammatory cytokine responses in particularly dysregulated il- -dependent acute phase responses associated with a decrease in cytotoxic t and natural killer (nk) cells. those findings explain why in addition to antiviral therapies, immunomodulatory therapies and passive immunisation strategies could potentially be considered to improve outcome in severely affected patients ( ) . consistent with this hypothesis is the report that in an endemic area, only children receiving immunosuppressant medication for liver transplantation developed sars-cov- infection and none were severe ( ) . the coronaviruses, sars-cov and mers-cov, were responsible for previous epidemics. although clinical and immunological data from these viruses cannot directly be translated to predict interventions in sars-cov- infections, it is noteworthy that thiopurine metabolites, -mercaptopurine and -thioguanine, have been shown to have direct antiviral activity by inhibiting the papain-like protease of both viruses ( ) as well as host proteins involved in antiviral response ( ) . systemic steroids, however, did not confer substantial clinical benefit ( ) . indeed, the safety of corticosteroids during covid- is unclear ( ) , but it seems that if used for short periods and at a low dose they are not related to a worse prognosis, even in patients with covid- pneumonia ( ) . there are no published data about the safety of monoclonal antibodies during this infection although anti-il- receptor antibody has been used in a few patients with covid- with promising results ( ) . current literature related to other viral infections does not indicate stopping these treatments or modifying therapeutic regimes ( ) . in light of the hyperinflammatory immune response seen in patients with covid- it is highly relevant that blockade of il- r with tocilizumab resulted in clinical improvement associated with normalisation of fever, lymphocyte counts, and crp in a retrospective group of adults with severe sars-cov- infection ( ) . locally active medications, such as anti-a b (eg, vedolizumab) or budesonide are unlikely to have a major impact on systemic nor pulmonary sars-cov- responses. despite the above, the ibd-related immunosuppressive treatment has raised concerns regarding the management of covid- with potential implications for treatment, isolation, and routine hospital attendance ( , ) . provisional reports from adult ibd centres in china are reassuring ( ) , and as some of the pulmonary damage may be caused by autoinflammatory response of the host, immunosuppressive medication have even been postulated to protect from severe disease ( , ) . nonetheless, children may have different recommendations than adults, given the overall milder course of the infection. in addition, ibd in children tends to be more extensive and severe than adults with consistently higher need for immunomodulators and biologics. we thus aimed to collate available data on paediatric ibd (pibd) and sars-cov- globally and to develop consensus statements for the management of pibd during the covid- pandemic. the consensus process included specialists in paediatric ibd from the paediatric ibd porto group of european society of paediatric gastroenterology, hepatology, and nutrition (espghan). following an open call to the members of the paediatric ibd porto group of espghan, international experts were selected as the writing group. in addition to porto group members, external paediatric experts from china (y.h. and y.z.) and south korea (b.k.), being the first endemic areas, were invited to participate, as well as a representative from the secure-ibd registry (m.k.). a redcap reporting system has been constructed to collect all covid- cases among children with ibd in the porto group-affiliated paediatric ibd centres in europe (and several beyond). we asked for cases with a virological confirmation of sars-cov- , but to avoid reporting bias of the more severe cases we allowed also highly suspected cases when testing was not available as per local testing policy since several countries permit testing only those with evidence of pneumonia (eg, france and spain). nonetheless, the suspected cases were labelled and justified individually based on both typical symptoms and close contact with a confirmed case. a -day follow-up was required to ensure capturing of the disease severity. the registry included demographic questions as well as pre-infection ibd clinical explicit details, treatments, and outcomes. the ethics committee of shaare zedek medical center, jerusalem, waived the need for approval, given the urgent need for reporting clinical experience and as the report was retrospective, anonymous, and without contacting patients. for the secure ibd cases, the unc-chapel hill office for human research ethics has determined that storage and analysis of deidentified data does not constitute human subjects research as defined under federal regulations ( cfr . and cfr . ) and does not require irb approval. a survey among members of the porto group on changes in common practice in this new situation was launched via online platform in march , . investigators working in tertiary centers around europe, israel, and canada completed the survey. different topics related to how the centers have adapted to covid- outbreak were collected, both regarding therapeutic strategies and in the logistic organization of the pibd units. a questionnaire was sent on march , to paediatric gastroenterology centers in china to evaluate the impact of covid- on the prognosis of ibd children. these centers were distributed across cities including shanghai, beijing, guangzhou, shenzhen, hangzhou, chongqing, chengdu, wuhan, changsha, zhengzhou, xi'an, xiamen, guiyang, nanjing, and shenyang, in total care for children with ibd. a questionnaire was sent on march , to tertiary centres in the metropolitan city of daegu and in gyeonsangbuk-do province where the majority of the total south-korean covid- infection cases have been confirmed. data regarding the changes in medication prescription by physicians, changes in hospital visits, medication administration, and disease exacerbation were collected since january , , the date of the first covid- occurrence in south korea. on the basis of the above collective data and literature review, the writing group formulated guidance points, which were sent to all members of the porto group of espghan for comments and electronic voting (appendix). it has been decided a priori that consensus is reached by at least % of voters. on the basis of the literature review, the initial global experience described below and the practice surveys, statements were formulated and agreed upon by the writing group. voting by the porto-group members and the authors retained of which (table ) postponing clinics except those experiencing flares or new diagnosis has been practiced by % of centres. of the centres, ( %) encourage their patients not to make any changes in current treatments in response to covid- ; drug doses and infusion intervals were not changed and combination therapy was continued. children requiring infusions (ie, infliximab, vedolizumab) continued with the same regimen. twenty-nine centers ( %), including endemic areas in europe, had no limitations in the administration of parenteral drugs. other actions implemented in the various centers included facilitating drug administration to the patients (eg, modifying pharmacy opening hours, and increasing the number of dispensed doses) and limiting or stopping nonurgent endoscopic procedures. seven children with ibd and covid- have been reported until march , by the sites affiliated with the paediatric ibd porto group of espghan ( table ) . all cases had a mild infection without the need for admission despite treatment with immunosuppressive medications, steroids, and/or biologics. the underlying ibd remained generally stable during the infection and the ibd-related medications were not held in any of the cases. surveillance epidemiology of coronavirus under research exclusion (secure-ibd) is an international registry to monitor and report on outcomes of covid- occurring in ibd patients. as of april , , adults and children with confirmed covid- infection have been reported. similar to the porto group cases, the paediatric patients had mild course of covid- and did not require hospitalization (known clinical data of the first case is added to table ). a total of adult patients have been hospitalised and died, all older than the age and of whom older than years of age. we provide the first document on the global impact of sars-cov- infection on paediatric ibd to date, from which we have generated guidance points for paediatric gastroenterologists in the era of this covid- pandemic. the general message of continuing current ibd treatments is supported by the chinese and south korean experience reported here of $ % disease exacerbation in children whose infliximab infusions were delayed. consensus rate . ibd per-se does not currently seem to be a risk factor for acquiring sars-cov- , nor for a more severe infection. % . for decreasing the risk of contracting sars-cov- in children with ibd, we recommend using the same measures as in the local population during the pandemic (eg, good hand hygiene, avoiding contact with anyone with respiratory symptoms and social distancing). . when possible by local situation and resources, children should continue follow-up visits to ensure appropriate monitoring of the disease. remote telemedicine consultations, along with the use of surrogate markers of inflammation (fecal calprotectin, c-reactive protein, patient-reported outcomes) may, however, be an alternative to face-to-face office visits during the epidemic, especially for those in remission. the option of delaying visits should be considered on an individual basis. . active ibd disease should be treated according to the standard guidance pibd protocols as before the epidemics, as the risk of ibd complications in active ibd outweighs any risk of covid- complications, especially in children. . there is currently no concrete evidence that any of the ibd treatments increases the risk for acquiring sars-cov- or for a more severe infection once infected. therefore, uninfected children should generally continue their medical treatment, including immunomodulators and biologic therapies, as the risk of a disease flare outweighs any estimated risk of sars-cov infection. this is especially true in children who have a much milder infection. specific considerations are listed below. . corticosteroids can be used to treat disease relapses, but as always recommended in children, the drug should be weaned as soon as possible. in crohn disease, exclusive enteral nutrition should be preferred. . switching from infliximab to adalimumab in a stable child should be discouraged unless impossible to provide intravenous infusions, as the risk of disease exacerbation after such a switch has been documented in the clinical trial setting. . there is no clear indication to stop ibd treatment during covid- infection, also because of the typical prolonged effect of ibd drugs. nonetheless, we recommend suspending immunosuppressive treatment during an acute febrile illness until fever subsides and the child returns to normal health, irrespective of the sars-cov- testing status. in case of positive sars-cov- testing in an asymptomatic child, the decision of therapeutic changes should be individualized. mesalamine should never be suspended. . elective surgeries and nonurgent endoscopies should be postponed during the epidemic. % all statements are limited to children and are based on the emerging but limited data available upon march ; it is possible that statements may change as data on pibd and covid- will accumulate. the following statements did not receive consensus of the porto group, and thus were removed: ''up to onethird of patients with covid- may present with gastrointestinal symptoms, mainly diarrhea or nausea. therefore, these symptoms during an active infection do not necessarily indicate a flare of the underlying ibd'' and ''in children with suspected symptoms of covid- , sars-cov testing is recommended before any therapeutic change''. covid- ¼ corona virus disease ; ibd ¼ inflammatory bowel disease; pibd ¼ paediatric inflammatory bowel disease. we provide the first case series of children with ibd who have sars-cov- infection, all cases were mild despite being treated with immunosuppressive medications. these reassuring cases are supported by the lack of symptomatic disease among children with pibd cases in china and south korea. as sars-cov- infection is often asymptomatic in children, it is likely that the true mild/minimal infection rate is higher than identified. among chinese children with covid- , nearly a quarter had no symptoms ( ) . the reason for the milder infection course in children, resulting in lower hospitalisation rate and mortality, is not yet clear. our observation of mild or minimal sars-cov- infection in children with ibd despite treatment with immunosuppressive medications is further supported by observations in children with liver disease on immunosuppression in northern italy where only / were documented to have sars-cov- infection and none with a severe course ( ) . the larger case series of adults reported from the secure-ibd registry show that current outcomes do not vary substantially from reports from the general population infected with sars-cov- . secure-ibd cases may be biased towards more severe cases as only confirmed infections have been reported and in many countries, asymptomatic and mild infections are not tested for the virus by local policy. careful continuous monitoring of the data is needed to base future possible adjustments to the current guidance. on the basis of currently (march ) available limited data presented here, we suggest the following: ibd children, with and without immunosuppressive and biological therapy, do not seem to carry a higher risk of sars-cov- infection, compared with the general population. we can cautiously suggest that currently there is no signal indicating worsening the covid- course by ibdrelated treatment. on the other hand, the risk of inappropriate management of ibd driven by the fear of the virus may have a significant impact on the health of ibd patients as indicated by increased flares with delayed therapy in china and south korea. therefore, there is presently no justification to support adaptation of therapies for children with ibd in the light of the currently ongoing sars-cov- pandemic, especially in children who have in general a more extensive and severe ibd on one hand and milder covid- course on the other. managing disease relapses in this period in epidemic areas can be difficult, thus it is crucial to advise patients to maintain their therapies, particularly when in remission. these interim conclusions may be adjusted in the future based on emerging data on covid- in children with ibd. espghan is not responsible for the practices of physicians and provides guidelines and position papers as indicators of best practice only. this guidance may be revised as necessary to account for changes in technology, new data, or other aspects of clinical practice. this guidance is intended to be an educational device to provide information that may assist clinicians in providing care to patients. they are not a rule and should not be construed as establishing a legal standard of care or as encouraging, advocating, requiring, or discouraging any particular treatment. clinical decisions in any particular case involve a complex analysis of the patient's condition and available courses of action. therefore, clinical considerations may require taking a course of action that varies from the suggestions made as part of this guidance. ¼ weighted paediatric cd activity index. the two suspected cases: household first-degree relative had concurrent confirmed infection but the child was not tested as per local testing policy as they has only mild presentation. none required admission. none of the ibd-related medications were stopped because of the sars-cov- infection. no worsening of the ibd has been reported in any of the children. patient numbers to did not suffer from other chronic disease; patient number had cardiovascular disease. cases reported from the porto group sites in: france, united kingdom, italy, spain, israel; case number was reported from the secure-ibd registry. covid- : gastrointestinal manifestations and potential fecal-oral transmission clinical characteristics of coronavirus disease in china clinical features of severe pediatric patients with coronavirus disease in wuhan: a single center's observational study coronavirus infections in children including covid- : an overview of the epidemiology, clinical features, diagnosis, treatment and prevention options in children epidemiological characteristics of pediatric patients with coronavirus disease in china chinese pediatric novel coronavirus study team. sars-cov- infection in children covid- : consider cytokine storm syndromes and immunosuppression covid- illness in native and immunosuppressed states: a clinical-therapeutic staging proposal dysregulation of immune response in patients with covid- in wuhan, china coronaviruses and immunosuppressed patients. the facts during the third epidemic thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of middle east respiratory syndrome coronavirus network-based drug repurposing for novel coronavirus -ncov/sars-cov- clinical evidence does not support corticosteroid treatment for -ncov lung injury on the use of corticosteroids for -ncov pneumonia potential benefits of precise corticosteroids therapy for severe -ncov pneumonia for the zhongnan hospital of wuhan university novel coronavirus management and research team, evidence-based medicine chapter of china international exchange and promotive association for medical and health care (cpam). a rapid advice guideline for the diagnosis and treatment of novel coronavirus ( -ncov) infected pneumonia (standard version) what should gastroenterologists and patients know about covid- ? clin gastroenterol hepatol effective treatment of severe covid- patients with tocilizumab. preprint server implications of covid- for patients with pre-existing digestive diseases protection of inflammatory bowel disease patients from the outbreak and rapid spread of covid- infection in wuhan korean society for antimicrobial t, korean society for healthcare-associated infection c, prevention helsinky . christian braegger, zurich . ola olén, stockholm . seamus hussey, dublin . dror shouval, tel aviv . amit assa, petach tiqva zagreb . lorenzo norsa, bergamo jerusalem chinese centres participating in the survey children's hospital of nanjing medical university wuhan children's hospital the children's hospital xiamen children's hospital shengjing hospital of china medical university hunan children's hospital sitang gong, guangzhou women and children's medical center chengdu women's & children's central hospital zhengzhou children's hospital beijing jing du children's hospital . xuemei zhong, children's hospital, capital institute of pediatrics the children's hospital of xi'an city beijing children's hospital peking university third hospital . zhongyue li, childen's hospital of chongqing medical university shenzhen children's hospital years received consultation fee or honorarium from abbvie, nestlé health science. p.l., advisory board and conferences for abbvie, pfizer, sandoz, nestlè, nutricia. m.d.k. for the last years received consultation fees from abbvie, janssen, takeda, and lilly; shareholder of johnson & johnson, research support from abbvie and janssen. jad received consultation fee from adacyte and honorarium for lectures from danone, ferrer. f.m.r. for the last years received consultation fee, research grant, or honorarium from janssen, pfizer, abbvie, takeda, celgene, nestlé health science, nestlé nutrition institute. c.s., y.h., and y.z. report no conflicts of interest. this article has been developed as a journal cme activity by naspghan.visit http://www.naspghan.org/content/ /en/continuing-medical-educa tion-cme to view instructions, documentation, and the complete necessary steps to receive cme credit for reading this article. copyright # by european society for pediatric gastroenterology, hepatology, and nutrition and north american society for pediatric gastroenterology, hepatology, and nutrition doi: . /mpg. key: cord- - yn c i authors: rimondi, alessandro; tontini, gian eugenio; mazza, stefano; caprioli, flavio; sangiovanni, angelo; lampertico, pietro; vecchi, maurizio title: fogging ibd management: an unusual case of ibd flare-up during the covid- outbreak date: - - journal: inflamm bowel dis doi: . /ibd/izaa sha: doc_id: cord_uid: yn c i nan we have read with interest the article by occhipinti and pastorelli, with particular reference to the management of inflammatory bowel diseases (ibd) relapse during the covid- outbreak. diagnostic challenges may arise in the presence of symptoms that overlap between active ibd and covid- , and concerns about the use of immunosuppressive drugs, mainly corticosteroids, which potentially lead to an increased risk for infections. lombardy, in northern italy where our clinic is located, has been severely hit by covid- since february . we hereby report on a representative case of how covid- has redefined priorities and changed our clinical approach to active ibd patients. in april , a -year-old man with a -year history of ulcerative colitis (uc) on maintenance with mesalamine was admitted to our clinic with a -week history of fever up to . °c, bloody diarrhea, dry cough, and ageusia. physical examination revealed tachycardia. blood tests showed neutrophilia and increased c-reactive protein. two consecutive nasopharyngeal plus one rectal swabs for sars-cov- tested negative. a contrast-enhanced chest-abdomen-pelvis ct scan revealed no signs of pneumonia but a widely thickened and hyper-enhancing colonic wall (fig. a) . abdominal imaging and gastrointestinal symptoms were consistent with ibd relapse. however, the persistence of fever, cough, and ageusia made it necessary to definitely rule covid- out. after a multidisciplinary discussion, a bronchoalveolar lavage was performed, which eventually tested negative for sars-cov- . ileocolonoscopy, performed days later, showed segmental cobblestone appearance and scattered aphthous erosions in the right and left colon (fig. b) , as opposed to a relative sparing of the rectum (fig. c) . histological examination was consistent with ibd colitis. a diagnosis of severe flare of ibd-unclassified was made, and corticosteroid therapy was initiated, with the subsequent rapid improvement of both gastrointestinal and respiratory symptoms. gastrointestinal manifestations have occurred in about half of covid- patients and may precede respiratory symptoms. therefore, the differential diagnosis between ibd relapse and sars-cov- infection has possibly proved challenging at the peak of the covid- outbreak. currently, ruling out covd- has become a priority for both clinical and public health reasons, and the timing of endoscopic examination, as well as treatment decisions, closely depend on the covid- diagnostic results. ageusia and anosmia have been reported in about one third of covid- patients, whereas they have rarely been observed in ibd patients. ageusia, as reported by our patient, contributed to increase the suspicion of sars-cov- infection and made the differential diagnosis trickier. challenges in the care of ibd patients during the covid- pandemic: report from a "red zone" area in northern italy aga clinical practice update on management of inflammatory bowel disease during the covid- pandemic: expert commentary gastrointestinal manifestations of sars-cov- infection and virus load in fecal samples from the hong kong cohort and systematic review and meta-analysis endoscopy in inflammatory bowel diseases during the covid- pandemic and postpandemic period selfreported olfactory and taste disorders in sars-cov- patients: a cross-sectional study ct scan image showing a cross-sectioned descending colon (white arrow) with wall thickening and mucosal hyperenhancement. b, left-colon image showing mucosal oedema, cobblestone appearance, and aphthous erosions. c, rectum image showing an endoscopically normal appearance of the mucosa marcello hinxman-allegri performed the linguistic revision. the patient gave informed consent. key: cord- -iyl ber authors: ghavami, shaghayegh baradaran; shahrokh, shabnam; hossein-khannazer, nikoo; shpichka, anastasia; asadzadeh aghdaei, hamid; timashev, peter; vosough, massoud title: ibd patients could be silent carriers for novel coronavirus and less prone to its severe adverse events: true or false? date: - - journal: cell j doi: . /cellj. . sha: doc_id: cord_uid: iyl ber inflammatory bowel diseases (ibds) are chronic disorders of the gastrointestinal tract. the goal of ibd treatment is to reduce the inflammation period and induce long-term remission. use of anti-inflammatory drugs including corticosteroids, immunosuppressants and biologicals, is often the first step in the treatment of ibd. therefore, ibd patients in pandemic of infectious diseases are considered a high-risk group. the public believes that ibd patients are at a higher risk in the current coronavirus pandemic. nevertheless, these patients may experience mild or moderate complications compared to healthy people. this might be because of particular anti-tnf-α treatment or any immunosuppressant that ibd patients receive. moreover, these patients might be silent carrier for the virus. inflammatory bowel diseases (ibds) are chronic inflammatory disorders of the gastrointestinal tract; ibds are categorized as crohn's disease (cd) and ulcerative colitis (uc) ( ) . the variation in the gut microbiota and certain genetic backgrounds as well as particular lifestyles are suggested as the main reasons for initiation and progression of these diseases ( ) . ibds include two clinical stages, flare-up and remission, and the main therapeutic measures involve establishment and extension of the remission phase and avoiding flare-up occurrence ( ) . the medications used for management of the condition, are -aminosalycilic acid ( -asa), immunosuppressants and biologicals targeting the immune system ( ) . therefore, ibd patients are considered a high-risk group in epidemic and pandemic of infectious diseases. interestingly, in the recent pandemic of coronavirus disease (covid- ) , and the sars-cov epidemic in , while the fecal samples of these patients were positive for the virus, they did not present any severe respiratory distress syndrome ( ). in patients infected with sars-cov- , the gastrointestinal symptoms such as diarrhea and nausea are more common compared to sars-cov patients. in addition, it was reported that while the sars-cov- test is negative for upper respiratory samples, stool samples remain positive for a few weeks after treatment ( ) . remarkably, the angiotensin-converting enzyme- (ace ) is the receptor for sars-cov- and it is expressed in different organs including the lungs, testis and ileum. this protein is also expressed on gut epithelial cells and secreted to the gut lumen ( ) . the following two questions should be addressed in this context. first, whether ibd patients show mild or moderate signs and symptoms of covid- compared to the others? second, in the recent pandemic situation, could ibd patients be considered "silent carriers" and might they increase the disease spread rate? the innate immune system has a crucial role in protecting body during viral infections. the innate immune system produces and releases interferon α (ifn-α), an essential cytokine that interferes with the viral replication, virulence and spread in the host during the early phases ( ) . one of the important activities of coronavirus is suppression of transcription and secretion of ifn-α ( ). moreover, it was shown that, sars-cov- can block the antiviral effects of ifn-α, in vitro. this is an essential mechanism for coronavirus to escape the host innate immune system ( ) . recently, it was considered that there is a regulatory cross-talk between ifn-α and tumor necrosis factor (tnf-α). of note, this cross-talk was reported between ifn-α and anti-tnf-α biologicals in clinic. banchereau et al. and palucka et al. showed that when rheumatoid arthritis patients were treated with anti-tnf-α biologicals (infliximab, adalimumab, and certolizumab pegol), the expression of ifn-α-regulated genes was increased in the peripheral blood mononuclear cell (pbmc) compared to the control group. moreover, it was shown that when the immune cells produce higher amounts of ifn-α, they are less prone to be infected with sars-cov- and other viruses ( ) . therefore, in patients with ibd who were treated with anti-tnf-α, there might be an increase in the production of ifn-α against viral infections. this could justify why ibd patients who were infected with sars-cov- , could probably present less severe symptoms compared to others ( ) . besides, in ibd patients, particularly those who are under anti-tnf-α treatment, the host innate immune system interferes more efficiently with viral replication cycle and the clinical presentations are more moderate ( , ) . ace regulates the renin-angiotensin system (ras) by cleaving several peptides (ang - ). this biological activity limits inflammation reflecting a protective role of the ace -masr pathway ( ) . ace is expressed on the epithelial cells in different organs including lungs, kidneys, liver, brain, blood vessels and particularly, on the cell membrane of the gut and illume epithelial cells ( ) . ace is known as the main receptor for spike (s) protein of sars-cov- and is crucial for colonization and entry of the virus into the target cells. the soluble form of ace (sace ) can act as a decoy molecule and cover the s protein on the virions and block colonization. this can prevent the successful binding of the viral particles to the surface of the cells ( ) . remarkably, patients with active uc and cd have higher numbers of ace in their affected tissues ( ) . since ibd patients are times more prone to viral infections such as cmv, ebv, varicella zoster virus, and hsv, possible correlations between immunosuppressive therapy/biological treatments and coronavirus infection in ibd patients, should be assessed ( ) . it was reported that ace could be a potential target for therapeutic protocols against covid- . therefore, human recombinant soluble ace (hrace ) could be considered a novel candidate for new treatment strategies ( , ) . additionally, in two studies, it was shown that soluble ace could block sars coronavirus replication ( , ) . ( ) . it was reported that upregulation of ang i-vii and ace might be a compensatory response to intestinal inflammation which could result in increased concentrations of circulating ace . in fact, in ibd patients, the soluble isoform of ace is found at higher levels in the peripheral blood, compared to normal individuals ( , ) . interestingly, the expression of ace and ang ( - ) are increased in terminal ileum and colon in cd and uc patients ( ) . though, ibd patients are quarantined and protected well during the pandemic, they could be "silent carrier" of the coronavirus. tnf-α converting enzyme (tace) is a protease enzyme that splits ace molecules from the surface of cells ( ) . blockage of tnf-α pathway induces tace activity and increases sace level. further, it was suggested that tace might be a potential target for antiviral compounds used for treatment of covid- patients ( ) . upregulation of tace activity increases detachment of the ace form the surface of epithelial cells. blocking tnf-α by monoclonal antibodies (mabs) may result in increased tace activity and higher rate of cleavage of ace from the surface ( ) . this was observed in ibd patients who are receiving different treatments such as immunosuppressants, corticosteroids and biological medications, for preventing relapse ( ) . the main cause of acute respiratory distress syndrome (ards) in covid- patients is the cytokine storm. the severity of covid- is associated with increasing serum levels of il- , il- , il- , granulocyte-colony stimulating factor (g-csf), ifn-γ, macrophage inflammatory protein -α (mip -α), and tnf-α. this situation increases recruitment of the immune cells into the lungs resulting in hyper inflammation in the patients and increasing adverse events and mortality ( ) . accumulating evidence revealed that anti-inflammatory treatments could control ards ( ) . a multicenter, randomized controlled trial, approved the use of tocilizumab (an il- receptor blocker licensed for cytokine release syndrome), in patients with covid- pneumonia. additionally, janus kinase (jak) inhibition could modulate both inflammation and viral entry into the cells in covid- infection ( ) . interestingly, ibd patients who regularly take cytokine blockers and immunosuppressant could control cytokine storm and the other related adverse events ( ) . based on the specific type of treatment employed for each ibd patient, the severity of inflammation in the lungs and the antiviral immune responses may vary. this might help ibd patients in combating covid- infection though the immune suppression can increase the risk of certain viral infections ( ) . it should be pointed out that further studies are required in this highly dynamic situation. there is no convincing evidence recommending that patients with ibd should stop their ibd-related medications ( ) . nevertheless, elder ibd patients suffering from other comorbidities like obstructive lung diseases, diabetes mellitus, coronary heart 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immunosuppression epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study baricitinib as potential treatment for -ncov acute respiratory disease advances in the research of cytokine storm mechanism induced by corona virus disease and the corresponding immunotherapies expression of sars-cov- entry molecules ace and tm-prss in the gut of patients with ibd covid- complications in ibd patients the authors would like to express their gratitude to royan institute and research institute for gastroenterology and liver diseases, shahid beheshti university of medical sciences, for their support throughout the course of this work. there is no conflict of interest. key: cord- -bpk xl x authors: dong, xiuli; ye, xiaohua; chen, xiangrong; chen, tanzhou; xie, saili; li, qinfan; lin, xiaoxiao; huang, zhiming title: intestinal and peripheral fibrinogen-like protein expression in inflammatory bowel disease date: - - journal: dig dis sci doi: . /s - - - sha: doc_id: cord_uid: bpk xl x background: fibrinogen-like protein (fgl ), a new member of the fibrinogen-like family, has recently been identified as a novel immunosuppressive molecule. aim: the purpose of this work was to investigate intestinal and peripheral expression of fgl in patients with inflammatory bowel disease (ibd), mainly ulcerative colitis (uc) and crohn’s disease (cd). methods: fgl expression in mucosal biopsies from three groups (uc group (n = ), cd group (n = ), and controls group (n = )) was detected by immunohistochemistry. concentrations of fgl in plasma from uc patients, cd patients, and controls were analyzed by enzyme-linked immunosorbent assay. western blot of fgl protein and real-time fluorescent quantitative pcr of fgl mrna expression by peripheral mononuclear cells was performed. correlations of fgl expression with disease type, activity, and location, and with measured laboratory data, including c-reactive protein (crp) and erythrocyte sedimentation rate (esr), were examined. results: intestinal and peripheral fgl protein data showed that fgl expression was significantly up-regulated in both uc and cd patients compared with controls (p < . ). expression of fgl was higher in uc and cd patients with active disease than in those with inactive disease (p < . ). moreover, fgl mrna expression was significantly higher in patients with active disease than in those with inactive disease (p < . ). expression of fgl protein was correlated with disease activity indices, crp levels, and esr levels. conclusion: expression of fgl was up-regulated in ibd patients with active disease. measurement of fgl may be used as a helpful biomarker for understanding immunopathogenesis and for assessment of ibd. inflammatory bowel disease (ibd) is a chronic relapsing and remitting inflammatory condition of the gastrointestinal (gi) tract that mainly includes two clinical entities, ulcerative colitis (uc) and crohn's disease (cd). both are frequently associated with systemic manifestations and with increased risk of colon cancer. in recent decades there has been a continuing trend of greater incidence and prevalence of ibd throughout the world, particularly in east asia [ ] , so more attention must be paid to effective diagnosis and treatment of ibd. although the exact causes and mechanisms of ibd have not been completely elucidated, the prevailing hypothesis of ibd pathogenesis is that the disease occurs in genetically susceptible individuals as the result of a complex interaction among environmental factors, microbial factors, and the intestinal immune system [ ] [ ] [ ] . furthermore, accumulated evidence indicates that an inappropriate and persistent immune response is central to the development of both major types of ibd [ ] [ ] [ ] [ ] . an excess of inflammatory stimuli and mediators, and an inadequately low function or number of regulatory components that downregulate the mucosal immune response can lead to a chronic, progressive inflammatory condition, and eventually to intestinal tissue damage [ , ] . fibrinogen-like protein (fgl ), also known as fibroleukin, was first cloned from cytotoxic t lymphocytes and was classified as a member of fibrinogen superfamily because of its homology ( %) with fibrinogen b and c chains [ , ] . originally described as an immune coagulant with the ability to generate thrombin directly, fgl has been implicated in the pathogenesis of several inflammatory disorders, including viral hepatitis and experimental arthritis [ , ] . it has recently been demonstrated that fgl is an effector molecule on regulatory t cells (treg cells) [ ] [ ] [ ] [ ] . fgl may bind to fc gamma receptors (fccr), which are expressed on antigen-presenting cells (apc), and then inhibits maturation of dendritic cells (dc) and induces apoptosis of b cells, eventually resulting in reduced ability to induce alloreactive t cell proliferation [ , ] . all these findings collectively indicate fgl has potent immunosuppressant properties. because there has been no suggestion fgl is involved in the immunopathogenesis of ibd, we felt it was necessary to investigate expression of fgl in patients with ibd and discuss its involvement. the study was performed at the department of gastroenterology and hepatology of the first affiliated hospital of wenzhou medical university from december to december . mucosal biopsy specimens were obtained from macroscopically inflamed areas of patients with uc (n = ) or cd (n = ), and from normal controls (nc, n = ), i.e. from the normal areas of healthy subjects or patients with colonic polyp (table ) . matched peripheral blood was available from patients with uc (n = ) or cd (n = ) at department of gastroenterology and hepatology of first affiliated hospital of wenzhou medical university who were enrolled from january to april (table ). these were compared with samples from normal controls, who were recruited from healthy blood donors, visitors of hospital wards, and normal hospital personnel. diagnosis of uc and cd was based on standard criteria [ ] . uc and cd patients with active disease enrolled in our study had not received any immunomodulatory medications for their disease. most uc patients with inactive disease were undergoing maintenance treatment with lowdose -aminosalicylic acid; others had stopped all drugs. cd patients with inactive disease were undergoing maintenance treatment with low-dose azathioprine, or low-dose azathioprine and methylprednisolone. disease activity in uc patients was evaluated by use of the truelove-witts criteria [ ] , for statistical purposes, the classification was left sided colitis (uc)/colon (cd) total colitis (uc)/ ileum ? colon (cd) left sided colitis (uc)/colon (cd) total colitis (uc)/ ileum ? colon (cd) quantitatively modified [ ] . active disease was defined as score [ and inactive disease as score b . for patients with cd, the severity of the disease was classified in accordance with the cdai score [ ] . active disease was defined as cdai score c and inactive disease as cdai score \ . disease activity was evaluated at the time of sample collection. standard laboratory data, including red and white blood cell count, hemoglobin, hematocrit, platelet count, erythrocyte sedimentation rate (alifax test ; italy) and c-reactive protein (beckman coulter detection kit; japan) were routinely measured for all patients with uc and cd. mucosal biopsy specimens were fixed with % paraform, processed into paraffin, and sectioned for immunohistochemical staining of fgl . the sections were initially deparaffinated in xylene and rehydrated through ethanol to water. nonspecific binding was blocked by sequential incubation of the sections in citrate buffer for min at °c and min at °c, then in % hydrogen peroxidase solution for min followed by % normal goat serum in pbs at °c for min. thereafter sections were incubated with mouse anti-human fgl monoclonal antibody (abnova, taiwan) at a dilution of : in pbs at °c for h. after washing with pbs, sections were incubated with immunoperoxidase-conjugated rabbit igg fraction to mouse igg fc (zhongshan, beijing, china) at °c for min, followed by three washes in pbs. finally, the sections were incubated with , -diaminobenzidine chromagen and counterstained with hematoxylin. a negative control was used in the experiment. for evaluation of fgl expression, ten random fields across each section were selected for semi-quantitative analysis of mean absorbance at a magnification of . all blood samples were drawn between and am, after fasting. after a resting period of min, non-traumatic venipuncture was performed in a standardized manner by trained operators. blood ( . ml) was drawn into test tubes with . ml citrate. within h of sample collection, the samples were centrifuged for min ( , g) at °c and stored at - °c in plastic tubes. before serial analysis, the plasma was thawed immediately in a water bath at °c for min. in accordance with the manufacturer's instructions, plasma concentrations of fgl were measured by use of a commercially available enzyme-linked immunosorbent assay (elisa; biolegend, usa). peripheral blood was obtained and peripheral blood mononuclear cells (pbmc) were isolated by use of ficoll density gradients (solarbio, shanghai, china) for western blot analysis. lysate protein ( lg) extracted from pbmc was loaded on to % sds-polyacrylamide gels. after separation, the proteins were transferred to a nitrocellulose (nc) membrane. the membrane was blocked and probed with a monoclonal antibody against fgl (abnova, taiwan) at a dilution of : in % milk in tbst. after washing with tbst, the blot was incubated with secondary antibodies conjugated to horseradish peroxidase (biosharp, hefei, china). immunoreactive bands were detected with the enhanced chemiluminescence (ecl) reagent (pierce biotechnology, shanghai, china). protein levels, normalized against gapdh, were determined by densitometric analysis using quantity one version . total rna was isolated from pbmc by use of trizol reagent (invitrogen, shanghai, china) in accordance with the manufacturer's procedure. the concentration and purity of rna were determined by measurement of absorbance at and nm. subsequently, the cdnas were synthesized (taigen biotechnology, china). the nucleotide sequences of the primers for pcr amplification of the bp fragment of fgl were: sense primer, -actgt gacatggagaccatg- , and antisense primer, -tcc ttactcttggtcagaag- . the amplified bp fragment of gapdh was used as an internal control to ensure equal loading with forward primer, -tcccat caccatcttccagg- and reverse primer, the protocol of this study was approved by the clinical research ethics committee of the first affiliated hospital of wenzhou medical university. all the subjects enrolled in our study had been informed and had given written consent. all results were expressed as mean ± sd. statistical analysis was conducted with spss . software. continuous measurements among the three diagnostic groups were compared by one-way anova. post-hoc multiple comparisons were performed by use of dunn's test. the same tests were used for comparisons of disease activity or disease location among the different groups. the association between fgl expression and disease activity indices or other laboratory data, including crp levels and esr levels, was examined by non-parametric correlation (spearman's r). a level of p \ . was considered to be statistically significant. (fig. b, c) . disease location in uc and cd patients was no different. there was an association between fgl levels and clinical indices of activity-truelove-witts for uc and cdai for cd (r = . , p \ . ; r = . , p \ . , respectively). furthermore, the enhanced fgl levels were correlated with crp levels for both uc and cd patients (r = . , p = . ; r = . , p = . , respectively), and with levels of esr (r = . , p \ . ; r = . , p \ . , respectively). in addition, two cd patients with active disease, both of whom underwent anti-tumor necrosis factor a (tnf-a) and azathioprine therapy, were followed-up regularly in our study. the levels of fgl in one patient were . , . , . , . , . , and . ng/ml during the period of treatment on the th, the nd, the th, the th, the nd and the th week respectively. the fgl levels of another patient were . ng/ml before the treatment, . ng/ml on the th week, . ng/ml on the nd week, . ng/ml on the th week of treatment. the decreased fgl levels were in parallel with the clinical cut-down cdai scores (fig. d) . western blot analysis revealed that fgl protein expression, normalized against gapdh, in pbmc was . ± . ( % ci . - . ) for patients with uc, and . ± . ( % ci . - . ) for patients with cd, compared with . ± . ( % ci . - . ) for controls. it was markedly elevated in patients with cd in comparison with normal controls (p = . ), and there was no statistically significant difference between patients with uc and controls (fig. a) . fgl mrna expression was significantly higher for uc and cd patients with active disease than for those with inactive disease (p \ . ) (fig. b, c) . however, fgl mrna expression was lower for patients with inactive disease than for controls (both inactive uc vs nc, inactive cd vs nc, p \ . ) (fig. b, c) . the intestinal lumen is home to more than strains of bacteria, so the gi mucosal immune system must strike a delicate homeostatic balance between maintaining tolerance toward the commensal microflora and remaining poised to mount an aggressive immune response against invading pathogens. under normal conditions, the epithelial barrier continually samples antigen from the lumen and presents it to lymphocytes in the peyer's patches to promote tolerance. when it encounters invasion of pathogens, apcs in the mucosa, including macrophages, dc, and intestinal epithelial cells, phagocytose the invading pathogens and present their components to naive cd ? t cells. the t cells then undergo activation and differentiation to an effector phenotype (i.e., th , th , or th ) or a regulatory phenotype (i.e., th or treg) [ ] . while effector t-helper cells mount specific immune responses by expression of unique cytokines to combat the invading pathogens, treg cells suppress inflammation and restore homeostasis to the mucosal tissues by expression of regulatory cytokines, for example interleukin- (il- ) and transforming growth factor (tgf-b). however, there is an inherent defect in the mucosal immune system of ibd patients [ ] . although the exact nature of this defect has not been identified, mounting evidence indicates that ibd occurs in the presence of dubious antigens, most likely normal resident luminal bacterial and/or food-derived, which initiate a dysregulated immune response within the intestinal mucosa and the eventual development of chronic intestinal inflammation [ ] . furthermore, there has been sustained interest in the importance of treg cells in ibd. it seems that fewer circulating treg cells are present in patients with active ibd, but insufficiently increased in the intestinal mucosa [ ] . subsequent research suggested the increased apoptosis of treg cells in ibd can be reversed by anti-tnf-a therapy, and this would be a critical factor in the recurrence of disease [ , ] . nevertheless, the clearly core mechanism of treg-mediated suppression is still controversial. although il- , tgf-b and some other molecules have been reported to account for the regulatory activity of treg cells, antibodies to these molecules in some cases had no effect or only minimally inhibited treg cells activity in vitro [ ] [ ] [ ] . recent findings have revealed the possible importance of the contribution of fgl to the suppressive activity of treg cells [ , , ] . fgl , consists of aa and contains a c-terminal fibrinogen-related domain (fred), which is a highly conserved region and is characteristic of proteins within the fibrinogen superfamily [ ] . these functionally diverse proteins, including fibrinogen, tenascin, angiopoietin, and ficolin, have been shown to have immunoregulatory activity [ , ] . similarly, much research has recently revealed that fgl , secreted by t cells, also has immunomodulatory activity [ ] . fgl mrna increased in cd ?cd ?foxp ? treg cells, along with mrna for several known treg suppression effector molecules [ , ] . furthermore, shalev et al. [ ] reported that the suppressive activity of fgl -/-treg cells was significantly impaired in fgl -/-mice, and antibody to fgl completely inhibited the activity of fgl ?/? treg cells in vitro. consistent with the contribution of fgl to the activity of treg cells, targeted deletion of the fgl gene led to an increase in immune reactivity of dc, t cells, and b cells, and the development of autoimmune glomerulonephritis in aged fgl -/-mice. the regulatory activity of fgl also contributes to inhibition of allograft rejection and the pathogenesis of experimental and human viral infections, including hiv, severe acute respiratory syndrome (sars), hepatitis b virus, and hepatitis c virus [ , , , ] . all this evidence strongly supports the hypothesis that fgl is an effector molecule of treg cells. the mechanism whereby fgl exerts its immunomodulatory activity has been demonstrated by liu et al. [ ] . fgl may bind to fccriib, which is expressed on apcs, and then inhibit dc maturation and induce b cells apoptosis, eventually resulting in reduced ability to induce alloreactive t cell proliferation. given that ibd shares immunologic features with autoimmune diseases to some extent, it is logical to hypothesize that the immunomodulatory activity of fgl may also be involved in the immunopathogenesis of ibd. however, our study demonstrated that intestinal and peripheral expression of fgl was significantly higher in uc and cd patients with active disease, and decreased in inactive disease. moreover, expression of fgl was positive correlated with disease activity indices, crp levels, and esr levels. up-regulation of fgl in both uc and cd patients with active disease may be an insufficient compensation secreted by treg cells which fails to counter chronically activated effector t cells, and which leads to inappropriate immune responses in ibd. reduced fgl expression in patients with inactive disease may be because successful treatment, including immune modifiers, anti-tnf-a therapy, and other anti-inflammatory treatment, made up for the deficiency of treg cells, or reversed the deficiency to exert sufficient suppression against other subsets of t lymphocytes through multiple other suppression molecules. this assumption is in agreement with the results of maul et al. [ ] , which reported a decrease of peripheral treg cells and an insufficient increase in intestinal lesions, and also consistent with therapeutic strategies in which immune modifiers [ ] or anti-tnf-a therapy [ ] are used. in conclusion, the results of our study of fgl expression in ibd have important theoretical implications for our understanding the immunopathogenesis of ibd, and practical implications for its therapy. fgl may be a helpful biomarker of the pathogenesis of ibd and for assessment of the disease. to confirm this, we have now initiated further investigation of the involvement of fgl among patients with ibd. world gastroenterology organization practice guidelines for the diagnosis and management of ibd in genetics and pathogenesis of inflammatory bowel disease role of the microbiota in inflammatory bowel diseases intestinal homeostasis and its breakdown in inflammatory bowel disease pathagenesis and immune mechanisms of chronic inflammatory bowel diseases immunopathogenesis of inflammatory bowel disease epigenetics: concepts and relevance to ibd pathogenesis inflammatory bowel disease innate and adaptive immune responses related to ibd pathogenesis regulatory t cells and mechanisms of immune system control structure of a cytotoxic t-lymphocyte-specific gene shows a strong homology to fibrinogen beta and gamma chains sequence of a human transcript expressed in t-lymphocytes and encoding a fibrinogen-like protein molecular and functional analysis of the human prothrombinase gene (hfgl ) and its role in viral hepatitis the prothrombinase activity of fgl contributes to the pathogenesis of experimental arthritis a function for interleukin in foxp -expressing regulatory t cells characterization of human fibroleukin, a fibrinogen-like protein secreted by t lymphocytes soluble fibrinogen-like protein /fibroleukin exhibits immunosuppressive properties: suppressing t cell proliferation and inhibiting maturation of bone marrow-derived dendritic cells targeted deletion of fgl leads to impaired regulatory t cell activity and development of autoimmune glomerulonephritis the fgl -fcgammariib pathway: a novel mechanism leading to immunosuppression classification of inflammatory bowel disease cortisone in ulcerative colitis; final report on a therapeutic trial platelet factor and beta-thromboglobulin in inflammatory bowel disease and giant cell arteritis development of a crohn's disease activity index. national cooperative crohn's disease study peripheral and intestinal regulatory cd ?cd (high) t cells in inflammatory bowel disease apoptosis of regulatory t lymphocytes is increased in chronic inflammatory bowel disease and reversed by anti-tnfalpha treatment reciprocal changes of foxp expression in blood and intestinal mucosa in ibd patients responding to infliximab tgf-beta production by cd ?cd ? regulatory t cells is not essential for suppression of intestinal inflammation normal pathogen-specific immune responses mounted by ctla- -deficient t cells: a paradigm reconsidered factors regulating apoptosis and homeostasis of cd ?cd (high) foxp ? regulatory t cells are new therapeutic targets the novel cd ?cd ? regulatory t cell effector molecule fibrinogen-like protein contributes to the outcome of murine fulminant viral hepatitis the novel immunoregulatory molecule fgl : a potential biomarker for severity of chronic hepatitis c virus infection the tenascin gene family yeilding, angiopoietin- and - coiled coil domains mediate distinct homooligomerization patterns, but fibrinogen-like domains mediate ligand activity cd ?cd ? t regulatory cells dependent on icos promote regulation of effector cells in the prediabetic lesion targeted deletion of fgl- /fibroleukin in the donor modulates immunologic response and acute vascular rejection in cardiac xenografts acknowledgments the authors are grateful to chengcheng chen, silu wang, and fajing yang, department of surgery laboratory, the first affiliated hospital of wenzhou medical university, for assistance with technical support and consulting. this work was supported by the science and technology project of wenzhou city, china (y ).conflict of interest none. key: cord- -r qgdgeu authors: dipasquale, valeria; cucchiara, salvatore; martinelli, massimo; miele, erasmo; aloi, marina; romano, claudio title: challenges in paediatric inflammatory bowel diseases in the covid- time date: - - journal: dig liver dis doi: . /j.dld. . . sha: doc_id: cord_uid: r qgdgeu nan the outbreak of coronavirus disease (covid- ), caused by severe acute respiratory syndrome coronavirus (sars-cov- ), first reported in china in december , now involves the whole world [ , ] . as of march , , more than cases and more than deaths in over countries have been reported ( https://www.worldometers.info/coronavirus/ ). few cases of covid- in children and adolescents have also been described [ ] [ ] [ ] , with a null death rate in the group - years, and . % in the group - years. the majority of the most severe clinical courses and deaths have occurred among the elderly and those with chronic comorbidities, often receiving immunosuppressive and/or immunomodulatory treatment [ , ] . it is well known that patients with inflammatory bowel disease (ibd) have an increased risk of infections, particularly opportunistic infections, due to a multifactorial immunological impairment [ , ] . the novel coronavirus seems to dysregulate immune response in infected individuals, mainly by acting on lymphocytes, especially t-cells [ ] . whether patients with ibd may be more susceptible to covid- is a reasonable concern. currently no cases of ibd patients infected by sars-cov- have been reported. international ibd non-profit organisations issued online advices and preliminary recommendations regarding covid- management in adults with ibd [ , ] . we would like to emphasize the importance of addressing main concerns from ibd paediatric patients during the covid- epidemic, taking into consideration their peculiar disease characteristics and comorbidity spectrum. beyond immunological dysregulations commonly underlying ibd, risk factors for infections in children with ibd are represented by rare monogenic disorders, including primary immunodeficiencies, poor nutritional status and, above all, in comparison to adults, the more frequent need for early and/or combined immunosuppressive and biologic therapies [ , ] . currently no evidence supports treatment suspension in mild or moderate cases of covid- , neither in children living in an endemic area, also due to the washout period of most immunomodulators (such as azathioprine, methotrexate) and biologics. an exception could be raised by steroid treatment. in paediatric ibd, different studies have shown steroids carrying higher risk of infections compared with anti-tumor necrosis factor (tnf)-α, while the risk of immunomodulators and anti-tnf-α agents, given alone, seems to be comparable [ , ] . in addition, steroids are not effective for the treatment of lung injury or shock in adults' sars-cov- infection [ ] . in line with these observations, discontinuation (or at least tapering) of steroid treatment during covid- epidemic may be a reasonable option. as the risk of infections is generally higher in case of combination therapy (biologics plus immunomodulators) in adult and paediatric patients with ibd [ , ] , caution should be used with this therapeutic strategy during the covid- epidemic. in a population-based study of ibd paediatric patients treated with infliximab, infections arose in those patients under combination therapy with immunomodulators [ ] . whether this is the most appropriate time to start an immunomodulatory treatment is also on discussion. the immunosuppressive effect of these drugs should not be overlooked, since it may eventually increase the risk of infectious complications and promote the spreading of covid- ; however, active ibd itself has been thought to be a potential risk factor for sars-cov- infection. overall, it seems wise to apply an individual risk assessment, avoiding the postponement of new treatment (or increasing in dose an ongoing treatment) in case of severe disease flares. for patients on biologics, switching to subcutaneous self-injection medication (i.e. adalimumab) at home could be encouraged, aiming to limit hospital appointments, or if intravenous medication (i.e. infliximab) is not available. several reports suggest the possible involvement of the gastrointestinal system in sars-cov- infection [ - , ] . indeed, although the most common clinical presentation of covid- is a moderate-to-severe respiratory illness, abdominal pain, diarrhoea ( . %), nausea, and vomiting ( %) can also occur [ ] . in the retrospective analysis of hospitalized children carried out from january to january , in wuhan, china, covid- was detected in previously healthy children, presenting with vomiting [ ] . a -year old male presented with diarrhoea at the onset of covid- [ ] . however, current clinical evidence does not support covid- as a cause of ibd flares, and therefore a systematic exclusion of covid- in the setting of an ibd flare is not recommended. in cases with suspected symptoms of covid- , prompt medical evaluation and careful followup are crucial. only where exposure to confirmed covid- occurs (viral rna detection from nasal and pharyngeal swab specimens, according to the world health organization), discontinuation of all immunosuppressive and biological treatment could be taken into consideration, as recommended during any severe infection [ , ] . in conclusion, while waiting for more specific data concerning the risk of covid- in children with ibd and, more generally, in paediatric patients on immunosuppressive therapy, it seems reasonable to carefully weigh the risks/benefits ratio of treatment with immunomodulators and biologics, especially in areas of high infection rate or outbreaks. in addition, all ibd children and their household contacts should be encouraged to practice good hygiene and to fulfil all the other preventive measures in order to reduce the risk of sars-cov- exposure. clinical features of patients infected with novel coronavirus in wuhan, china china medical treatment expert group for covid- . clinical characteristics of coronavirus disease in china a novel coronavirus from patients with pneumonia in china characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention detection of covid- in children in early pharmacological treatments and infectious diseases in pediatric inflammatory bowel disease infections and malignancies risks related to tnf-α-blocking agents in pediatric inflammatory bowel diseases. expert dysregulation of immune response in patients with covid- in wuhan, china crohn's colitis foundation crohn's & colitis uk. novel coronavirus (covid- ) advice clinical evidence does not support corticosteroid treatment for -ncov lung injury long-term outcome of treatment with infliximab in pediatric-onset crohn's disease: a population-based study sars-cov- induced diarrhoea as onset symptom in patient with covid- none. none declared. key: cord- - r vm authors: grossberg, laurie b; pellish, randall s; cheifetz, adam s; feuerstein, joseph d title: review of societal recommendations regarding management of patients with inflammatory bowel disease during the sars-cov- pandemic date: - - journal: inflamm bowel dis doi: . /ibd/izaa sha: doc_id: cord_uid: r vm nan coronavirus disease , caused by severe acute respiratory syndrome coronavirus (sars-cov- ), emerged in wuhan, china in december , and has rapidly expanded globally. patients who develop this disease typically present with fever and respiratory symptoms and less commonly experience a change in their bowel habits or other gastrointestinal symptoms. the world health organization declared this coronavirus outbreak as a public health emergency of international concern on january , , and as a pandemic on march , . as of june , , there were almost million confirmed cases and almost , deaths from the disease worldwide. inflammatory bowel disease (ibd), consisting of crohn's disease (cd) and ulcerative colitis (uc), affects millions of people worldwide. patients with ibd are often prescribed immunosuppressive or biologic medications for treatment, which may predispose them to an increased risk of infection. although data in patients with ibd contracting covid- are still limited, both providers and patients have particular concerns regarding the risk of infection with sars-cov- and how to manage their medications during the covid- pandemic. the centers for disease control and prevention states that patients who are immunocompromised may be at higher risk for severe illness. there are inadequate data for the optimal management of patients with ibd in the setting of covid- , and patients and gastroenterologists have been left to rely on societal consensus statements and expert opinion to guide management during these unprecedented times. , given the limited evidence, we evaluated gastrointestinal society and ibd organization websites and the literature for consensus statements and advice regarding the management of ibd during covid- and compared their recommendations. we conducted a search of the medical literature using pubmed to assess for recommendations or consensus statements regarding the management of ibd during covid- . articles were identified using terms including "inflammatory bowel disease" or "crohn's disease" or "ulcerative colitis" and "coronavirus" or "covid- ." the search was restricted to english language. titles were reviewed and all relevant articles were examined in detail. in addition, we also searched gastroenterology society and ibd organization websites for consensus statements and recommendations for patients and providers during covid- . information regarding risk factors, prevention, routine care (including office visits, testing, endoscopy, and surgery), and medication management of patients with ibd in the setting of covid- was collected from each reference and is summarized in the results. there is no evidence that patients with ibd are at increased risk of infection with sars-cov- or covid- disease compared with the general population. data from china did not show immunomodulator use to be a risk factor for severe disease, and a large ibd group in france, with % of patients on anti-tumor necrosis factor agents, did not report any patients with severe covid- . , the international organization for the study of inflammatory bowel disease (ioibd) states that patients with ibd do not have an increased risk of infection with sars-cov- ; however, it is uncertain if patients with ibd who are exposed to sars-cov- have a higher risk of developing covid- or have a higher mortality than patients without ibd. , other organizations, including the american gastroenterological association doi: . /ibd/izaa published online july (aga), the gastroenterological society of australia, and the european crohn's and colitis organisation (ecco), agree that there are no data to support an increased risk of infection among patients with ibd. , , the centers for disease control and prevention cites older age, comorbidities, pregnancy, and smoking tobacco as possible risk factors for infection, and therefore many organizations note that patients with ibd and these characteristics are at increased risk of more severe covid- . , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] despite the paucity of data, most groups agree that immunosuppressive medications increase the risk of covid- among patients with ibd, [ ] [ ] [ ] [ ] [ ] [ ] and several ibd societies have added that ibd disease activity and malnutrition may be risk factors for infection with sars-cov- . - groups from canada and the united kingdom classify patients with ibd into low-, medium-or moderate-, and high-risk groups using these variables ( table ) . sars-cov- infection is thought to be spread from person to person by droplet transmission and possibly airborne inhalation of aerosolized particles. in addition, viral particles have been identified in the stool suggesting possible fecal transmission, but this has not been verified. [ ] [ ] [ ] [ ] [ ] [ ] all societal recommendations agree that patients with ibd should, at a minimum, take the same precautions as the general population to prevent infection and should follow local public health advice. recommendations for prevention of covid- among patients with ibd according to risk category are summarized in table . health care workers with ibd face challenges during the covid- pandemic. the ioibd states that it is uncertain if health care workers with ibd on immune-modifying medications working in an environment with patients with known or suspected covid- should continue working in that same environment. , although the recommendations for personal protective equipment (ppe) for health care workers with ibd do not differ from those for the general population, ibd societies agree that considerations should be made to redeploy vulnerable health care personnel to duties with reduced exposure to patients with confirmed or suspected covid- . , , in addition, the crohn's & colitis foundation suggests that all health care workers with ibd consider wearing a face mask and gloves and practice social distancing in the workplace. vaccinations, smoking cessation, and avoidance of nonsteroidal anti-inflammatory medications are recommended as routine health maintenance among patients with ibd to reduce the risk of disease flare and poor outcomes from infections. australian and european ibd societies highlight the importance of these measures to reduce the risk of severe covid- , although the logistics of vaccination during the pandemic must be considered. , , outpatient clinics, laboratory testing, and imaging many ibd societies agree that in-person appointments should be avoided and switched to telephone or video visits when possible and that contact with health care facilities for nonurgent testing should be minimized (table ). , [ ] [ ] [ ] [ ] providers should consider using clinical disease scores and fecal calprotectin levels to guide decision-making as their initial testing instead of endoscopy. , , other testing modalities such as radiology or capsule endoscopy can be considered, although access to these services during the pandemic may be limited and influence the choice of investigation for patients with ibd. infusion suite services should be maintained as a priority area or essential service. , , patients should continue to receive infusions at an infusion center, assuming that the infusion center has a screening protocol in place and that extra precautions are taken to minimize the risk of exposure to covid- (table ) . , , , , , , the ecco task force suggests that postponing infusions of infliximab to every weeks and infusions of vedolizumab by an additional weeks may be possible in a select group of patients in remission; however, the original schedule is probably the best strategy. other societies also suggest possibly extending infusion intervals when feasible; however, this extension must be done cautiously to avoid loss of response. , , the crohn's & colitis foundation proposes that patients schedule infusions at off-peak hours or consider home infusion, whereas the aga and ioibd specifically recommend against home infusion because of safety and logistical issues. , , , endoscopy and surgery on march , , the american college of surgeons released a statement recommending that hospitals and health systems review all elective procedures with a plan to minimize, postpone, or cancel elective surgeries, endoscopies, or other invasive procedures to support the expected surge in critical patient care needs and to minimize the use of essential items needed to care for patients including ppe, cleaning supplies, ventilators, and intensive care unit beds. soon after the statement was released, gastrointestinal and ibd organizations issued opinion statements supporting these recommendations and advising appropriate ppe (table ) . , however, it has been proposed that patients with ibd should continue to undergo endoscopy in certain situations: to diagnose new severe ibd, to exclude cytomegalovirus in acute severe uc if noninvasive tests are equivocal, to direct surgical intervention in patients with severe disease or suspected cancer, to assess patients with partial obstructive symptoms for balloon dilation, or to intervene in a dominant biliary stricture in a patient with concomitant primary sclerosing cholangitis and cholangitis. , iacucci et al propose algorithms for the timing of endoscopy in each of these situations. patients with ibd and urgent perianal sepsis should undergo a day-case procedure. however, complex ibd surgeries should be deferred if possible, and the timing should be regularly reviewed at a multidisciplinary team meeting. emergency surgeries, such as colectomy for acute severe uc and intestinal resection for penetrating disease in cd, should continue as part of routine care; however, the choice of postoperative therapy to prevent recurrence needs to be considered in context of covid- . all patients follow general precautions to prevent infection *, †, ‡, §, ¶,‖,**, † †, ‡ ‡, § § frequent hand washing with soap and water or hand sanitizer (> % alcohol) avoid touching eyes, nose, and mouth cover your mouth when coughing or sneezing with a flexed elbow or tissue avoid close contact with anyone who is sick stay home as much as possible distance yourself from others by feet † wear a cloth face cover when going out in public † clean and disinfect frequently touched surfaces daily ensure vaccination against flu and pneumococcus ‡ ‡, § § avoid nonsteroidal anti-inflammatory drugs † † stop smoking † †, ‡ ‡ maintain adequate supply of medication † †, ‡ ‡, § § moderate risk avoid in-person meetings, public transport, and public spaces ¶ ¶ discontinue any nonessential travel §, ¶ work at home or discuss options for modified duties with employer ‖ perform only essential self-care tasks (i.e. food shopping) ** use services for vulnerable people to avoid contact with others ‖ high risk self-isolate or "shield" (avoid all contact with others) ‖,** use services for vulnerable people to avoid contact with others ‖ leave home for infusion treatment only ¶ ¶ family members should also work from home, use services for vulnerable people to avoid contact with others, and keep a clean residence as best as possible ¶ ¶ all gastrointestinal organizations we researched recommend that patients should stay on their ibd maintenance medications as prescribed during the covid- pandemic. , , , , [ ] [ ] [ ] [ ] [ ] [ ] clinicians maintain that the risk of disease flare off medications is greater than that of contracting sars-cov- . this risk is related to the potential need for high-dose steroids, increased contact with a health care facility for evaluation, hospitalization, or surgery. moreover, delaying biologics increases the risk of immunogenicity and loss of response. a summary of society recommendations per medication class is outlined in table . enteral nutrition, probiotics, -aminosalicylates, antibiotics, and local steroids do not suppress the immune system and are safe to continue or start for treatment. , , societies are overall in agreement with the recommendations to taper steroids and continue ibd therapy as prescribed, although the british society of gastroenterology suggests stopping thiopurines in patients aged > years or with comorbidity who are in sustained remission. patients with ibd who have been in close contact with someone with proven covid- should self-isolate and follow local recommendations. the ecco task force states that it is not necessary to stop medications on the basis of exposure alone. the surveillance epidemiology of coronavirus under research exclusion (secure-ibd) is a database that was established to monitor and report outcomes of covid- occurring in patients with ibd. providers for ibd are encouraged to report all confirmed cases, regardless of severity, to this database. the recommendations for medication management of patients with ibd who have confirmed sars-cov- or covid- are summarized in table . although the ioibd states that it is uncertain if biologics should be held in patients with confirmed sars-cov- , the aga notes that it is reasonable to delay the next dose for weeks to monitor for the onset of symptoms. , , for patients with covid- and quiescent ibd, biologics, immunomodulators, and small molecules should be delayed until after symptoms resolve, typically - days or after nasopharyngeal polymerase chain reaction tests are negative. , [ ] [ ] [ ] for patients involved in clinical trials, the ioibd recommends that patients stop treatment if they test positive for sars-cov- or develop covid- . , although the ioibd recommends that patients with moderate to severely active cd or uc (both new disease or relapsing) should be treated with the same therapies as one would have chosen in the pre-covid- era, other organizations propose special considerations. chinese ibd societies recommend against a new prescription or increased dose of an immunosuppressant in epidemic areas, and the ecco task force suggests that providers postpone the start of treatment based on individual risk assessment. , however, for patients requiring new therapy, ibd societies propose therapeutic options that limit risk and reduce patient contact with a health care facility (table ) . for example, providers should consider using budesonide over corticosteroids, avoiding immunomodulators and janus kinase inhibitors, and using monotherapy with antitumor necrosis factor drugs rather than combination therapy locally acting steroids include budesonide and budesonide mmx. corticosteroids include prednisone. immunomodulators include thiopurines ( -mercaptopurine, azathioprine), methotrexate, and cyclosporine. anti-integrin includes vedolizumab. the janus kinase inhibitor includes tofacitinib. anti-il- / indicates anti-interleukin / -includes ustekinumab; anti-tnfs, anti-tumor necrosis factors-include infliximab, adalimumab, certolizumab pegol, and golimumab; budesonide mmx, budesonide multi-matrix system; -asas, -aminosalicylates-refers to oral or rectal mesalamine. when possible (table ) . , , , furthermore, providers should consider subcutaneous biologics for new treatment starts to avoid a burden on infusion units and decrease the risk of exposure. , in patients with active ibd and confirmed sars-cov- and/or covid- , the risks and benefits of treatment must be weighed against the severity of covid- . in outpatients with mild symptoms of covid- , safer ibd therapies such as vedolizumab should be prioritized. in patients who are hospitalized with severe covid- , the choice of therapy for covid- should take into account the coexisting ibd if feasible. on the other hand, if patients are hospitalized for severe ibd and test positive for sars-cov- or have mild covid- , then standard algorithms for hospitalized patients with ibd should be applied with a focus to limit intravenous steroids to no more than days. , the covid- pandemic has swept across the world, and gastroenterologists are now managing patients with ibd in an unprecedented way. providers are commonly using telemedicine, limiting nonurgent laboratory and imaging evaluation, and reserving endoscopy only for select situations that will guide management decisions. as data regarding covid- in patients with ibd are sparse, gastroenterologists are faced with complex decision-making and must rely on expert opinions and consensus. in this study, we summarize the available recommendations and consensus statements from organizations throughout the world on the management of ibd during covid- . at the time of this writing, confirmed cases of covid- in patients with ibd have been reported to the secure-ibd registry internationally. preliminary analysis of publicly reported data shows worse outcomes in patients who are older, have more comorbidities, and are prescribed corticosteroids. however, many questions remain on how to best manage patients with ibd in the setting of the covid- pandemic. it is unknown if patients with ibd are at higher risk of severe covid- or worse outcomes. it is hypothesized that patients with ibd may be at increased risk of covid- because the covid- receptor, the angiotensin converting enzyme , is highly expressed in the terminal ileum and colon of patients with ibd. however, a recent study shows that patients with ibd do not have higher expression during inflammation, and some biologic therapies are associated with lower levels of ace . moreover, early data from wuhan of patients with ibd during a local outbreak of disease did not report any covid- , although all biologic and immunosuppressive therapy was held in this patient population. it is not clear if treatment needs to be held in patients who test positive for the virus but do not have symptoms, or in patients who have very mild symptoms. despite the lack of evidence, most current recommendations and consensus statements propose holding biologics, immunomodulators, and small molecules for patients who test positive for sars-cov- without covid- and in all patients with covid- , regardless of the severity. although this practice may result in some patients losing efficacy of their therapy, prioritizing patient safety in the setting of limited data should be considered. future research on ibd during covid- should focus on the following: • risk stratification for severe covid- among patients with ibd. • the development of evidence-based algorithms for medication management in patients with confirmed sars-cov- without symptoms, mild covid- , and severe covid- . • long-term outcomes for patients with covid- and ibd. • the role for sars-cov- vaccination in patients with ibd and its prioritization. as many practices and institutions shift focus toward reopening endoscopy units and resuming surgery as the number of new covid- cases decreases, new policies are needed. the american society for gastrointestinal endoscopy recommends screening of all patients with a covid- questionnaire within hours of procedure or testing for covid- , careful preparation and cleaning of rooms, physical distancing by patients and staff when possible, and appropriate ppe worn by all staff. all procedures should be ranked based on a tier system of urgent, semi-urgent, or elective, and patients whose state might rapidly worsen or become more vulnerable to covid- if endoscopy is deferred should be prioritized. this includes patients with ibd who have the potential to deteriorate or require empiric high-dose steroids if procedures are delayed. caring for patients with ibd during covid- has posed unique challenges for both patients and providers, and decisions regarding the evaluation and treatment of disease have become more complex. the pervasive concerns surrounding the novel coronavirus require providers to present patients with evidence-based information regarding the risks of covid- and the best treatment of their ibd. analysis of the secure-ibd registry and additional research are needed to further our knowledge about covid- in patients with ibd and to help better advise patients and guide management in an unprecedented time. clinical characteristics of coronavirus disease in china gastrointestinal manifestations of sars-cov- infection and virus load in fecal samples from a hong kong cohort: systematic review and meta-analysis world health organization. coronavirus disease (covid- ) pandemic the global, regional, and national burden of inflammatory bowel disease in countries and territories, - : a systematic analysis for the global burden of disease study concerns related to covid- pandemic among patients with inflammatory bowel disease and its influence on patient management centers for disease control and prevention. coronavirus (covid- aga clinical practice update on management of inflammatory bowel disease during the covid- pandemic: expert commentary british society of gastroenterology guidance for management of inflammatory bowel disease during the covid- pandemic european crohn's and colitis organisation. th interview covid- ecco taskforce surveys/ th_interview_covid- _ecco_taskforce_published ioibd. ecco update on covid- and ibd management of patients with crohn's disease and ulcerative colitis during the covid- pandemic: results of an international meeting european crohn's and colitis organisation. st interview covid- ecco taskforce publication/ _ _surveys/ st_interview_covid- _eccotaskforce_published implications of covid- for patients with preexisting digestive diseases crohn's & colitis foundation. coronavirus (covid- ): what ibd patients should know about crohn's & colitis: covid- and ibd. https:// crohnsandcolitis.ca/about-crohn-s-colitis/covid- -and-ibd covid- ): faqs for people with crohn's and colitis crohn's & colitis australia. coronavirus (covid- ) response management of inflammatory bowel disease patients in the covid- pandemic era: a brazilian tertiary referral center guidance joint gi society message on covid- aerosol and surface stability of sars-cov- as compared with sars-cov- evidence for gastrointestinal infection of sars-cov- prolonged presence of sars-cov- viral rna in faecal samples persistence and clearance of viral rna in novel coronavirus disease rehabilitation patients characteristics of pediatric sars-cov- infection and potential evidence for persistent fecal viral shedding detection of sars-cov- in different types of clinical specimens virological assessment of hospitalized patients with covid- european crohn's and colitis organisation. th interview covid- ecco taskforce acg clinical guideline: preventive care in inflammatory bowel disease european crohn's and colitis organisation. nd interview covid- ecco taskforce publication/ _ _surveys/ nd_interview_covid- _ecco_taskforce_pub-lished covid- : recommendations for management of elective surgical procedures joint gi society message on ppe during covid- aga institute rapid recommendations for gastrointestinal procedures during the covid- pandemic endoscopy in inflammatory bowel diseases during the covid- pandemic and post-pandemic period etiology and management of lack or loss of response to anti-tumor necrosis factor therapy in patients with inflammatory bowel disease coronavirus and ibd reporting database strategies for the care of adults hospitalized for acute ulcerative colitis covid- and immunomodulation in ibd expression of sars-cov- entry molecules ace and tmprss in the gut of patients with ibd prevention of covid- in patients with inflammatory bowel disease in wuhan, china guidance for resuming gi endoscopy and practice operations after the covid- pandemic key: cord- - yn hg authors: d’amico, ferdinando; peyrin-biroulet, laurent; danese, silvio title: inflammatory bowel diseases and covid- : the invisible enemy date: - - journal: gastroenterology doi: . /j.gastro. . . sha: doc_id: cord_uid: yn hg nan a new beta-coronavirus, called severe acute respiratory syndrome coronavirus (sars-cov- ) was recently identified in wuhan, china . the virus proved to be transmitted from person to person and to be highly contagious, causing the onset of a pandemic in a short time . sars-cov- infection can be asymptomatic or cause coronavirus disease (covid- ) , which mainly includes respiratory and gastrointestinal symptoms and in severe cases it can be fatal . several measures have been taken to prevent the further spread of the virus, including the use of masks, gloves, and disinfectants, and restrictions to avoid traveling to risk areas and having close contact with other people . moreover, many countries have imposed quarantine and lockdown of schools and all non-essential activities . in this context, particular attention should be paid to patients treated with immunosuppressive drugs or biologics, as they have a higher risk of infection . other risk factors identified for the development of covid- are the elderly age, the presence of chronic diseases such as hypertension and diabetes, some professional categories (e.g. health personnel, policemen, and supermarket clerks) and exposure to infected people . in a recent work published in lancet , precautions taken by a wuhan center for inflammatory bowel diseases (ibd) were reported. all biological and immunosuppressive treatments were discontinued, visits in person were replaced by online consultations, and patients were daily recommended to wash their hands frequently, to reduce the time spent outside the home and to use masks outside the home . it is important to underline that a relevant percentage of evaluated patients had risk factors of infection: chronic diseases ( . %), immunosuppressive agent therapy ( . %), elderly ( . %), high-risk professional categories ( . %), and biological therapy ( . %) . recommendations were followed by % of subjects and no cases of covid- infection were detected after a period of approximately months . finally, the authors stressed the importance of daily warnings and recommendations, which favored a wide adherence of patients and contributed to avoid viral contagion . the covid- health emergency has forced physicians to deal with problems never before and to take quick decisions in consideration of the urgent needs. in addition, the limited knowledge regarding transmission modalities, clinical symptoms, and natural history of the virus infection has made the management of ibd patients and the adoption of preventive measures even more complex and difficult. currently, the main source of transmission seems to be mediated by air droplets , although ever-increasing evidence supports the possibility of a fecal-oral infection identify the best way to prevent covid- in ibd patients, to understand whether ibd patients are at increased risk of being infected, and to clarify whether they should be considered as a high-risk population as well as the impact of ibd-related drugs on covid- . another aspect suggested by an et al. is the relevance of the continuous assistance to ibd patients also at home. home patient management has been called "telemedicine" and it is a well-accepted approach by patients, as evidenced by the high adherence to home therapies or recommendations from physicians , . telemedicine has also been associated with a reduction in gastroenterological consultations and hospitalizations and could be a valid alternative to improve the quality of ibd patient care during the covid- outbreak . follow-up of this cohort is essential to confirm the real impact of the preventive strategies. furthermore, the biggest challenge for western countries will be to extrapolate chinese success and overcome cultural differences that could hinder adhesion rates. in conclusion, the coronavirus pandemic is straining the existing health systems and will leave an indelible mark on both patients and physicians, but it is providing new insights and could be a starting point for investing in new approaches and increasingly personalizing patient care. a novel coronavirus from patients with pneumonia in china who director-general's opening remarks at the media briefing on covid- - clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china q&a on coronaviruses (covid- ). available at the positive impact of lockdown in wuhan on containing the covid- outbreak in china comparative risk of serious infections with biologic and/or immunosuppressive therapy in patients with inflammatory bowel diseases: a systematic review and meta-analysis protection of inflammatory bowel disease patients from the outbreak and rapid spread of covid- infection in wuhan aerosol and surface stability of sars-cov- as compared with sars-cov- covid- : gastrointestinal manifestations and potential fecal-oral transmission imbalance of the renin-angiotensin system may contribute to inflammation and fibrosis in ibd: a novel therapeutic target? gut increased incidence of systemic serious viral infections in patients with inflammatory bowel disease associates with active disease and use of thiopurines ioibd update on covid for patients with crohn's disease and ulcerative colitis | ioibd management of ibd during the covid- outbreak: resetting clinical priorities withdrawal of immunosuppressant or biologic therapy for patients with quiescent crohn's disease evolution after anti-tnf discontinuation in patients with inflammatory bowel disease: a multicenter long-term follow-up study european crohn's and colitis organisation topical review on treatment withdrawal facing covid- in italy -ethics, logistics, and therapeutics on the epidemic's front line fair allocation of scarce medical resources in the time of covid- implications of covid- for patients with pre-existing digestive diseases feasibility and acceptance of a home telemanagement system in patients with inflammatory bowel disease: a -month pilot study telemedicine platform myibdcoach reduces hospitalisations and outpatient gastroenterology visits in patients with ibd amico declares no conflict of interest. l peyrin-biroulet has served as a speaker, consultant and advisory board member for merck danese has served as a speaker, consultant, and advisory board member for schering-plough fd wrote the article. lpb critically reviewed the content of the paper and supervised the project. sd conceived and critically revised the manuscript. the manuscript was approved by all authors. key: cord- - alfvda authors: galmozzi, e.; lampertico, p. title: letter: does the ifnl gene discovery really provide a causal role for the il b haplotype blocks? date: - - journal: aliment pharmacol ther doi: . /apt. sha: doc_id: cord_uid: alfvda nan sirs, we read with great interest the comprehensive review study of mouli and ananthakrishnan. the authors have evaluated and discussed current evidence regarding the roles of vitamin d deficiency in the pathogenesis and progression of inflammatory bowel disease (ibd). in this regard, different cellular and molecular mechanisms are discussed in this review. however, as mentioned by the authors, there is a strong role for environmental factors in the pathogenesis and progression of ibd. there is growing evidence that psychological factors, including psychological stress and depression, are influential in the pathogenesis and clinical course of ibd. , growing evidence also indicates the importance of sleep disorders in the clinical course of ibd. it has been shown that sleep disturbance increases the risk of disease flares in patients. [ ] [ ] [ ] it is therefore interesting that vitamin d deficiency has been shown to play a role in both psychological and sleep disorders in non-ibd populations, although the underlying mechanisms are still unknown. there is no study till now evaluating the effects of vitamin d deficiency on psychological health or sleep quality in ibd patients. although several factors can affect psycho-logical health and sleep quality in ibd patients including demographic-and disease-related factors, , a role for vitamin d deficiency is also plausible. therefore, the 'optimal role of vitamin d supplementation as a therapeutic modality in patients with ibd' is not only the induction and maintenance of remission as mentioned by mouli and ananthakrishnan but may also include treatment of psychological and sleep disorders in ibd patients. these issues should be added to the remaining unanswered questions regarding the role of vitamin d in ibd, and warrant evaluation in future studies. letter: does the ifnl gene discovery really provide a causal role for the il b haplotype blocks? while ifnl variant was strongly associated with a sustained virological response in both hcv genotypes and , and thus confirmed previous findings by prokunina-olsson et al. and bibert et al., the study confirms a strong linkage between ss and rs variants (q = . ) compared with the modest correlation between rs and ifnl (q = . ). these findings therefore do not support an additional clinical benefit of ifnl in the prediction of a response to peg/ rbv in caucasian patients. recently, the finding that recombinant ifnl protein exerts a potent antiviral activity against both hcv and human coronaviruses works against the assumption that the same protein does inhibit clearance of hcv, i.e. negatively interfering with hcv infection, while being endowed with a strong anti-hcv activity. this suggests the existence of a complex relationship between ifnl and hcv in humans, similar to that recently described for ifn- and lymphocytic choriomeningitis virus in mice, where chronic ifn-i signalling due to persistent infection drives immunosuppression and disease progression. although validation studies in large cohorts of patients, like the one investigated by st€ attermayer et al., may help understand the clinical utility of ifnl , these studies have so far failed to demonstrate any causal rela-tionship between ifnl protein and nonresponse to interferon therapy in chronic hcv patients. among other approaches, the assessment of the missense variants previously described in the ifnl coding region, may help unravel this issue. review article: vitamin d and inflammatory bowel diseases psychological stress in ibd: new insights into pathogenic and therapeutic implications environmental triggers for inflammatory bowel disease sleep disturbances and inflammatory bowel disease: a potential trigger for disease flare? sleep disturbance and risk of active disease in patients with crohn's disease and ulcerative colitis assessment of the relationship between quality of sleep and disease activity in inflammatory bowel disease patients vitamin d deficiency and depression in adults: systematic review and metaanalysis the link between vitamin d metabolism and sleep medicine polymorphisms of interferon-k and il b -effects on treatment response to interferon/ribavirin in patients with chronic hepatitis c a variant upstream of ifnl (il b) creating a new interferon gene ifnl is associated with impaired clearance of hepatitis c virus il b expression depends on a novel tt/-g polymorphism which improves hcv clearance prediction interferon lambda signals via the ifnk receptor to regulate antiviral activity against hcv and coronaviruses blockade of chronic type i interferon signaling to control persistent lcmv infection acknowledgement declaration of personal and funding interests: none. declaration of personal and funding interests: none. sirs, we thank dr galmozzi and dr lampertico for their comment on our recently published paper on the effect of the dinucleotide frameshift variant in ss in the interferon (ifn)-k gene on interferon/ribavirin treatment and its relationship with the two commonly used single nucleotide polymorphisms (snp) in il b (rs , rs ). , we agree that our study does not provide insights on the causal relationship between ifnl and treatment response in patients with chronic hepatitis c virus (hcv) infection. nevertheless, our study was designed to investigate the clinical usefulness of the different snps in il b and ifnl in a large cohort of caucasian patients infected with chronic hcv.hamming et al. demonstrated that ifnl encodes an active type iii interferon with potent anti-viral activity against both hcv and coronaviruses. galmozzi and lampertico therefore conclude that this strong anti-viral activity works against the assumption of an inhibition of the ifnl protein of treatment-induced hcv clearance. we would like to remind them that up-regulation of intrahepatic interferon-stimulated genes (isg) is associated with treatment failure in patients with chronic hcv, and levels of isg are differently distributed according to different il b genotypes. furthermore, prokunina-olsson and her collaborators demonstrated that ifnl induces isg expression in hepg hepatoma cells. thus, high baseline isg levels might be associated with poor response to exogenous ifn, by exhausting the ifn response pathways. key: cord- -cww ha j authors: manolakis, anastassios c; christodoulidis, gregory; kapsoritakis, andreas n; georgoulias, panagiotis; tiaka, elisavet k; oikonomou, kostas; valotassiou, varvara j; potamianos, spyros p title: α -heremans-schmid glycoprotein (fetuin a) downregulation and its utility in inflammatory bowel disease date: - - journal: world j gastroenterol doi: . /wjg.v .i . sha: doc_id: cord_uid: cww ha j aim: to investigate the impact of inflammatory bowel disease (ibd) on α -heremans-schmid glycoprotein (ahsg/fetuin a) and potential associations with disease and patient characteristics. methods: ahsg serum levels were determined in treatment-naïve newly-diagnosed patients, with ulcerative colitis (uc), with crohn's disease (cd), with diarrhea-predominant or mixed irritable bowel syndrome (ibs, d- and m- types) and healthy controls (hc), by an enzyme linked immunosorbent assay (elisa). all patients were followed for a minimum period of years at the gastroenterology department of the university hospital of larissa, greece. c-reactive protein (crp), anti-glycan antibodies, anti-saccharomyces cerevisiae mannan antibodies igg, anti-mannobioside carbohydrate antibodies igg, anti-laminariobioside carbohydrate antibodies igg and anti-chitobioside carbohydrate antibodies iga were also determined via immunonephelometry and elisa, respectively. results: the mean ± se of serum ahsg, following adjustment for confounders, was . ± . g/l in ibd, . ± . g/l in cd and . ± . g/l in uc patients, significantly lower than in ibs patients ( . ± . g/l) and hc ( . ± . g/l) (p < . , in all cases). ahsg levels were comparable between the cd and uc groups. based on ahsg levels ibd patients could be distinguished from hc with about % sensitivity and specificity. further adjusted analysis verified the inverse association between ahsg and penetrating, as well as stricturing cd (partial correlation coefficient: - . and - . , respectively) (p < . ). after adjusting for confounding factors, inverse correlations between ahsg and crp and the need for anti-tnfα therapy or surgery, were found (partial correlation coefficients: - . , - . , - . , respectively, p < . , in all cases). finally, ibd individuals who were seropositive, for at least one marker, had ahsg levels falling within the two lower quartiles (or = . , %ci: . - . , p < . ) while those with at least two serological markers positive exhibited ahsg concentrations within the lowest quartile (or = . , %ci: . - . , p < . ), after adjusting for age, sex and smoking. conclusion: ahsg can be used to distinguish between ibd and ibs patients or hc while at the same time "predicting" complicated disease behavior, need for therapy escalation and surgery. moreover, ahsg may offer new insights into the pathogenesis of ibd, since it is involved in key processes. inflammatory bowel disease (ibd) has been long ago recognized as a systemic inflammatory entity and as such, it is anticipated to induce changes exceeding the boundaries of bowel mucosa, being reflected in a broader spectrum of tissues, including blood [ ] [ ] [ ] [ ] [ ] [ ] [ ] . examples of such changes are the fluctuations in the levels of c-reactive protein (crp), tumour necrosis factor alpha (tnf-α), interleukins, s proteins, metalloproteinases, angiogenins, etc [ ] [ ] [ ] [ ] . since a different task is carried out by each of these biomarkers, it has been proposed that ibd induces multifarious responses which in turn, may affect the levels of different compounds via feedback mechanisms, consumption or reprioritisation of synthesis [ ] [ ] [ ] [ ] [ ] . these observations along with the inflammatory nature of ibd itself seem to encourage the study of novel proteins that tend to become affected by the establishment of systemic inflammation [ , ] . such a candidate is fetuin a or α -heremans-schmid glycoprotein (ahsg), a substance synthesized in the liver, bone marrow and fetal organs, exhibiting properties similar to those possessed by negative acute-phase proteins [ ] . ahsg has been shown to carry out various immunologic tasks, including regulation of macrophage-related lipopolysaccharide-triggered opsonisation, tnf-α and transforming growth factor beta (tgf-β) levels [ ] [ ] [ ] [ ] . ahsg is also an inhibitor of ectopic tissue calcification such as that taking place in coronary arteries and heart valves, while at the same time promoting mineralization within fibrils and subsequently, proper bone formation [ ] [ ] [ ] . other recently discovered properties of ahsg include a binding ability to insulin receptor, modifying its sensitivity, participation in wound healing as well as tumor growth processes, through the induction of a more effective cell migration [ , ] . increased ahsg levels have been recorded in fetal organs (probably reflecting an additional role for ahsg in normal organ development), in adults with either metabolic syndrome or increased insulin resistance alone, patients infected with the severe acute respiratory syndrome-coronavirus as well as individuals at high risk for future cardiovascular events: stroke and acute myocardial infarction [ , , ] . low levels of ahsg have been recorded in patients on hemodialysis, those with cirrhosis, hepatoma or rheumatoid arthritis (ra), an entity sharing common inflammatory pathways with ibd, and have been linked to vascular -excessive valvular and coronary artery calcification, ischemic events-and skeletal disorders -osteopenia [ , , ] . moreover, similar phenomena: upregulation of tnf-α and tgf-β, remodelling in intestinal microvessels, both on an acute and chronic basis, as well as manifestations linked to mineral homeostasis i.e., osteoporosis and urolithiasis, are all exerted in ibd. when bearing in mind these characteristics, the idea of examining ahsg levels in ibd, seems more than tempting [ , [ ] [ ] [ ] [ ] [ ] . among several patients visiting er wards, outpatient clinics or being hospitalised for chronic diarrhea, a total of patients were recruited: diagnosed with ulcerative colitis (uc), with crohn's disease (cd) and with irritable bowel syndrome (ibs). all patients were followed for a minimum period of years at the department of gastroenterology at the university hospital of larissa, greece. another group consisting of healthy individuals was also formed (hc). study groups were age and sex matched (p > . ) with one exception: the cd and uc groups, differed significantly (p = . ) as anticipated, since cd is often associated with younger age. all individuals participating in the study lacked any known disease i.e., end-stage renal disease, ra, cirrhosis, hepatoma, liver metastases potentially affecting ahsg levels, with the exception of diabetes mellitus (dm), which was treated as a confounding factor and was embedded in the models used for multivariate testing. the demographic and clinical characteristics of patients and hc are presented in table . the diagnosis of ibd and ibs was established upon the co-evaluation of findings originating from clinical and endoscopic procedures, imaging studies, histopathology and laboratory analyses. ibs patients were also diagnosed and classified as d-ibs or m-ibs ( and individuals, respectively) according to the rome Ⅲ criteria [ ] . disease activity in the ibd group was documented using conventional indices: crohn's disease activity index (cdai) and the clinical activity index (cai), for uc [ , ] . a cdai score greater than january , |volume |issue | wjg|www.wjgnet.com and a cai score exceeding , on a - scale, were considered as active cd and active uc, respectively. disease location and behavior, in cd, were determined using the vienna classification whereas for disease extent, in uc, the montreal classification was used [ , ] . no animals were used for the present study. blood samples were collected upon presentation of patients in our hospital, in serum separator tubes and were allowed to clot for min. all samples were then centrifuged and the obtained serum was stored at - ℃ for later analysis. the pre-analytical phase, including sampling and handling methods (sampling tubes, storage conditions etc.) was identical in all cases. ahsg assay: for ahsg determinations, a two-site "sandwich" enzyme-linked immunosorbent assay (elisa) was performed, using a commercially available human fetuin a elisa kit (biosource europe sa, belgium). assay calibrators, controls and prediluted patient serum samples ( μl initially) containing human ahsg were added to microplate wells, coated with a high affinity polyclonal goat anti-human ahsg antibody. during incubation period, the antibody could capture human ahsg in the sample. unbound proteins were then washed away and a horseradish peroxidase (hrp) conjugated polyclonal anti-human ahsg antibody was added to each well, so that a "sandwich" of "capture antibody-human ahsg-hrp conjugated detecting antibody" could be formed. after additional washing, incubation with a substrate solution took place, the reaction was stopped and the developed colour was quantified spectrophotometrically. the enzymatic activity of the detecting antibodies, bound to the ahsg on the wall of the microwells, was directly proportional to the amount of ahsg in the sample. a calibration curve which was generated by plotting the absorbance vs the respective human ahsg concentration for each calibrator, allowed sample ahsg determination. c-reactive protein assay: for the determination of c-reactive protein (crp), immunonephelometry was performed using the behring nephelometer analyzer Ⅱ, as well as the n high sensitivity commercially available kit (dade behring gmbh, germany). the control and standard sera were provided by the same company and used according to the manufacturer's instructions. anti-glycan antibodies assay: serum levels of anti-saccharomyces cerevisiae mannan antibodies (gasca) igg, anti-mannobioside carbohydrate antibodies (amca) igg, anti-laminariobioside carbohydrate antibodies (alca) igg and anti-chitobioside carbohydrate antibodies (acca) iga were also determined in ibd ( cd and uc) patients, using commercially available elisa kits (ibdx, glycominds ltd., israel). cut-off levels for positivity were set at , , , and u/ml for gasca igg, amca igg, alca igg, and acca iga, respectively, as instructed by the manufacturer. normality (kolmogorov-smirnov) test was initially carried out and since the normality assumption was satisfied for the comparison of means between two groups, student's t-tests were used. for comparisons between multiple groups, one-way anova and tukey's post-hoc tests were applied. variables are expressed as mean ± sd or mean ± se. for variables without comparable variations, welch's correction has been applied. ahsg was tested for its ability to predict ibd, uc and cd, separately, using receiver operating characteristic (roc) curves, while area under the curve (auc) and cut-off values, with the optimal sensitivity and specificity, were also calculated. for the simple correlation studies, pearson's rank test was used. statistical significance was set at p < . . whenever statistical significance or trend ( . < p < . ) was recorded in univariate analysis, multivariate testing was also performed. using multiple linear regression and a backward selection process independent variables affecting ahsg levels -confounders -were identified. as candidate confounding factors were initially considered age, sex, smoking, dm, treatment modalities, disease duration and behaviour, age at onset. for the associations originating from multiple linear regression, partial correlation coefficients -quantifying the relationship between two variables while controlling for other factors-are reported. whenever a categorical parameter was treated as dependent variable, logistic regression analyses, simple and multiple, were applied and odds ratios (ors) as well as %cis, unadjusted/adjusted for confounding, were calculated. adjusted means were also calculated using analysis of covariance. statistical analyses were conducted using graphpad prism ( . and . ) and the medcalc . . . statistical softwares. statistical review of the study was performed by a biostatistician. the study was approved by the university of thessaly medical school ethics committee. informed consent was obtained from all study participants, along with a verbal permission for the use of the acquired samples for scientific research. the mean ± se of ahsg in serum was . ± . g/l for ibd, . ± . g/l for ibs patients and . ± . g/l for hc. the recorded difference between the ibd and control groups was statistically significant (p < . ) and this was also the case when cd and uc were compared separately with ibs patients and hc. ahsg levels in the cd group were . ± . g/l, significantly lower than those of ibs patients and hc (p < . ). likewise, uc patients also exhibited lower ahsg levels ( . ± . g/l) compared to ibs patients and hc (p < . ). these differences remained significant between ibd ( . ± . g/l), cd ( . ± . g/l) or uc patients ( . ± . g/l), ibs patients ( . ± . g/l) and hc ( . ± . g/l), after adjustment for age and sex (p < . , for all comparisons). when ahsg levels were compared between uc and cd patients or between ibs patients and hc, no significant differences were observed (p > . , in both cases) ( figure ). roc curve analysis showed that the optimal cut-off of ahsg for the prediction of ibd was . g/l ( % sensitivity and specificity). similarly, an ahsg value of . g/l could distinguish cd patients from non-ibd individuals (ibs and hc) with a sensitivity of % and a specificity of . %, while a value of . g/l could discriminate uc patients and non-ibd subjects with . % sensitivity and . % specificity. the auc was . ( %ci: . - . ), . ( %ci: . - . ) and . ( %ci: . - . ) for the prediction of ibd, uc and cd, respectively (p < . , in all cases) ( figure ). all ibd patients exhibited active disease when blood was drawn for later analysis. in our study, in patients with active disease the ahsg levels were marginally associated with cdai and cai scores (r = - . , p = . ). when disease location (cd) and extent (uc) were taken under consideration no statistically significant differences were observed (p > . in all cases). another part of the present study included the comparison of ahsg serum concentrations among cd patients with diverse disease behavior: stricturing, penetrating and non-stricturing non-penetrating (ns/np). the performed analysis showed that patients with stricturing or penetrating disease had lower ahsg levels ( . ± . g/l and . ± . g/l, respectively), compared to patients in the ns/np subgroup ( . ± . g/l). these differences were statistically significant between cd patients with stricturing and ns/np (p < . ) or between the penetrating and ns/np disease subgroup (p < . ) but not between the stricturing and penetrating subgroups (p > . ) (figure ). further analysis verified the inverse association between ahsg and penetrating, as well as stricturing cd, both before (r = - . and - . , respectively, p < . ), as well as after adjustment (partial correlation coefficient: - . and - . , respectively, p < . ) for age, sex and smoking status. in order to perform additional testing of the link between lower ahsg levels and complicated disease behavior, logistic regression was applied, while considering penetrating or stricturing cd, as dependent, and ahsg concentrations, in quartiles -lowest, low, high, highest -as independent variables. the results originating from this analysis showed that ahsg levels, in the lowest quartile, were associated with both penetrating as well as stricturing disease, before (or = . , %ci: . - . and or = . , %ci: . - . , respectively) and after adjustment (or = . , %ci: . - . and or = . , %ci: . - . , respectively) for age, sex and smoking status (p < . , in all cases). ahsg levels were also examined with respect to the presence of one or more ibd-related extraintestinal manifestations. ibd patients exhibiting extraintestinal manifestations had comparable ahsg levels ( . ± . g/l) to the ibd subgroup without such disorders ( . ± . g/l, p = . ). interestingly, ibd patients with a history of recurrent urolithiasis had ahsg levels at the lowest quartile, this result, however, did not reach statistical significance. all data on ahsg variations according to the already described disease characteristics are presented in table . the levels of serum ahsg were studied with respect to gender of ibd patients, so that potential differences could be highlighted. both male and female ibd patients exhibited comparable ahsg levels ( . ± . g/l and . ± . g/l, respectively) (p = . ). likewise, ahsg levels were similar between ibd patients, while taking into account age at onset and smoking habits (table ) . serum ahsg concentrations were examined with regard to treatment modalities adequate for inducing and maintaining remission during the -year follow up period: -aminosalicylates ( -asa), corticosteroids, immunosuppressants, anti-tnfα agents or surgery. during comparison, ibd patients requiring surgical intervention or the use of anti-tnfα therapy exhibited lower ahsg concentrations ( . ± . g/l and . ± . g/l), compared to those adequately treated with -asa ( . ± . g/l) or corticosteroids ( . ± . g/l) (p < . , in both cases). further evaluation of the recorded associations, using simple linear regression analysis, showed that ahsg levels were inversely associated with the need for anti-tnfα treatment (r = - . , p < . ) and surgery (r = - . , p < . ). after multivariate analysis -also considering age, sex, activity, duration, smoking status-the inverse association between ahsg and need for anti-tnfα therapy or surgery remained statistically significant (partial correlation coefficients: - . and - . , respectively -p < . , in both cases). since these results are suggestive of a link between a more profound downregulation of ahsg levels and the need for anti-tnfα treatment or surgical intervention, ahsg concentrations were classified into quartiles. using logistic regression, ahsg levels in the lowest quartile were found to be an independent predictor of the need for anti-tnfα treatment, in a model adjusted for other treatment modalities, age, sex, smoking status and disease duration (or = . , %ci: . - . , p < . ). similarly, by applying the same adjusted model, it was shown that need for surgery could be independently predicted by the presence of ahsg levels within the lowest quartile (or = . , %ci: . - . , p < . ). ibd patients had higher crp levels (median: . mg/dl, range: . - . mg/dl) compared to ibs (median: . mg/dl, range: - . mg/dl) and hc groups (median: . mg/dl, range: - . mg/dl) (p < . ). a correlation study of ahsg with the levels of the inflammatory marker crp was performed revealing marginal association (r = - . , p = . ). multivariate analysis, considering as confounding variables age, sex and smoking on the other hand, revealed a closer association between the two substances with a - . partial correlation coefficient and a p = . level of significance. positivity rates for serological markers were % for gasca, . % for alca, % for amca and alca while actual median concentrations are presented in table . additional associations were investigated by examining positivity for these serological markers and january , |volume |issue | wjg|www.wjgnet.com ahsg (in quartiles). interestingly, an inverse association between serology and ahsg levels was recorded. ibd individuals who were seropositive, for at least one marker, had ahsg levels falling within the two lower quartiles (or = . , %ci: . - . , p < . ) while those with at least two serological markers positive exhibited ahsg concentrations within the lowest quartile (or = . , %ci: . - . , p < . ). further analysis also considering potential confounding factors such as age, sex and smoking did not alter the reported associations significantly: or = . ( %ci: . - . ) for single and or = . ( %ci: . - . ) for multiple seropositivity (p < . , in both cases). in accordance with a preliminary report from our team in , ahsg levels were downregulated in patients with ibd thus, allowing discrimination from ibs and hc individuals [ ] . furthermore, a more profound downregulation of ahsg was very well associated with complicated disease behavior and the need for biological anti-tnfα treatment or surgery. additional associations with crp and anti-glycan antibodies offered better insight into ahsg's link with acute-phase response and january , |volume |issue | wjg|www.wjgnet.com ibd course. the finding of an ibd-induced downregulation of ahsg is nothing but surprising since, ahsg levels decrease in the presence of robust inflammation [ , , ] . moreover, an interplay had already been documented between ahsg and the "notorious", in ibd, tnf-α and tgf-β [ ] [ ] [ ] . an additional finding further confirming the tight link of ahsg with tnfα, was the discovery of a binding site for tnfα, within the ahsg gene. as a consequence of tnfα's binding on this region, the expression of ahsg gene is suppressed, leading to decreased ahsg production while other substances are favored-repriorisation of liver synthesis [ , [ ] [ ] [ ] . due to the magnitude and rather ibd-selective character of this suppression, however, an evaluation of ahsg's ability to differentiate between entities has been performed. when a cut-off of . g/l was used, ahsg levels were shown to discriminate ibd from ibs and hc with a sensitivity and specificity of %. when magnitude of disease activity, as expressed through cdai and cai scores, was taken into consideration marginal differences were detected. according to the study by ma et al [ ] significant differences in ahsg levels exist between ibd patients with active or inactive disease. on the other hand, a correlation between ahsg and crp was confirmed, in our study. this may be, at least in part, due to limitations of the indices themselves although one should bear in mind that apart from the negative acute phase protein properties attributed to ahsg, its downregulation is also a part of a more complex liver deregulation, resulting from an excessive uptake and processing of signals [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . perhaps, possible associations of ahsg with other indices should also be evaluated [ ] [ ] . at this point, however, an asset of the present study has to be underlined: the ahsg levels recorded in ibd patients correspond to a prior-to-therapy status and therefore, could not have been altered by any of the known drugs used to treat ibd. a different set of results was obtained while examining ahsg levels with respect to uc extent and cd location. the recorded ahsg levels in this study could not predict disease location or extent and therefore, could not be used for this purpose. this was not the case when disease behavior was taken into account. a further downregulation of ahsg was observed, in cd patients with stricturing or penetrating disease, compared to those with the ns/np subtype. this is not surprising as a more diffuse transmural inflammation would exert a stronger proinflammatory effect [ ] . moreover, in many organs inflammatory stress leads to fibrosis and subsequently tissue calcification [ ] . from another pathogenetic perspective, a reason for the observed link between ahsg and stricturing disease could also be the reduced ahsg-induced counteraction of tgf-β's fibrogenic and antiproliferative potential [ , , ] . likewise, microvascular calcification, reduced blood flow and microthrombosis, exerted phenomena in the presence of low ahsg levels are also major findings during a chronic phase in the intestinal vasculature of ibd patients, leading to diminished intestinal perfusion and in turn to ulceration and fibrosis [ , ] . another scopus of the present study was to test whether there is a link between ahsg levels and the presence of extraintestinal manifestations. the performed analysis did not reveal any associations between ahsg serum concentration and the presence of any extraintestinal manifestations. in this case, a limitation exists: the rather small number of ibd patients manifesting specific disorders i.e., urolithiasis. a study focusing on the characteristics of ibd patients and ahsg levels was also performed. ahsg concentrations were compared among patients of different gender, smoking status and age at onset. none of these characteristics -male or female sex, current smoking or non-smoking habit and age at onset-could be linked with significant ahsg variations. this was not the case when ahsg levels were evaluated with respect to treatment modalities adequately inducing and sustaining disease remission during the three-year follow-up period. a robust downregulation of ahsg levels correlated with the need for anti-tnfα therapy. this finding is within reason since, a more profound and persisting upregulation of tnfα, requiring subsequently the use of an anti-tnfα agent, would result in a greater ahsg decrease. as far as the link between ahsg levels and need for surgery, is concerned, one should also bear in mind that this subgroup predominantly consisted of cd patients with stricturing and penetrating disease. in the case of a refractory uc, requiring colectomy, safe conclusions could not be drawn due to the small number of colectomised patients, in our study. the evidence mentioned above seems to make rather appealing the idea of ahsg's use as a diagnostic or even "predictive" tool rather tightly linked to ibd behavior as well as with markers related to it, such as the anti-glycan antibodies [ ] . a prerequisite for this type of use is that its lack of a disease-specific nature, with similar fluctuations also reported in other disorders, is taken into account [ , , , ] . a combined clinical and ahsg-based algorithm for the diagnosis of ibd, on the other hand, could help overcome this limitation. setting aside the already-performed diagnosticallyoriented interpretation of current results, a careful examination of ahsg's potential implication in the pathogenesis and complications of ibd, should also be performed. as already mentioned, ahsg is an inhibitor of unwanted, ectopic, tissue calcification and its decreased levels, as in this case, have been linked with excessive valvular and arterial calcification [ ] . although, initial reports originated from animal studies and those recruiting dialysis patients, similar results were obtained in patients with ischemic heart disease, alone [ ] . since a decrease in circulating ahsg is present in ibd, it would be logical to hypothesize that an accompanying reduced inhibition of vascular and valvular calcification would also be present. as this study was not designed to "tackle" these matters, the actual contribution of low ahsg levels in vascular changes and related acute or chronic ischemic events in ibd [ ] , remains to be clarified. apart from the implication of ahsg in vascular pathology, another role, that of bone mineralisation has been identified [ ] [ ] [ ] . under the influence of ahsg, a "reliable", stress-resistant bone structure is formed, while in the absence of ahsg, minerals deposit outside fibrils, leading to defective osseous formation and finally osteoporosis [ ] . as ibd patients are at high risk for osteoporosis, resulting from corticosteroids, malnutrition and inflammation-induced osteopenia [ ] the examination of possible contribution of ahsg suppression for the onset of osteopenic manifestations might prove fruitful. in a rather similar manner, a defective coordination in mineral use, predominantly that of calcium, due to decreased ahsg levels, may lead to urolithiasis. indeed, in the study of stejskal et al [ ] lower levels of ahsg in urine have been associated with the presence of urolithiasis. although, in the case of ibd, wellestablished mechanisms-i.e., hyperoxaluria, reduced citric acid etc [ ] -for urinary compications exist, when bearing in mind that still % of urinary stones consist of calcium salts it is within reason to test for additional candidates involved in calcium homeostasis and urinary manifestations, such as ahsg [ ] . conclusively, ahsg seems to emerge as a molecule of potential diagnostic and perhaps predictive value in ibd, as its downregulation shows a tight link with acute-phase. as well as with chronic inflammatory responses in both cd and uc. this link also encompasses the more challenging cases, those with complicated disease behavior, as well as those requiring advanced treatment strategies such as biological agents or surgery. as far as the pathogenetic role of ahsg in ibd is concerned, further studies designed to assess the impact and the association of low ahsg levels with respect to micro-and macro-vascular changes, skeletal and urinary complications recorded in patients with cd and uc, are needed. laboratory evaluation of inflammatory bowel disease diagnostics of inflammatory bowel disease the immunology of inflammatory bowel disease inflammatory mediators and acute phase proteins in patients with crohn's disease and ulcerative colitis noninvasive markers in the assessment of intestinal inflammation in inflammatory bowel diseases: performance of fecal lactoferrin, calprotectin, and pmn-elastase, crp, and clinical indices matrix metalloproteinases in inflammatory bowel disease: boon or a bane? vascular endothelial growth factor in inflammatory bowel disease fetuin-a: a multifunctional protein alpha -hs glycoprotein: a protein in search of a function fetuin (alpha -hs-glycoprotein) opsonizes cationic macrophagedeactivating molecules fetuin, a negative acute phase protein, attenuates tnf synthesis and the innate inflammatory response to carrageenan fetuin/alpha -hs glycoprotein is a transforming growth factorbeta type ii receptor mimic and cytokine antagonist alpha -hs glycoprotein/fetuin, a transforming growth factor-beta/bone morphogenetic protein antagonist, regulates postnatal bone growth and remodeling vascular calcification and osteoporosis--from clinical observation towards molecular understanding mineralization by inhibitor exclusion: the calcification of collagen with fetuin increased fetuin a levels in helicobacter pylori infection: a missing link between h. pylori and insulin resistance? human fetuin/ alpha hs-glycoprotein level as a novel indicator of liver cell function and short-term mortality in patients with liver cirrhosis and liver cancer decreased levels of circulating alpha -heremans-schmid glycoprotein/fetuin-a (ahsg) in patients with rheumatoid arthritis fibrogenesis in crohn's disease nonimmune cells in inflammatory bowel disease: from victim to villain paradox of simultaneous intestinal ischaemia and hyperaemia in inflammatory bowel disease osteoporosis in inflammatory bowel disease renal stone formation in patients with inflammatory bowel disease guidelines--rome iii diagnostic criteria for functional gastrointestinal disorders development of a crohn's disease activity index. national cooperative crohn's disease study coated mesalazine ( -aminosalicylic acid) versus sulphasalazine in the treatment of active ulcerative colitis: a randomised trial a simple classification of crohn's disease: report of the working party for the world congresses of gastroenterology toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: report of a working party of the montreal world congress of gastroenterology inflammatory bowel disease-induced changes in circulating levels of a -heremans-schmid glycoprotein (fetuin a) decreased serum fetuin-a levels and active inflammatory bowel disease how accurate are clinical activity indices for scoring of disease activity in inflammatory bowel disease (ibd)? a systematic prospective comparison of noninvasive disease activity indices in ulcerative colitis effects of early reperfusion on the mechanical and biochemical characteristics of ischemic myocardium the cytological spectrum of chronic pancreatitis a review of electron beam computed tomography: implications for coronary artery disease screening new serological markers for inflammatory bowel disease are associated with earlier age at onset, complicated disease behavior, risk for surgery plasma fetuin-a levels and the risk of myocardial infarction and ischemic stroke risk of arterial thrombotic events in inflammatory bowel disease urine fetuin-a values in relation to the presence of urolithiasis genetics of hypercalciuric stone forming diseases the authors would like to thank the mathematician/ statistician ms aikaterini nikolaidou, msc for checking and verifying statistical analysis data. background α -heremans-schmid glycoprotein (ahsg/fetuin a), a negative acute-phase protein with multiple functions, becomes downregulated in the presence of inflammation. an ahsg downregulation in inflammatory bowel disease (ibd) patients has been initially reported by our research team in . another independent study, later verified this result. a major confounding factor, that of ibd-specific treatment existed in both studies. this is the first study assessing ahsg levels in treatment-naive patients. the low levels of ahsg found in ibd patients are not recorded in control subjects-ibs patients and healthy controls-and are well-associated with complicated disease behavior, as well as the need for anti-tnfα treatment or surgery. additional links with the acute phase protein c-reactive protein and serological markers linked to ibd course, the anti-glycan antibodies, have been found. ahsg could serve as an additional marker for ibd diagnosis and prediction of a more challenging course requiring treatment with biological agents or surgery, during the three-year period following initial diagnosis. the paper is valuable. it finds ahsg can be used to distinguish between ibd and irritable bowel syndrome, and may predict complicated disease behavior, and foretell the need for intensified therapy and surgery. moreover, ahsg may offer new insights into the pathogenesis of ibd. manolakis ac et al . fetuin key: cord- -rjseio s authors: sim, winnie h; wagner, josef; cameron, donald j; catto‐smith, anthony g; bishop, ruth f; kirkwood, carl d title: expression profile of genes involved in pathogenesis of pediatric crohn's disease date: - - journal: j gastroenterol hepatol doi: . /j. - . . .x sha: doc_id: cord_uid: rjseio s background and aim: expression profiling of genes specific to pediatric crohn's disease (cd) patients was performed to elucidate the molecular mechanisms underlying disease cause and pathogenesis at disease onset. methods: we used suppressive subtractive hybridization (ssh) and differential screening analysis to profile the mrna expression patterns of children with cd and age‐ and sex‐matched controls without inflammatory bowel disease (ibd). results: sequence analysis of clones enriched by ssh identified functionally annotated human genes, represented by clones. the genes have potential involvement in gene networks, such as antigen presentation, inflammation, infection mechanism, connective tissue development, cell cycle and cancer. twenty‐eight genes were previously described in association with cd, while were new genes not previously reported in the context of ibd. additionally, of the genes have been previously implicated in bacterial and viral infections. quantitative real‐time reverse transcription polymerase chain reaction performed on ileal‐derived rna from cd and nine non‐ibd patients confirmed the upregulation of extracellular matrix gene mmp (p = . ), and cell proliferation gene reg a (p = . ) in our pediatric cd cohort. conclusion: the retrieval of genes previously reported in association with adult cd emphasizes the importance of these genes in the pediatric setting. the observed upregulation of reg a and mmp , and their known impact on cell proliferation and extracellular matrix remodeling, agrees with the clinical behavior of the disease. moreover, the expressions of bacterial‐ and virus‐related genes in our cd‐patient tissues support the concept that microbial agents are important in the etiopathogenesis of cd. crohn's disease (cd) is a chronic inflammatory disorder of the bowel. the cause of cd is unclear and a complex interplay between genetic, environmental and immune components has been implicated. the prevailing hypothesis for the pathogenesis of cd is that an aberrant immune response, generated against microbial agents in genetically susceptible hosts, results in chronic intestinal inflammation. thus far, genes have been implicated in cd based on genome-wide association studies, and include genes involved in autophagy, maintenance of mucosal barrier integrity and immune regulation. , the nod /card on chromosome was the first locus implicated, mutations of which are thought to affect bacterial recognition. subsequently, four genes, il ra, il rb, psmg and tnfrsf b, have been linked to pediatric cd. , the polygenic nature of cd suggests that direct targeting of individual disease susceptibility genes is unlikely to be therapeu-tically effective. key molecules in pathophysiology, downstream of regulatory events induced by different causative factors are more likely targets for therapeutic interventions. insights into key gene-environmental interactions relevant to disease pathogenesis could help identify causative stimuli (e.g. infectious agents) based on molecular signatures of the host response. to date, microarray studies carried out on intestinal tissue of cd patients have identified several molecular biomarkers relating to inflammation, abnormal immunoregulation and cell biology, metabolism, signaling, transcription, electrolyte transport and extracellular matrix structure. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the suppressive subtractive hybridization (ssh) technique provides a complementary, non-biased approach to the identification of new genes or pathogens associated with cd. in ssh, suppression pcr normalizes the representation of rare and abundant cdna within the target population, and the subtraction step removes common nucleic acid sequences between the target specimen and its matched control. this results in an enriched pool of sequences specific to the target population. the advantage of this approach is that no assumed knowledge of gene identity is required, as it does not rely on a defined set of gene library or conserved sequence signatures as probes for gene identification. hence ssh complements microarray studies by identifying potentially important genes that may not be represented on the array platforms utilized by inflammatory bowel disease (ibd) microarray studies. ssh has been successfully used in the discovery of novel viruses, and the transcriptome profiling of human hepatoma and bone regeneration. [ ] [ ] [ ] in the present study, we used ssh to analyze the differential expression profile in ileal biopsies from children with cd compared with age-and sex-matched non-ibd control children. the purpose of this study was to examine the initial events occurring during cd pathogenesis. tissue selection. ileal biopsy specimens ( - mm ) were obtained from patients (aged - ) with symptoms suggestive of ibd and undergoing initial diagnostic endoscopy at the royal children's hospital, melbourne, australia. all tissue specimens were stored in rnalater (ambion, melbourne, australia) at - °c until nucleic acid extraction. the diagnosis of cd was established using standard clinical endoscopic and histopathological criteria according to the montreal classification. patients with esophagitis, mild non-specific gastritis or no known pathological diagnosis were used as non-ibd controls. none of the patients had received antibiotics or immunosuppressive drugs prior to endoscopy. demographic and clinical details of patients assayed by suppressive subtractive hybridization and real-time reverse transcription polymerase chain reaction (rt-pcr) are presented in tables and , respectively. each biopsy was mechanically homogenized, the supernatant harvested, and rna extracted using the allprep dna/rna mini kit (qiagen, melbourne, australia) according to the manufacturer's protocol. all extractions were conducted in a biological safety cabinet class ii. the cdspecific subtractive library was constructed using the pcr-select cdna subtraction kit according to the user manual provided (clontech, palo alto, ca, usa). an overview of the ssh technique is described in figure s . ileal rna were obtained separately from four cd and four non-ibd patients, then pooled into cd and non-ibd groups for the ssh assay. the patient groups were matched based on sex, mean age and common genotypes associated with cd, to minimize heterogeneity. differential screening. the library of differentially expressed cdna specific to the cd population was constructed using the topo ta cloning kit (invitrogen, melbourne, australia). five thousand randomly selected clones from the cd-specific subtractive library were spotted onto hybond nylon membrane endoscopic presentation of ileal region where biopsy is taken. where two biopsies taken from separate ileal locations of a patient differ in presentation, both are described here. § genotyping of patients based on single-nucleotide polymorphism were performed for an earlier study. major alleles are del, c, g, g, c and a; for nod leu fsinc, arg trp, gly arg; il , atg l and tlr respectively. cd, crohn's disease; ssh, suppressive subtractive hybridization. (amersham biosciences, sydney, australia) in ¥ by arrays by the australian genome research facility (agrf), melbourne. cd-specific sequences were detected by reverse hybridization with digoxigenin (dig)-labeled probes (roche, sydney, australia) synthesized directly from cdna of the cd and non-ibd subtractive library, according to manufacturer's protocol (dig applications manual for filter hybridization, roche). clones with greater than three times hybridization affinity to the cd-library-specific probes as compared to non-ibd-library-specific probes were selected for sequencing. the primers used are detailed in table s . quantification of cdna by real-time pcr was performed using the sybr greener qpcr super mix for abi prism (invitrogen), in accordance with manufacturer's instructions. analysis of real-time rt-pcr reactions and quantification of rna was determined using the system sequence detection software version . (applied biosystems). each sample was analyzed in triplicate. gene expression levels for individual patient samples were normalized relative to the expression of ribosomal protein l (rpl ) housekeeping gene. calculations were based on the pfaffl method, a mathematical method based on the real-time pcr efficiencies. the origene clone cdna ( fg) of each gene was used as the calibrator in every assay to allow for direct comparison of gene expression for all samples analyzed across multiple assays. statistical analysis. the mann-whitney u-test was used to compare the difference in median values between gene expression in cd and non-ibd patient samples. a p-value of less than . was considered statistically significant. all statistical tests were performed using sigmastat, version . (systat software inc., san jose, ca, usa). this study received ethics approval from the human ethics committee of the royal children's hospital (ehrc no. ). written and informed consent was obtained from each individual, parent or guardian prior to enrolment in the study. sequence analysis of differentially expressed clones from the cd subtraction library identified clones with high homology to genbank sequences. these included clones, which had matches to human mrna sequences representing annotated genes. the remaining clones had sequence similarity to mitochondrial and ribosomal genes, hypothetical proteins, expressed sequence tag (est), human chromosomes, bacterial and animal genes. the annotated genes were assigned to eight functional clusters based on information obtained from the ucsc genome browser and ncbi entrez gene database. the map location, gene function and frequency of ssh clone representation for each gene is listed in table s . we noted an enrichment of immune function genes and inflammatory mediators (cluster i and ii); extracellular matrix, remodeling, and ion transport coding genes (cluster iii); metabolic enzymes and signal transducers (cluster iv); genes involved in cell-cycle regulation (cluster v); cancer-related genes (cluster vi); transcription factors and post-transcription modifiers (cluster vi) and genes with unknown function (clusters viii). to assess the quality of the ssh data, genes representing different clone abundance levels were selected for real-time rt-pcr quantification on ileal biopsies. three genes were selected based on their representation of the ssh detection frequency range (high: > ; moderate: - ; low: < ), and also on potential functional interest with respect to cd pathogenesis. reg a ( clones) was selected based on its cell proliferative function and earlier reports of upregulation in colonic tissue of adult cd patients. , mmp ( clones) is involved in wound healing and has been proposed to have a protective role in colitis by regulating barrier function and vascularisation. anpep ( clones) has previously been reported to be a receptor for coronavirus. real-time rt-pcr analysis of the three genes was conducted on ileum-derived rna from cd and nine non-ibd patients, in triplicate. for cd patients cd , cd and cd , biopsies taken from both endoscopically affected and unaffected ileal locations were used in the analysis. individual gene expression levels for each sample were represented as fold change ratios relative to the expression of positive controls (origene clones for mmp , anpep and reg a). the individual expression levels (fold change value) of each gene for the biopsy samples of the cd and nine non-ibd patients are depicted in figure . using the mann-whitney statistical test for non-parametric and unpaired populations, the transcript expression levels of mmp were found to be significantly higher in cd ileal biopsies as compared to non-ibd ileal biopsies (p = . ). the cd population had a trend towards a higher level of reg a transcript expression, although the difference was not statistically significant (p = . ). there was no significant difference in anpep transcript expression between cd and non-ibd patient samples (p = . ). the real-time rt-pcr results validated that genes represented by > clones enriched by subtractive hybridization were expressed in higher abundance in cd as compared with non-ibd ileal biopsies. reg a, mmp and anpep expression. analysis of reg a, mmp and anpep gene expression across the cd patient samples revealed interesting patterns of expression. using a fold change ratio of as reference, four cd ileum samples (cd , cd , cd , cd un) with high levels of mmp expression, had low or negligible reg a and anpep expression (fig. ) . this inverse pattern of expression was also observed in the cd ileum samples where mmp gene expression was high. to contextualize our ssh findings, we compared our results with the data tables from seven microarray studies published previously, that had reported differential expression of genes between inflamed biopsies of cd and non-inflamed biopsies of non-ibd controls. [ ] [ ] [ ] [ ] [ ] [ ] [ ] of the annotated genes, genes have been previously analyzed by microarray ( table ) . the genes were either reported to be upregulated (n = ), downregulated (n = ) or variable (n = ) depending on biopsy site assayed. there were genes identified in this study that have not been previously described in the context of ibd investigations. to identify biological and functional networks based on potential gene interactions among the ssh enriched genes, we utilized the "core" program of the ingenuity pathway analysis software. the majority of the genes were classified into six networks comprising the following functions: (i) antigen presentation, inflammatory response, cancer; (ii) cancer, cell cycle, cellular compromise; (iii) connective tissue development and function, tissue morphology, developmental disorder; (iv) infection mechanism, genetic disorder, nutritional disease; (v) cell signaling, cellular assembly and organization, cellular function and maintenance; and (vi) amino acid metabolism, molecular transport, small molecule biochemistry (table ). network contained the highest number of ssh genes. interestingly, / genes in this network have been previously reported in microarray studies. the five newly identified genes within this network are cathepsin (ctss), dopa decarboxylase (ddc), integrin beta (itgb ), poly adp-ribose polymerase (parp ) and prothymosin alpha (ptma). figure depicts a schematic representation of this gene network. ctss and itgb appear to be involved in multiple pathways, including several direct and indirect associations with the previously reported genes. to elucidate evidence for microbial pathogenesis, the functionally annotated genes were individually searched against the ncbi entrez gene database for reported functional associations with viral or bacterial infections. a total of genes associated with microbial pathogenesis were identified (table ). the pathogenesis of cd is thought to involve a complex interplay between the microbiome, the environment and multiple genetic factors. to gain further insights into the gene regulation processes involved, several gene array analyses have been performed using surgical resections or endoscopic biopsies of the colon obtained during treatment of adults with known ibd. [ ] [ ] [ ] [ ] [ ] however, the chronicity of the disease process and variability of treatments used are likely to have influenced gene expression profiles in these patients. our study used tissue obtained at initial diagnosis in treatment-naive children with early onset disease. to date, there have been very few studies of events at the genetic level during early disease onset in children. a recent study examining the genome-wide expression profile of pediatric ibd patients was conducted using colonic tissue. our study extends these initial gene expression profile studies by comparing ileal biopsies from a pediatric cohort of cd and non-ibd patients. ssh analysis led to the identification of functionally annotated genes, specific to the cd cohort. comparison of our ssh data with existing microarray studies revealed that of these genes are novel and genes have been previously identified by microarray to be either upregulated or downregulated in the cd population. gene networks. the antigen presentation, inflammatory response and cancer gene network (network ) comprise one-third figure the relative expression levels of reg a, mmp and anpep in ileal biopsies from crohn's disease (cd) and nine non-inflammatory bowel disease (ibd) patients. the relative expression ratio of each gene was calculated based on real-time reverse transcription polymerase chain reaction (rt-pcr) efficiency and the crossing point deviation of the target patient sample versus the internal rpl control, according to pfaffl. of the genes identified by ssh, with a high proportion of genes previously identified to be differentially expressed in cd. this is partially attributable to acute inflammation of the biopsies of cd patients as compared with the non-inflamed biopsies of non-ibd controls. differences in gene expression profiles between inflamed and non-inflamed cd terminal ileum have been recently described. relative to non-ibd controls, the gene expressions of il- and saa were reportedly much higher in inflamed cd terminal ileum as compared to non-inflamed cd terminal ileum. new genes identified within this network include ctss, ddc, itgb , parp and ptma. based on the molecular interactions depicted in this network, ctss and itgb appear to be involved in , and with other genes previously reported as upregulated in cd population. ctss is mainly expressed in antigen-presenting cells and is required for the degradation of mhc-class-iiassociated invariant chains, necessary for proper mhc class ii antigen presentation. , integrins, which include itgb , are membrane receptors involved in cell adhesion and several processes, including immune response. itgb is expressed during hypoxic conditions, and can serve as an indicator of intestinal wound repair, which occurs only in a hypoxic environment. the reg a gene is involved in regulation of cell proliferation, and has been proposed to function as a mitogenic and/or an anti-apoptotic factor in ulcerative colitis (uc)-colitic cancer progression. its high expression levels have been correlated with the severity of intestinal inflammation in patients with uc, and microarray studies have reported its upregulation in the colon of adult ibd patients. , , similarly, we identified an upregulation of reg a in the terminal ileum of pediatric cd patients. this was however contrary to a recent study comparing the expression of reg a in the terminal ileum of adult cd and non-ibd controls, which reported a downregulation in reg a expression. the difference in reg a expression could indicate a distinction between the pathogenesis of early onset cd and adult-onset cd. based on the knowledge that reg a gene expression is associated with cancer development, the high level of reg a expression in the terminal ileum of some cd pediatric patients could indicate an increased risk for colorectal cancer development. individuals with early onset cd have been previously described to have an increased risk of developing colorectal cancer. the increased levels of mmp observed in cd ileum are consistent with previous studies conducted on colonic tissue where mmp is highly expressed in the intestinal epithelia during ibd. , other studies have suggested the involvement of mmp in the regulation of epithelial barrier function. since epithelial barrier dysfunction plays a central role in the pathogenesis of intestinal inflammation, the increased expression of mmp may serve as a response to counteract tissue damage, hence protecting against colitis. the fluctuation in reg a and mmp gene expression between ileal biopsies of different patients and also between biopsies taken at different ileal locations of the same patient, suggest a spatialtemporal nature of gene regulation during early cd pathogenesis. this finding is consistent with the clinical nature of cd, with its patchy distribution. glycoprotein processing (man a ); packaging (tgoln , eef a ) and possibly release (canx). evidence of response to bacterial infection is reflected by the enrichment of receptors for adherent invasive escherichia coli and helicobacter pylori (ceacam , cd ). , the enrichment of mmp , serpina , otud , macf , pls , muc and clca transcripts suggests the presence of infectious agent(s) early in disease pathway as these genes have previously been reported to be upregulated during bacterial or viral infections. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the involvement of slc a and sf b gene products in the impairment of intestinal glucose absorption and apoptosis due to hiv- -induced glucose channel mis-sorting and cell cycle arrest suggest the occurrence of viral activities in early cd pathogenesis. , the psme , ptma, hla-dra, lrrc and xrn genes or gene products have been previously reported to be associated with defense against viral and bacterial infections. [ ] [ ] [ ] [ ] it is possible that these genes are differentially expressed in cd patients in response to infectious triggers. our study recognizes the limitation of the ssh technique whereby the cd subtraction library contained clones that are not differentially expressed, as shown by the anpep expression data. this limitation was also observed in previous studies. preliminary ssh data presented in this study were verified either by real-time pcr quantification or comparison to microarray data from studies performed on individuals with and without ibd. several of the genes anecdotally identified in the context of cd by our study have roles in microbial pathogenesis, promoting inflammation, epithelial remodeling, vesicular transport or cell differentiation and proliferation. these processes are relevant to cd pathogenesis, hence future investigations into the association between these novel gene candidates and cd could contribute to the understanding of the disease. suppressive subtractive hybridization method. restriction endonuclease-digested tester dna was split into two pools and ligated with adaptor or adaptor r. two successive rounds of hybridization with excess restriction endonuclease-digested driver dna followed. thereafter, single-stranded components of the adaptors were filled in. exponential amplification of tester-specific sequences is used to enrich for potential differentially expressed genes. type a molecules are significantly enriched, differentially expressed sequences, while cdna that are not differentially expressed form type c molecules with the driver. the concentration of high-and low-abundance sequences is equalized, whereby highly abundant molecules re-anneal to form type b and d molecules. during the second hybridization, remaining equalized and subtracted single-stranded tester cdna reassociate to form type e hybrids, with different ends corresponding to sequences of adaptor and adaptor r (adapted from clontech pcr-select cdna subtraction kit user manual [bd biosciences]). primers used for real-time reverse transcription polymerase chain reaction quantification of anpep, reg a, mmp and rpl table s differentially expressed genes specific to crohn's disease (cd) ileum. genes within each functional category are listed in order of clone abundance please note: wiley-blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. any queries (other than missing material) should be directed to the corresponding author for the article. wh sim et al. mechanisms of disease: pathogenesis of crohn's disease and ulcerative colitis genome-wide meta-analysis increases to the number of confirmed crohn's disease susceptibility loci the genetics of crohn's disease mapping of a susceptibility locus for crohn's disease on chromosome loci on q and q are associated with pediatric-onset inflammatory bowel disease inflammatory bowel disease and mutations affecting the interleukin- receptor new technologies, human-microbe interactions, and the search for previously unrecognized pathogens activation of an il- :stat -dependent transcriptome in pediatric-onset inflammatory bowel disease dissection of the inflammatory bowel disease transcriptome using genome-wide cdna microarrays analysis of mucosal gene expression in inflammatory bowel disease by parallel oligonucleotide arrays regulation of gene expression in inflammatory bowel disease and correlation with ibd drugs: screening by dna microarrays ulcerative colitis and crohn's disease: distinctive gene expression profiles and novel susceptibility candidate genes characterization of intestinal gene expression profiles in crohn's disease by genome-wide microarray analysis genome-wide gene expression differences in crohn's disease and ulcerative colitis from endoscopic pinch biopsies: insights into distinctive pathogenesis suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cdna probes and libraries identification of herpesvirus-like dna sequences in aids-associated kaposi's sarcoma transcriptional profiling of bone regeneration. insight into the molecular complexity of wound repair differentially profiling the low-expression transcriptomes of human hepatoma using a novel ssh/microarray approach toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: report of a working party of the montreal world congress of gastroenterology interaction of crohn's disease susceptibility genes in an australian paediatric cohort source: a unified genomic resource of functional annotations, ontologies, and gene expression data the ucsc genome browser database: update primer on the www for general users and for biologist programmers a new mathematical model for relative quantification in real-time rt-pcr selective ablation of matrix metalloproteinase- exacerbates experimental colitis: contrasting role of gelatinases in the pathogenesis of colitis aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev human coronavirus e: receptor binding domain and neutralization by soluble receptor at degrees c ceacam acts as a receptor for adherent-invasive e. coli, supporting ileal mucosa colonization in crohn disease integrin alpha beta (cd c/ ) is a cellular receptor for kaposi's sarcoma-associated herpesvirus (kshv/hhv- ) entry into the target cells helicobacter pylori binds to cd on gastric epithelial cells and stimulates interleukin- production cellular protein ttrap interacts with hiv- integrase to facilitate viral integration gp promotes transcytosis of human immunodeficiency virus type in genital tract-derived cell lines and primary endocervical tissue hiv- -mediated apoptosis of neuronal cells: proximal molecular mechanisms of hiv- -induced encephalopathy genetic and pharmacologic alteration of cathepsin expression influences reovirus pathogenesis htlv type i tax activation of the cxcr promoter by association with nuclear respiratory factor activity of lysosomal exoglycosidases in saliva of patients with hiv infection the hepatitis delta virus rna genome interacts with eef a , p (nrb), hnrnp-l, gapdh and asf/sf human herpesvirus- induces mvb formation, and virus egress occurs by an exosomal release pathway the measles virus (mv) glycoproteins interact with cellular chaperones in the endoplasmic reticulum and mv infection upregulates chaperone expression hiv- interaction with human mannose receptor (hmr) induces production of matrix metalloproteinase (mmp- ) through hmr-mediated intracellular signaling in astrocytes helicobacter pylori infection and short-term intake of low-dose aspirin have different effects on alpha- antitrypsin/alpha- peptidase inhibitor (alpha -pi) levels in antral mucosa and peripheral blood hiv- promotor insertion revealed by selective detection of chimeric provirus-host gene transcripts single-nucleotide polymorphisms associated with symptomatic infection and differential human gene expression in healthy seropositive persons each implicate the cytoskeleton, integrin signaling, and oncosuppression in the pathogenesis of human parvovirus b infection investigating the human immunodeficiency virus type -infected monocyte-derived macrophage secretome two atypical enteropathogenic escherichia coli strains induce the production of secreted and membrane-bound mucins to benefit their own growth at the apical surface of human mucin-secreting intestinal ht -mtx cells lps-induced mucin expression in human sinus mucosa can be attenuated by hclca inhibitors adenovirus infection inactivates the translational inhibitors e-bp and e-bp inhibitory effect of hiv- tat protein on the sodium-d-glucose symporter of human intestinal epithelial cells cleavage of poly(a)-binding protein by poliovirus c proteinase inhibits viral internal ribosome entry site-mediated translation human immunodeficiency virus type vpr induces g checkpoint activation by interacting with the splicing factor sap the role of the proteasome activator pa in mhc class i antigen processing novel function of prothymosin alpha as a potent inhibitor of human immunodeficiency virus type gene expression in primary macrophages hla and hepatitis b infection s rna narnavirus defies the antiviral activity of ski /xrn in saccharomyces cerevisiae human cathepsin s, but not cathepsin l, degrades efficiently mhc class ii-associated invariant chain in nonprofessional apcs essential role for cathepsin s in mhc class ii-associated invariant chain processing and peptide loading selective induction of integrin beta by hypoxia-inducible factor: implications for wound healing possible role of reg ialpha protein in ulcerative colitis and colitic cancer reg a expression is a prognostic marker in colorectal cancer and associated with peritoneal carcinomatosis increased risk of large-bowel cancer in crohn's disease with colonic involvement matrix metalloproteinase levels are elevated in inflammatory bowel disease genes expressed in pediatric crohn comparable expression of matrix metalloproteinases and in pouchitis and ulcerative colitis matrix metalloproteinases in inflammatory bowel disease: boon or a bane? upregulation of reg alpha and gw in the epithelium of inflamed colonic mucosa stability of housekeeping genes in alveolar macrophages from copd patients we would like to thank the children and families for their participation in this study. this project was supported by research grants from the murdoch children's research institute, the cass foundation, the lynne quayle charitable trust, equity trustees ltd, glaxosmithkline australia, the victorian government's operational infrastructure support program, and by a national health and medical research council (nhmrc) research grant. dr kirkwood is supported by an nhmrc rd wright research fellowship ( ). key: cord- - z rkzq authors: aysha, al‐ani; rentsch, clarissa; prentice, ralley; johnson, doug; bryant, robert v.; ward, mark g.; costello, samuel p.; lewindon, peter; ghaly, simon; connor, susan j.; begun, jakob; christensen, britt title: practical management of inflammatory bowel disease patients during the covid‐ pandemic: expert commentary from the gastroenterological society of australia inflammatory bowel disease faculty date: - - journal: intern med j doi: . /imj. sha: doc_id: cord_uid: z rkzq the covid‐ pandemic, caused by the novel coronavirus sars‐cov‐ , has emerged as a public health emergency and challenged healthcare systems globally. in a minority of patients, sars‐cov‐ manifests with a severe acute respiratory illness and currently there are insufficient data regarding the virulence of covid‐ in inflammatory bowel disease patients taking immunosuppressive therapy. this review aims to summarise the current literature and provide guidance on the management of inflammatory bowel disease (ibd) patients in the context of the covid‐ pandemic in the australasian setting. the covid- pandemic, caused by the novel coronavirus sars-cov- , has emerged as a public health emergency and challenged healthcare systems globally. it has rapidly spread across the world without regard for borders, manifesting as an acute respiratory illness that ranges broadly in severity from asymptomatic carriage to mild, non-specific symptoms to severe pneumonia, sepsis and death. mortality is estimated to be between % and % with disease severity associated with advanced age, chronic respiratory illness, hypertension, diabetes and other comorbidities. there is limited information on the impact of covid- on immunosuppressed patients, in particular, those with inflammatory bowel disease (ibd). ibd is a relapsing and remitting inflammatory condition of the bowel. a significant proportion of ibd patients are treated with long-term immunomodulator/immunosuppressive therapy which potentially places them at increased risk of infections and associated complications. practitioners and patients alike are therefore concerned about the risk and implications of covid- infection in the ibd patient, despite a paucity of evidence supporting an altered predisposition to disease or more severe disease course. as higher quality evidence gradually accumulates, this article aims to provide an interim practical guide for ibd management during this uncertain time. wuhan, china, in december and is transmitted via direct contact and exhaled droplets from an infected individual. human-to-human transmission is enabled by the interaction of the sars-cov- spike (s)-protein with human angiotensin-converting enzyme (ace ) receptor. ace is expressed on multiple cell types throughout the body including alveolar type (at ) cells in the lungs and enterocytes of the small intestine and colon. once the virus is attached to ace it uses the host serine protease tmprss for s priming allowing fusion of viral and cellular membranes and viral entry into the cell. the median incubation period of covid- is - days, with the majority of patients developing symptoms within weeks. the most commonly reported symptoms include fever, dry cough and shortness of breath. gastrointestinal symptoms include diarrhoea in - . % of patients and vomiting in . - . % of patients. gastrointestinal symptoms in covid- are important to note, as there is a subgroup of patients with mild disease who initially present with diarrhoea rather than respiratory symptoms, and this can lead to a delay in diagnosis. the pathophysiology of diarrhoea in covid- has not been elucidated; however, virus rna has been detected in up to % of stool specimens and stool can remain persistently positive after clearance of respiratory tract samples in approximately % of patients. in fact, the australian government is currently looking at methods of testing sewerage for sars-cov- rna as part of the australian wide monitoring programme to predict future spread and act as an early warning signal for imminent covid- outbreaks. therefore, it is possible that enteric symptoms are caused by invasion of sars-cov- into ace expressing enterocytes of the gastrointestinal tract. the implications of gastrointestinal shedding are unknown, as a polymerase chain reaction (pcr) positive stool sample does not equate to viable virus, and whether the disease is transmissible via the faecal-oral route remains unclear. furthermore, whether gastrointestinal symptoms are more prevalent in patients with ibd is ill-defined, but if an ibd patient presents with worsening diarrhoea, especially in the context of respiratory symptoms and/or fevers, excluding sars-cov- infection is prudent. in suspected cases, diagnosis of covid- is via nucleic acid amplification testing (naat) of nasopharyngeal and oropharyngeal swabs. serology testing and stool testing for sars-cov- are not currently widely available in australia. a suspected case of covid- can only be cleared following two consecutive negative covid- pcr swabs due to the potential of false-negatives. despite concerns regarding immunosuppression and consequent predisposition to infection, there is no evidence to suggest increased infection rates of covid- in ibd patients to date. reports from china and italy suggest very low infection rates in ibd patients and, at the time of writing, an international covid-ibd registry reported only ( crohn disease (cd); ulcerative colitis (uc)/unspecified) cases worldwide, despite almost million confirmed covid- cases. , hence, expert consensus currently is that patients with ibd do not appear to be at increased risk of sars-cov- infection compared with the general population. importantly, this should not negate attempts to minimise infection in ibd patients, particularly those with comorbidities including cardiovascular disease, hypertension, chronic pulmonary disease, diabetes and cancer which place them at increased risk of significant morbidity and mortality with covid- infection. more specific risk factors for severe infection in immunocompromised patients include age > years, receipt of corticosteroids, lymphopenia and neutropenia. mechanisms to protect these particularly vulnerable patients are crucial and include: • proactively reducing transmissionpatients should practice good hand hygiene by washing hands with soap and water for at least s or use an alcohol based hand sanitiser, apply social distancing including working from and staying at home where possible and standing at least . m apart from people, as well as avoiding nonessential travel. • managing hospital and healthcare facility exposureevidence from china and italy suggest attendance at hospitals and healthcare facilities for non-covid- reasons may increase the risk of covid- exposure. to reduce this risk, outpatient appointments should be moved to telehealth where possible and non-urgent pathology requests limited. elective surgery and endoscopy should be postponed, but when an urgent endoscopic procedure needs to occur, pre-screening for symptoms and exposure to covid- prior to endoscopy should occur. endoscopy should still be undertaken for acute severe ulcerative colitis, confirmation of a new diagnosis of ibd, cholangitis in primary sclerosing cholangitis and in an unresolved partial bowel obstruction. to limit pharmacy visits, prescriptions can be delivered via post; in australia there is currently a free delivery service available for vulnerable members of the community. • infusion accesspatients should continue to have access to infusions. to reduce the risk of transmission within infusion centres, patients should be screened for symptoms or risks of covid- prior to presenting and on attendance to the centre, with a temperature check on arrival. where possible the infusion centre should be moved to an 'off-site location' or 'clean' covid- free area of the hospital with a separate entrance. in addition, there should be m between patient chairs and if feasible a single nurse per patient arranged to prevent infection spread. finally, infliximab infusions should be converted to a - min protocol where safe to do so to limit the duration patients are in the infusion centre. • optimising nutritionmalnutrition significantly increases the risk of infection in patients with ibd. therefore nutritional status should be optimised and preferential use of exclusive enteral nutrition (een) for treatment of cd flares where appropriate and acceptable to patients should be considered. low vitamin d levels may increase susceptibility to covid- hence supplementation if levels are low is reasonable. • reducing disease activitythere is evidence that moderate to severe disease activity increases the risk of infection in ibd patients. active disease may also lead to corticosteroid use which can increase susceptibility and severity of covid- and/or hospitalisation which may inadvertently lead to covid- exposure. therefore, optimisation of medical therapy to maintain tight disease control is optimal. • smoking cessationsmoking may increase the chance of developing severe covid- symptoms, therefore smoking cessation encouragement and support should be a priority. • vaccinationto reduce co-infection with influenza and other respiratory infections, influenza (quadrivalent inactivated vaccine) and pneumococcus (pcv and ppsv ) vaccination should be provided and maintained in accordance with schedule recommendations. recommendations for the management of ibd medications during the pandemic are summarised in table and a more detailed analysis can be found in a recently published review on the prevention, diagnosis and management of covid- in the ibd patient. overall, medications should not be ceased without careful consideration of risk of disease flare, and corticosteroids should be avoided or exposure minimised. • there are no reports of increased risk of infection including serious or opportunistic infections with -asa medications. • considered safe to start and continue. corticosteroids • prednisolone and other systemic corticosteroids are associated with substantial increased risk of respiratory tract infections and other infections in ibd patients. • they have also been associated with worse outcomes when used to treat middle eastern respiratory syndrome (mers), severe acute respiratory syndrome (sars) and influenza. • avoid commencing prednisolone where possible. if induction agent required, alternatives include budesonide or een for cd and budesonide multimatrix (mmx) system for uc as well as topical steroids for distal disease. • if systemic steroids are required, we recommend rapid tapering where possible but balance this against risk of flare and advise against sudden cessation. • council patients to avoid self-commencing corticosteroids to control ibd symptoms, alternatively supporting ready to access medical advice. immunomodulatorsthiopurines and methotrexate • thiopurines (azathioprine and mercaptopurine) are associated with an increased risk of serious and opportunistic infections and reduce immune response to viruses. however, there is limited evidence to suggest an increased risk of either upper respiratory tract infections or pulmonary infections, and mercaptopurine has actually been shown to inhibit one of the proteases essential to viral maturation of mers-cov in vitro. although no further animal based models exist and the studies have not been replicated for covid- , it does raise the possibility that thiopurine use may not necessarily pose an increased risk from covid- . • of note, thiopurines can cause lymphopenia. patients with lymphopenia caused by sars-cov- have a worse prognosis and have an increased risk of death associated with the virus. hence, blood counts should be carefully monitored and thiopurine doses altered accordingly, where necessary. • a recent systematic review found that in the nonrheumatoid arthritis inflammatory disease population, methotrexate does not increase the risk of infection including respiratory infections. • overall there is limited evidence of increased risk of infection with the use of immunomodulators. they appear to be relatively safe at the doses typically used for immune-mediated disease, and most patients will not require dose modification. anti-tumour necrosis factor (tnf) therapies (infliximab, adalimumab, golimumab) • anti-tnf have been shown in multiple studies to increase the risk of upper and lower respiratory tract infections, as well as serious and opportunistic pulmonary infections. , • serious infection risk is most evident in patients using combination therapy with an immunomodulator and steroids. • overall, anti-tnf are considered safe to continue during the pandemic. • in older patients in deep remission with good biologic levels, consider drug-holiday from immunomodulator if on combination therapy. • when initiating therapy, consider monotherapy with therapeutic drug monitoring and utilising subcutaneous formulations where possible to reduce hospital exposure. • it is not recommended to switch from intravenous to subcutaneous formulations due to risk of loss of response and consequent flare. anti-interleukin- therapies (ustekinumab) • the risk of severe respiratory tract infections, severe infections or opportunistic infections does not appear to be increased in long term follow-up studies of ustekinumab in both ibd and psoriasis. • ustekinumab is considered safe to start and continue during the pandemic. anti-integrin (vedolizumab) • vedolizumab is a monoclonal antibody to the α β integrin that modulates gut lymphocyte trafficking. hence, there is a theoretical risk of increased susceptibility to gastrointestinal infections. however, respiratory tract infections, severe infections or opportunistic infections do not appear to be increased in long term follow-up studies of vedolizumab in ibd. • vedolizumab is considered safe to start and continue during the pandemic. jak-kinase inhibitor (tofacitinib) • tofacitinib impairs immunity to viral infections in long-term extension trials. • doses of mg twice daily are associated with increased serious infection risk when compared to mg twice daily. • continue tofacitinib but ideally at the lower dose of mg twice daily. • avoid commencing tofacitinib unless no other alternatives are available, to avoid potential side effects and frequent pathology monitoring. patient management if they are exposed to or develop covid- there are currently no evidence-based guidelines on medical management of ibd in a patient who becomes covid- positive. table summarises our recommendations. based on the lack of data, in most patients we recommend temporary withholding of immunesuppressing therapies until the resolution of active infection as would be typical in the setting of any serious infection. in those exposed but with no symptoms, this should be weighed up against the risk of ibd flare. it is also important to note that many ibd medications may take months to be eliminated from the body so the utility of cessation in the short-term is likely to be limited. (table ). there is currently no evidence to guide the recommencement of medication following exposure to or infection with sars-cov- . most patients will develop symptoms within days of exposure or testing positive for disease. of note, prodromal asymptomatic infection is increasingly being recognised in outbreak settings with a recent study of nursing home patients demonstrating that of ( %) asymptomatic patients who tested positive for sars-cov- developed symptoms within the subsequent days. therefore we recommend that ibd medications can be restarted after days in exposed or asymptomatic infected patients provided they have not developed symptomatic illness. in those who develop covid- , testing for clearance of virus before recommencing medications or accessing infusion centres may have limited utility as some patients shed virus for extended periods of time despite not being actively infected or infectious. relying on testing to clear these patients may result in unnecessary delays to ibd treatment. in australia, covid- patients are cleared from isolation and considered no longer infectious days from symptoms onset if they have clinical improvement and no fever for at least days. european guidelines, however, suggest waiting days in those who have been on immunosuppressive therapies due to the potential for prolonged shedding of virus. therefore, in patients with mild to moderate covid- , recommencing therapy after at least days in those who are currently asymptomatic or have clinical improvement with no fever for at least days is likely safe. in more severe cases, clinical judgement should be used, and a greater window between covid- improvement and medication recommencement may be prudent. serology testing is currently not widely available to help guide these decisions and the impact of immunosuppressive medications on seroconversion is as yet unknown. however, these tests may be utilised in the future. supporting the nutritional requirements of covid- affected patients is critical. this is of increased importance in the ibd patient secondary to the increased probability of premorbid malnutrition. early dietician review is therefore warranted. nutritional interventions are particularly important in those unable to maintain adequate oral nutritional intake due to a need for prolonged intubation or non-invasive respiratory support, and in those with increased gastrointestinal losses. the latter may occur in the setting of active ibd, or as a consequence of the virus itself. enteral feeding via nasogastric tube (ngt) remains the preferred first line option, and should be considering within h of admission for those requiring intensive care support. delayed gastric emptying with elevated residual gastric volumes can occur in critically unwell covid- patients, increasing the risk of aspiration and therefore continuous rather than bolus feeding is preferable. , in addition, prone positioning is often required for respiratory care and treatment in severe covid- and may hinder ngt feeding. where necessary, nasojejunal tubes and parenteral nutrition are valid routes for nutritional support, although the former carries the risks of endoscopic placement and the latter requires intensive dietician input with risk of hyperglycaemia, refeeding syndrome and central line infections. , of note, ngt insertion is considered an aerosol generation procedure and thus appropriate personal protective equipment with full airborne precautions is recommended. maintaining quality of care during the covid- pandemic the unintended immediate and longer term consequences of the covid- pandemic may be a loss of ibd control and an increased rate of flare. inappropriate cessation of medications poses risks to patients with ibd, further compounded by a potential lack of access to healthcare. maintaining quality ibd care is imperative. engagement with ibd services can be facilitated through ibd helplines, telemedicine clinics, as well as dissemination of accurate information via a regular ibd newsletter. while access to endoscopy for disease activity assessment is limited, objective monitoring may be undertaken using non-invasive tools such as faecal calprotectin and gastrointestinal ultrasound. utilisation of ibd-specific smart-phone applications as well as decision-aid tools may also be helpful where available and the covidsafe app, accessible in australia, may be useful to notify ibd patients of close contact with a covid- case. many patients with ibd are treated with long-term immunomodulating therapy and there is concern amongst patients and clinicians alike that this may predispose to an increased risk of covid- . however, available data are reassuring and despite understandable anxiety, patients with ibd do not appear to be at increased risk of covid- . in order to prevent covid- infection and its complications, optimisation of disease activity, nutrition, co-morbidities, smoking and vaccination status is important and where possible, corticosteroids should be avoided. if a patient with ibd does develop covid- , ibd medications can be temporarily withheld and recommenced with timing of recommencement dependent on severity of covid- and baseline ibd disease. the mental health of medical workers in wuhan, china dealing with the novel coronavirus review article: prevention, diagnosis and managment of covid- in the ibd patient sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor review article: gastrointestinal features in covid- and the possibility of faecal transmission clinical characteristics of covid- patients with digestive symptoms in hubei, china: a descriptive, cross-sectional, multicenter study evidence for gastrointestinal infection of sars-cov- first confirmed detection of sars-cov- in untreated wastewater in australia: a ibd - © royal australasian college of physicians proof of concept for the wastewater surveillance of covid- in the community secure-ibd database public data update protection of inflammatory bowel disease patients from the outbreak and rapid spread of covid- infection in wuhan international organization for the study of inflammatory bowel disease. management of patients with crohn's disease and ulcerative colitis during the coronavirus disease- pandemic: results of an international meeting british society of gastroenterology guidance for management of inflammatory bowel disease during the covid- pandemic risk factors for opportunistic infections in patients with inflammatory bowel disease sulphasalazine and mesalazine: serious adverse reactions re-evaluated on the basis of suspected adverse reaction reports to the committee on safety of medicines the effect of corticosteroids on mortality of patients with influenza pneumonia: a systematic review and meta-analysis clinical evidence does not support corticosteroid treatment for -ncov lung injury thiopurine analogs and mycophenolic acid synergistically inhibit the papainlike protease of middle east respiratory syndrome coronavirus risk of infection with methotrexate therapy in inflammatory diseases: a systematic review and metaanalysis comparative risk of serious infections with biologic and/or immunosuppressive therapy in patients with inflammatory bowel diseases: a systematic review and metaanalysis the safety of vedolizumab for ulcerative colitis and crohn's disease real-world experience with tofacitinib in ibd at a tertiary center presymptomatic sars-cov- infections and transmission in a skilled nursing facility european centre for disease prevention and control. technical report: guidance for discharge and ending isolation in the context of widespread community transmission of covid- -first update nutrition management for critically and acutely unwell hospitalised patients with covid- in australia and new zealand pdf bapen's nasogastric tube safety special interest group. bapen statement on covid- and enteral tube feeding safety characteristics and outcomes of critically ill patients with covid- in washington state nasogastric (ngt)/nasojejunal tube (njt) placement and aerosol generation (agp) key: cord- -y agnh authors: nan title: oral research communications of the (nd) ecvim‐ca congress date: - - journal: j vet intern med doi: . /jvim. sha: doc_id: cord_uid: y agnh nan sidestream dark field imaging (sdf) is a technically relatively simple method to visualize the microcirculation. however, current gold-standard, or 'consensus' analysis (ca) of sdf films takes approximately hour per film limiting the application of sdf in a clinical setting, and leading to increased expense due to analysis of low quality films. we developed a subjective point of care scoring system (bedside evaluation of the microcirculation, bem) that could provide real-time intervention points and optimize care for the critical patient. the objective of this study is to evaluate whether the bem-score correctly identifies films of sufficient and insufficient diagnostic quality as defined by ca.twenty variable-length microcirculation films taken at the level of the canine tooth were selected from a database of films with available ca. before the study, three observers were trained using an instruction video to evaluate five quality parameters: stability, content, illumination, focus and pressure. the bem was performed by viewing and scoring each film four times in immediate succession. all five quality parameters were scored ( perfect, sufficient and insufficient) according to ca analysis. only the fourth viewing was considered for analysis. bem quality analysis was only considered sufficient if no parameter was scored insufficient. repeatability and reproducibility were assessed by assessing all films in a random order three times daily for three days.bem pass-fail assessment matched ca . % of the time with individual observer agreement of . - . %. agreement of bem with ca did not change over the study period ( . %, . % and . % on days , and respectively) indicating accurate quality analysis after a single bem-score of the training video. the mean cumulative bem quality score ( . , sd . ) was very similar to ca (mean . , sd . ). however mean individual bemparameter scores differed from ca reflecting differences in observer interpretation.high levels of inter-observer agreement and the strong correlation with ca for pass-fail assessment demonstrate that bem quality evaluation can produce repeatable and reliable results. variation in individual parameter scores may reflect systematic erroneous assignment of certain parameters or individual bias towards assessment of certain features. nevertheless, this did not impact on the overall evaluation of film quality. this study provides a platform to investigate whether rapid semi-quantitative analysis of the microcirculation itself is similarly feasible. sidestream dark field imaging (sdf) is a straightforward technique to evaluate microcirculation. however, current 'consensus' analysis (ca) of sdf films is time-consuming, thereby restricting the clinical application of sdf. a subjective bedside scoring system (bem) is proposed that could rapidly provide a semi-quantitative assessment. we have previously shown that observers could accurately evaluate film quality parameters. the objective of the present study is to assess the correlation of bem with ca for quantitative microcirculation parameters. three observers were trained using an instruction video to evaluate four quantity parameters: total vessel density (tvd), capillary vessel density (cvd), perfused vessel density (pvd) and microvascular flow index (mfi). fifteen variable-length microcirculation films of sufficient quality, taken at the level of the canine tooth, were selected from a database of films with available consensus analysis. each parameter was scored ( lowest - highest) . the bem was performed by viewing and scoring each film four times in immediate succession with the final score being considered for analysis. bem was performed on each film in random order three times daily for three days. ca scores were divided into quintiles for each parameter and mean bem score were calculated for each. conversely, for each bem score, mean ca scores were calculated, per observer or per day.the mean tvd and pvd bem scores for the % quintile were lower than mean bem scores for other quintiles. mean bem score for the - % quintile was the highest mean bem score for pvd only. mean cvd, pvd and mfi values for bem score were lower than mean values for other bem scores. in parallel, although tvd never received a bem score of , the mean ca value for films with bem score were lower than for higher bem scores. the mean tvd and mfi values for bem score of were higher than mean values for bem scores [ ] [ ] [ ] [ ] . similar values were obtained on each individual study day.this study demonstrates that rapid semi-quantitative assessment of the microcirculation using the bem-score can produce repeatable and reliable results, although significant overlap exists. further studies are required to evaluate the value of this technique in a clinical setting. objective: compare two non-invasive blood pressure (nibp) measurement devices (petmap and doppler) with invasive blood pressure (ibp) measurement in normotensive, anesthetised and awaken dogs. design: prospective clinical study animals: ten female dogs aged between to months (average . months), and weighting . to . kg (average . kg) undergoing a routine spay. interventions: blood pressure measurement procedures: after the induction of general anesthesia, a catheter ( g) was placed in the dorsal pedal artery and invasive systolic (sap i ), diastolic (dap i ) and mean (map i ) arterial blood pressures were obtained. the nibp cuffs were placed on the ipsilateral front limb. five consecutive measurements were obtained with each indirect device and considered as a mean measure. the ibp was obtained simultaneously to the five nibp measurements and also considered as a mean measurement. measurements on awaken dogs were obtained four hours after surgery. doppler's systolic (sap d ) and petmap's systolic (sap o ), diastolic (dap o ) and mean (map o ) arterial blood pressure were evaluated and compared to the corresponding ibp using the bland-altman analysis. the percentage of paired measurements with a mean difference of and mmhg was also evaluated for the sap o , dap o and sap d . results: agreement between ibp and nibp measurements obtained with the petmap and the doppler was assessed with the bland-altman analysis. on anesthetised dogs, both indirect devices underestimated all direct blood pressures. the petmap bias (standard deviation) were - . mmhg ( . mmhg), - . mmhg ( . mmhg), and - . mmhg ( . mmhg) for sap o , dap o and map o respectively. the doppler bias was - . mmhg ( . mmhg) for sap d . on awaken dogs, the petmap underestimated sap i and overestimated dap i and map i . the bias were - . mmhg ( . mmhg), . mmhg ( . mmhg) and . mmhg ( . mmhg) for sap o , dap o and map o respectively. the doppler underestimated the ibp and the bias was - . mmhg ( . mmhg).on anesthetised and awaken patients, the percentage of values lying within and mmhg of the ibp was higher (or equal) for the petmap compared to the doppler. conclusion: results suggest a better performance of petmap device to predict the ibp in normotensive-anesthetised and normotensive-awaken dogs. intracranial hypertension (ich) is associated with high morbidity and mortality in canine veterinary medicine, yet remains difficult to clinically diagnose. the lack of an easily feasible and available diagnostic method has prevented clinical studies on the prevalence, cause and treatment of ich. recently, transcranial doppler ultrasonography (tcd) has been reported as a noninvasive diagnostic method in humans. however, only a few preliminary reports on the use of this technique in dogs have been published. this study evaluated the repeatability and reproducibility of tcd of the basilar and left and right cranial, cerebral arteries in healthy beagle dogs.tcd was performed using a standard ultrasound machine at the level of the basilar artery, and left and right cranial cerebral artery, in six adult beagle dogs. systolic, diastolic and average velocity, resistance (ri) and pulsatility indexes (pi) were assessed for each vessel. repeatability was evaluated by calculating the intra-class correlation coefficient (icc) between three separate, consecutive measurements in every dog. an icc was also determined for the reproducibility of these measurements on three consecutive days. during the procedures, ecg, blood pressure and clinical parameters were monitored.statistical analysis showed a highly significant repeatability of all measured parameters (systolic, diastolic and average velocity, ri and pi) for all blood vessels (n = , icc = . - . , p < . ). systolic velocity, ri and pi were also significantly reproducible for the basilar artery (n = , icc = . - . , p < . ), and the left and right cranial cerebral arteries (n = , icc = . - . , p < . ). however, no significant correlation was found between basilar and cerebral blood flow velocities.measurement of pi and ri of the basal and cranial cerebral arteries using tcd appeared to have high intra-operator repeatability and reproducibility. however, measurements performed on the basilar artery had higher between day reproducibility. since the technical skill required to assess these parameters subjectively appeared to be less complicated at the level of the basilar artery, tcd in a clinical setting is probably advocated at the basal artery.in conclusion, tcd is characterized by high intra-observer repeatability and reproducibility, making this technique a promising tool for the measurement of pi and ri, which have both been reported to be correlated with intracranial pressure. intracranial hypertension (ich) is associated with high morbidity and mortality in canine veterinary medicine, yet remains difficult to clinically diagnose. furthermore, many patients suspected to suffer from ich are treated with sedatives or anticonvulsants. besides the impact of these molecules on the patients' neurological examination, they variably influence the cardiovascular system. this study evaluated the effect of various sedatives and anticonvulsants on the pulsatility and resistance index (pi) and (ri), as measured by transcranial doppler ultrasonography (tcd) in healthy beagle dogs. additionally, we evaluated the effect of ivabradine, a specific negative chronotrope, on tcd findings. tcd was performed at the level of the basilar artery in six adult, beagle dogs. this technique was performed prior to the injection of molecules (v ), and after administration of acepromazine (ace, lg/kg iv), diazepam (dia, . mg/kg iv), medetomidine (med, lg/kg im) or ivabradine (iva, mg/kg iv). a wash-out period of at least hours was respected between the administration of each drug. during the procedure, ecg, heart rate (hr) and blood pressure (bp) were monitored. results were analyzed using repeated measures anova. correlations between the clinical parameters, and pi and ri were investigated using pearson's correlation. results were considered significant when p < . . significantly lower pi and ri values [(mean pi; % ci), (mean ri; % ci)] were obtained for med [( . ; . - . ( . ; . - . ) ]. pi for dogs after iva was significantly increased compared to dia, but did not differ significantly from other groups.hr was significantly lower after med compared to other groups, and after iva, compared to v . bp was significantly lower after ace than after iva, med or on v . these clinical parameters were not significantly correlated with pi or ri. dia and ace did not significantly influence ri or pi, however med significantly decreased ri and pi. these findings agree with previous reports describing a decrease in icp provoked by med, as pi is positively correlated with icp. this effect appears to be independent of the effect of med on hr, since iva also significantly decreased hr, yet did not significantly affect pi or ri. in conclusion, blood pressure and heart rate did not significantly affect pi and ri. hypertrophic cardiomyopathy (hcm) is the most commonly diagnosed heart disease in cats with little documentation of the effects of treatment on outcome in cats with preclinical disease. therefore, this prospective cohort study was undertaken to evaluate the effects of treatment with atenolol on outcome in cats with asymptomatic hcm. we hypothesized that ) -year mortality would be increased in cats with hcm compared to a matched control group of healthy cats, and ) administration of atenolol would reduce -year cardiac mortality in occult feline hcm.cats were enrolled over a -year time period ( to ) in a prospective, open-label, observational study. diagnosis of hcm was based on transthoracic echocardiography. cats were either treated with atenolol ( . to . mg/cat, q h, po) or did not receive treatment. decision to treat was based on owner preference, suggestion by the clinician, and animal compliance with regard to pill administration. baseline echocardiograms were analyzed, and morbidity and mortality were monitored at and months and thereafter by annual rechecks, and by phone interviews of referring veterinarians and owners over a -year time period. groups were compared by a non-paired t-test, introduction: systolic anterior motion of the mitral valve (sam) is a dynamic left ventricular outflow obstruction frequently observed in feline hypertrophic cardiomyopathy (hcm). however, several cases of sam have also been observed in cats without left ventricular hypertrophy (lvh) and this finding could be interpreted as an early stage of the disease not yet accompanied by lvh. alternatively, it could be speculated that the dynamic outflow obstruction could cause intraventricular pressure overload sufficient enough to induce lvh at a later stage. this study was conducted to test the hypothesis that sam can potentially cause myocardial stress and myocardial damage by measuring plasma ntprobnp and serum troponin-i levels in cats with sam not associated with lvh. methods: the study was based on a retrospective analysis of cats who underwent cardiac investigation of a heart murmur, performed by a boarded cardiologist. these cats were diagnosed with sam not associated with lvh (ivsd and lvfwd < . mm, both on bmode and mmode measurements) via colour doppler echocardiography. plasma ntprobnp (cardiopet test, idexx laboratories, inc.) and serum high sensitivity troponin-i (hsctni hs, idexx laboratories, inc) were measured in these patients, as well as serum urea, creatinine and thyroxin concentrations. the association between biomarkers (ntprobnp and hsctni) concentration and echocardiographic values (myocardial thickness, left atrial dimension and aortic peak velocity) was determined by using the spearman correlation coefficient (rho). results: all cats were euthyroid and did not show evidence of renal disease. mean hsctni measurement was not performed in one cat due to an unsuitable sample. eleven out of cats ( %) showed ntprobnp values above the normal reference limit ( pmol/l) with a median concentration of pmol/l (± , range - ) . five out of cats ( %) showed hsctni values above the normal reference limit ( . ng/ml) with a median concentration of . ng/ml (± . , range . - . ). there was a significant positive correlation between aortic peak velocity and ntprobnp (rho= . , p= . ). conclusion: ntprobnp is increased in cats with sam without lvh, suggesting the presence of myocardial stress, which appears to be proportional to the degree of outflow obstruction derived by the aortic peak velocity. some of these cats also present a degree of myocardial insult suggested by the increased serum hsctni. whether the increased biomarker concentrations are related to sam-induced pressure overload or to an early stage of primary myocardial disease needs to be evaluated with appropriate longitudinal studies. heart murmurs are caused by turbulent blood flow or by vibration of cardiac structures. turbulent blood flow in young animals may originate from congenital structural heart disease or from physiological phenomena. the prevalence of heart murmurs and congenital heart disease in the general (i.e. non referral) feline population are unknown. the aims of this prospective study were to determine the prevalence of heart murmurs in young cats and to determine the prevalence of congenital heart disease in young cats with heart murmurs.in total domestic shorthair cats aged to months underwent a routine physical examination prior to vaccination between may st until march st . cats were from adoption programs run by either dierenasiel breda e.o. ( / ) or dierenopvangcentrum tilburg ( / ). in cats with murmurs, the murmur was timed, graded and the point of maximum intensity was determined. subsequently, d, m-mode and doppler transthoracic echocardiography with continuous ecg monitoring was performed.heart murmurs were detected in animals ( . %). congenital heart disease was detected in animals ( . %), acquired heart disease in animals ( . %) and no identifiable heart disease in animals ( . %) with murmurs.in the animals without heart disease dynamic right ventricular outflow tract obstruction was the cause of the murmur in cases, turbulence within the left ventricle was the cause in cases.in animals with acquired heart disease, pulmonary hypertension associated with a. abstrusus was diagnosed.in the cats with congenital heart disease, isolated defects were found in cats, being tricuspid valve dysplasia in cats, dynamic left ventricular outflow tract obstruction associated with mitral valve dysplasia in cats, ventricular septal defects in cats, double chambered right ventricle in cats and pulmonic stenosis in cat. combination defects were found in cats, being ventricular septal defect and tricuspid valve dysplasia in , ventricular septal defects and double chambered right ventricle in , ventricular septal defect and atrial septal defect in cat, a ventricular septal defect and pulmonic stenosis in cat and mitral and tricuspid valve dysplasia in cats.in conclusion in this prospective study we found the prevalence of heart murmurs to be . % and the prevalence of congenital heart disease to be . % with atrioventricular valve abnormalities and ventricular septal defects being the most common. dynamic left ventricular outflow tract obstruction (dlvoto) is a common cause of heart murmurs in adult cats. the obstruction is caused by systolic anterior motion (sam) of the mitral valve. sam is typically observed in adult cats and humans with hypertrophic cardiomyopathy (hcm). many studies support the concept that structural deformities of the mitral valves and the papillary muscles could be primary causes of sam. congenital malformations of the mitral valve causing sam and dlvoto have been observed in dogs and people and are believed to exist in cats, but have not yet been reported. the aim of this study was to report the clinical and echocardiographic findings in young cats with dlv-oto.thirteen domestic shorthair kittens, between - weeks of age, presented with a systolic heart murmur between may st until march st . all cats underwent a physical examination preceding d-, m-mode and doppler echocardiography. kittens were from adoption programs run by either dierenasiel breda e.o. (n= / ) or by dierenopvangcentrum tilburg e.o. (n= / ) nine kittens were male, four were female. all kittens were asymptomatic. dlvoto caused by sam was present in all animals, uninterruptedly in eleven, only at high heart rates in two. seven cats had normal left ventricular dimensions (nlvd), six cats had concentric left ventricular hypertrophy (clvh). in five cats papillary muscle abnormalities were noted being an accessory papillary muscle in two and enlarged papillary muscles in three, two of which had clvh.twelve animals were reexamined - months after the initial examination. all animals were still asymptomatic. a heart murmur and dlvoto could no longer be detected in nine animals, five with initial clvh and four with initial nlvd. in three animals a heart murmur and dlvoto were still present. two of the cats with nlvd developed clvh. one cat with initial clvh continued to have clvh.treatment with atenolol was instigated in three cats which continued to have dlvoto and clvh. two cats were reexamined. treatment with atenolol led to complete reversal of clvh in both cases. interventional radiology techniques have been used to palliate both malignant and non-malignant causes of vascular obstruction for both intrinsic and extrinsic lesions.three dogs presented for with large, non-resectable cardiac masses obstructing venous return to the right atrium. venous return to the heart was severely obstructed leading to congestion with subsequent ascites ( ) or head swelling and pleural effusion ( ) . due to the extensive nature of the disease, an interventional palliative approach was pursued. transatrial self-expanding metallic nitinol stents were placed from the cdvc to the crvc in order to restore venous return to the heart via blood flow through the stent interstices.in all cases, stent placement was successful and resolution of clinical signs achieved. two dogs required additional stent placement in months and months, respectively, for stent occlusion. in both cases, restenting resulted in ascites resolution or substantial reduction. one dog was euthanized months following initial stent placement for general systemic decline and return of moderate ascites. the second dog remains alive months following initial stent placement on diuretic therapy with moderate ascites present. the third died of undetermined causes / months later.to the authors' knowledge, this is the first report of longterm palliative transatrial stenting for cardiac tumors affecting venous return to the heart. the stents were well tolerated in these canine patients for which surgical options were not possible. the irish wolfhound (iw) dog has a high prevalence of heart diseases, particularly dilated cardiomyopathy (dcm) and atrial fibrillation (af).during a prospective longitudinal study, irish wolfhounds were investigated by one veterinary cardiologist ( ) between and . dcm was diagnosed in about % of dogs. in % of cases, dcm was accompanied by af. af without evidence of dcm was diagnosed in dogs ( %).for pathological investigations, hearts of iws were collected and fixed in % buffered formalin. based on the most recent results from cardiovascular examination, five groups were established: normal hearts (group , n= ), dcm with sinusrhythm (group ; n= ), dcm with af (group ; n= ), dcm with af and congestive heart failure (chf) (group ; n= ), af and left ventricular reverse remodeling (lvrr) due to medical therapy after diagnosis of dcm (group ; n= ).all hearts were evaluated by one pathologist ( ) who was blinded to the clinical diagnosis. gross inspection included measurments of weight, size, and architecture of left (lv) and right (rv) ventricles. three sites of each lv, rv, and interventricular septum (ivs) were histologically evaluated, and the extend of myocardial fibrosis, adipocyte infiltration, and angiosclerosis were graded semiquantitatively.iws with dcm and chf (group ) had died significantly younger ( . ± . years) than dogs with normal hearts ( . ± . years) (p= . ). concerning gross pathology findings in hearts with clinical dcm diagnosis, lv chambers were dilated in / cases of group , while in groups and , the papillary muscles appeared grossly prominent and not flattened as in group .histopathologically, in control dogs lv and rv myocardium showed no ( / ) or mild ( / ) interstitial collagen deposits, no or single adipocytes, and normal vessels. in contrast, heterogeneous findings were seen in groups - : most hearts ( / ) showed mild to moderate multifocal myocardial fibrosis and up to moderate diffuse infiltration of adipocytes within the lv myocardium, and mild angiosclerosis, but five hearts were histologically normal. only in one dog attenuated wavy fibers were seen in the apical region of lv. ivs was normal in out of cases. rv showed mild interstitial fibrosis and mild to moderate adipocytes in most cases ( / ) of all groups.in conclusion, pathological findings in hearts of iws affected with dcm are different from other breeds. furthermore, the gross and histological findings are variable and do not correspond to the clinical diagnosis in all cases. the irish wolfhound (iw) dog has a high prevalence of heart diseases, particularly dilated cardiomyopathy (dcm) and atrial fibrillation (af). during a prospective longitudinal study, irish wolfhounds were investigated by one veterinary cardiologist ( ) between and . dcm was diagnosed in about % of dogs. in % of cases, dcm was accompanied by af. in addition, af without evidence of dcm was diagnosed in dogs ( %).for pathological investigations, hearts of iws were collected and fixed in % buffered formalin. based on the most recent results from cardiovascular examination, three groups were established: dcm with af (group ; n= ), af without evidence of dcm (group ; n= ), normal hearts (group , n= ).aim of this study was to investigate the histopathological findings in left and right atria of iws with atrial fibrillation compared to normal hearts of iws. all hearts were evaluated by one pathologist ( ) who was blinded to the clinical diagnosis. hearts were inspected grossly and two cross sections of each left (laa) and right (raa) atrial appendage were embedded in paraffin wax and stained with h&e and picrosirius red. myocardial fibrosis and adipocyte infiltration in both atria were graded semiquantitavely.mean age ± sd of all dogs at time of death was , ± . yrs. the gross and histopathological findings of the left atrial appendage (laa) were not significantly different among groups, but dogs in both af groups had raa dilation ( . to . ml, median ml volume) compared to controls ( . to . ml volume, median . ml). histopathologically, raa in control dogs showed small amounts of interstitialcollagen and single adipocytes. in contrast, raa in dogs affected with af with andwithout evidence of dcm, had mild to moderate multifocal or diffuse myocardial fibrosis, and diffuse infiltration of adipocytes which was statistically significant different from normal hearts (p= . ). in general, fibrosis and number of adipocytes were significantly increased in raa compared to laa in both groups with af (p= . ).fibrosis and the accumulation of adipocytes within the myocardium are described toresult in electrical inhomogeneity predisposing to arrhythmia.on a cellular level, right atrial fibrosis and adipocyte accumulation might be the changes responsible for the development of atrial fibrillation and atrial dilatation in this breed of dogs with an exceptional high prevalence of af and dcm. asynchronous ventricular contraction causes deterioration in cardiac function and reduces the response to medical therapy in people with heart disease. various echocardiographic techniques have been used in humans for evaluation of left ventricular (lv) synchronization in order to assess whether cardiac resynchronization therapy (crt) would be beneficial. prior studies using tissue doppler imaging (tdi) derived strain imaging have not detected lv dyssynchronization in doberman pinschers with dilated cardiomyopathy (dcm). a newer d imaging modality, speckle tracking strain analysis, is more effective in detecting lv dyssynchrony in humans. this technique has not been previously evaluated in doberman pinschers with dcm. this study therefore aims to evaluate lv synchrony in doberman pinschers with dcm using d speckle tracking strain. client-owned doberman pinschers were included. standard echocardiography was used to evaluate systolic function and left ventricular dimension in order to categorize the dogs as normal or abnormal, depending on systolic and diastolic ventricular dimensions, ejection fraction and/or shortening fraction. the operator was blinded to the echocardiographic diagnosis of each dog. each parameter was measured in triplicate. radial (rs) and circumferential strain (cs), using right parasternal short axis at the level of the papillary muscles were calculated. synchrony was assessed by measuring the difference in the qrs onset time-to-peak strain between the septal anterior and posterior left ventricular segments. to evaluate differences between groups a nested effect anova (beat within group) was performed. a p< . was considered significant. based on the echocardiographic parameters mentioned above, / dogs were considered normal and / had dcm. significant differences were found in the time to peak rs (p= . ) between normal dogs and dogs affected with dcm. mean in time to peak rs was . ms (+/- . ) in normal dogs and . ms (+/- . ) in dogs with dcm. time to peak cs did not exhibit significant differences between groups (p= . ) and was . ms (+/- . ) in normal dogs and . ms (+/- . ) in dogs with dcm. the results of this study show that the delay between the anteroseptal-to-posterior wall peak radial strain is greater in doberman pinschers with dcm than in normal dogs. further studies would be required to evaluate whether there is a difference in synchrony between dogs in occult stage and overt dcm. ventricular dyssynchrony could negatively affect systolic dysfunction as it does in people, and crt may therefore be helpful for the treatment of dobermans with dcm. patent ductus arteriosus is often treated with intra-arterial coilembolization or implantation of an 'amplatz canine duct-occluder'. for both procedures an arterial access is necessary. because of the high risk of developing a fatal hemorrhage from the arterial puncture site after removal of the large-bore introducer sheath upon completing the intervention, cardiologists generally use surgical cut-down with subsequent ligation of the femoral artery instead of percutaneous arterial puncture using seldinger's technique. the effect of commercially available chitosan patch has been tested in experimental dogs with the approval of the institute's ethical committee. on the first beagles the committee required a terminal experiment. for the selection of an appropriate introducer-sheath (i.e. smaller than the arterial diameter), the femoral artery was first imaged with ultrasound. under general anesthesia introducer-sheaths were placed in both femoral arteries using seldinger's technique. after their removal a chitosan-patch was applied on the wound according to the manufacturer's instructions: -minute manual pressure. the dogs were monitored with direct arterial blood pressure measurement, ecg, pulse oxymetry and capnography. all dogs were kept under general anesthesia for several hours and the legs were moved vigorously every minutes to mimic movements of awake animals. no macroscopic hemorrhage was noticed on the puncture sites and no signs of severe subcutaneous bleeding was suspected as the blood pressure and heart rate remained stable. after several hours the dogs were euthanized. after having shown the effective working of chitosan-patch, a permission was granted for survival experiments in beagles. in of these dogs one femoral artery was punctured with an introducer whose thickness exceeded the diameter of the femoral artery. in both dogs an uncontrollable subcutaneous hemorrhage occurred immediately after the -minute manual compression time with severe drop of blood pressure and development of tachycardia. both dogs were euthanized during anesthesia. in another beagles the size of the introducer was smaller than the diameter of the artery ( - %). no relevant hemorrhage took place in these dogs and they recovered from the procedure without any complications. similarly good outcome was found in two -month-old boerboels. from this pilot study we concluded that chitosan-patch can effectively control hemorrhage from a femoral arterial puncture site if the introducer-sheath is thinner than the artery's lumen. using seldinger's technique allows a less invasive and quicker cardiac catheterization and preservation of the femoral artery for the leg's blood supply and for repeated intra-arterial interventions. objectives were to examine longitudinal change in echocardiographic left heart chamber dimensions and cardiomyopathy classification in cats with primary myocardial disease.clinical records from - were reviewed for cats with or more echocardiographic examinations at least months apart and a diagnosis of primary myocardial disease. for each study ( from each cat), left heart chamber dimensions were measured by a single blinded trained observer in random order, and the cardiomyopathy type was classified according to predefined criteria. paired comparisons were made using the wilcoxon signed rank test and fisher's exact test. cats met the inclusion criteria. cardiomyopathy type at entry consisted of hypertrophic cardiomyopathy (hcm, n= ), dilated cardiomyopathy (dcm, n= ), arrhythmogenic right ventricular cardiomyopathy (arvc, n= ) and normal cat. overall, median left ventricular (lv) diastolic diameter and median long axis left atrial (la) diameter both increased (p= . , . respectively) whereas lv diastolic wall thickness and lv fractional shortening did not change, but changes in dimensions of > % were seen in both directions. change in cardiomyopathy type was documented in cats: hcm to normal; normal to hcm; hcm to the endomyocardial form of restrictive cardiomyopathy.the inclusion criteria led to a bias towards clinically stable cats with hcm. despite this, changes in cardiomyopathy phenotype were observed. either our criteria for the different feline cardiomyopathies should be defined more precisely, or phenotypic expression in cats with cardiomyopathy changes over time. transesophageal echocardiography (tee) has proven useful in evaluating patent ductus arteriosus (pda) morphology thereby guiding appropriate device selection. additionally, tee, in combination with fluoroscopy, has been used to guide the transcatheter coil embolization and for deployment of amplatz canine ductal occluder (acdo) in dogs. recently, we described the use of transthoracic echocardiography (tte) guidance during transcatether pda occlusion with acdo without the use of fluoroscopy, but observed problems of deployment in patients with sub-optimal acoustic windows. however, tee, can overcome issues of suboptimal tte acoustic windows and provides higher image resolution of cardiac and vascular regions. therefore, we hypothesized that tee could be used to successfully visualize the vascular structures and interventional devices to safely perform pda occlusion with acdo without requiring fluoroscopy.we recruited dogs with patent ductus arteriosus (pda) for tee-guided percutaneous ductal occlusion with an acdo. dogs were anesthetized, positioned in right lateral recumbency and the right femoral artery was accessed percutaneously (modified seldinger technique). the tee probe was advanced to a midesophageal position with minimal force to obtain a long axis -chamber view (transverse plane). the probe was then retroflexed and withdrawn to a cranial esophageal position until a cross section of the descending aorta was seen. to visualize pda to the probe was slightly straightened and turned counterclockwise, and the ultrasonic beam was oriented between and degrees.in all dogs, the guide wire and a long introducer-sheath were guided from the aorta through the pda into the main pulmonary artery by tee monitoring. the acdo was advanced through the introducer-sheath until the flat distal disk was visualized within the main pulmonary artery by tee monitoring. the distal disk was positioned against the pulmonic ostium and the coupled proximal disk was deployed within the ductal ampulla while being monitored by tee visualization.the guide wires, long introducer-sheath and acdo appeared hyperechoic on tee images and tee guidance provided images of sufficient quality to clearly monitor the procedures in real-time. real-time monitoring also allowed for immediate corrections to guide wire, catheter or device positioning. the procedures were successful and without complications in all patients.we have demonstrated that tee monitoring, like tte monitoring, can guide every step of transcatheter acdo embolization procedures without requiring fluoroscopy, thereby avoiding radiation exposure, and provides an alternative to tte-based guidance, especially when tte visualization of the pda is insufficient for safe and timely acdo deployment. right ventricular (rv) dysfunction occurs in human patients with left-sided cardiac disorders because of the mechanism of ventricular interdependence. doppler echocardiographic indices of diastolic function of the right ventricle are good prognostic markers during left ventricular (lv) failure secondary to ischemic and dilated cardiomyopathy.the aims of the present study were: to assess lv and rv diastolic function by conventional doppler and pulsed-wave tissue doppler imaging (pw-tdi) in dogs with mitral valve disease (mvd), with or without pulmonary hypertension (ph); to test if echocardiographic parameters of lv and rv diastolic dysfunction correlate to the doppler-estimated pulmonary artery systolic pressure (pasp). dogs were prospectively evaluated, including dogs with mvd. for each dog, a complete echocardiographic evaluation was carried out. dogs with mvd were divided in groups according to the acvim classification of heart failure. using the cut-off value of tricuspid regurgitation (tr) peak velocity of . m/s, presence or absence of ph was considered, standard echocardiographic and mitral and tricuspid doppler parameters (e wave and a wave), and pw-tdi parameters (systolic wave, sa; early diastolic wave, e'; late diastolic wave, a'; e'/a' ratio; e/e' ratio) for lateral and septal mitral annulus, and lateral tricuspid annulus were measured. the echocardiographic data were compared by use of anova and multiple contrast t-test with bonferroni correction. the relationship between left-sided echocar-diographic parameters and rv diastolic parameters was examined by correlation analysis. the correlation of pasp with lv and rv diastolic parameters was examined by multiple linear regression. a value of p< . was considered significant.dogs were classified as follows: healthy dogs; dogs in class b ; dogs in class b ; dogs in class c and d. no differences were found among groups regarding rv conventional doppler and pw-tdi parameters. however, a significant, weak correlation was found between some left-sided echocardiographic parameters (left atrial dimension; end diastolic and end systolic volume indexes; peak e wave and a wave velocities and e:a ratio) and some rv pw-tdi parameters. ph was diagnosed in dogs, while dogs were deemed without ph. dogs with ph had significantly different trans-mitral e and a wave peak velocity and e/e' ratio of lateral and septal mitral annulus. these two latter parameters were also correlated with pasp (r = . and . , respectively).our findings highlight the importance of considering ventricular interdependence in dogs with mvd, particularly those with ph. during the cardiac cycle, the left ventricle (lv) undergo a complex deformation, consisting of overall shortening accompanied by increase in wall thickness and twisting due to the helical orientation of the myocardial fibers. using speckle tracking echocardiography (ste), the circumferential strain (cs) as well as the twisting motion of the lv, calculated as the net difference between lv apical and basal rotation angles during the cardiac cycle, can be quantified by post-processing -dimensional short axis images of the lv. thus, the aim of this study was to evaluate the global cs and the twisting motion in small-medium sized dogs with varying severity of mr attributable to myxomatous mitral valve disease (mmvd). using a vivid-i ultrasound system, all dogs underwent echocardiography including parasternal short axis views at the basal and apical level for offline analysis of rotation, and at the midpapillary muscle level for analysis of global cs, using commercially available software (echopac). twisting motion during systole (twist sys ), early (untwist early ) and late (untwist late ) diastole were calculated as well as their rates computed as the time derivatives. furthermore, time to onset of untwist, calculated from start of electromechanical activation until peak twist sys was measured. associations between twist and untwist variables, global cs and conventional echocardiographic indices of mr severity and lv remodeling were examined by multiple linear regression analyses including dog characteristics such as heart rate (hr), sex, breed, body weight and age. global cs increased with increasing mr (p < . ). untwist early (p = . ) and its rate (p = . ) also increased with increasing mr, albeit in co-variation with certain baseline dog characteristics. time to onset of untwist increased with mr (p = . ), left atrium to aortic root ratio (p = . ) and lv internal diameter in diastole (p < . ). in conclusion, untwist early and global cs gradually increased and the onset of untwist appeared to be delayed with increasing mr severity in small-medium sized dogs with spontaneous mmvd. this hyperdynamic stage with a delay in untwist may represent lv adaptation to loading conditions in mmvd, but might also indicate mid-and subepicardial compensation for an early lv dysfunction, as reflected by delayed onset of relaxation, as timing of contraction-relaxation cross over is the most vulnerable period of myocardial fiber mechanics. systolic anterior motion (sam) of the mitral valve is the mechanical correlate of left ventricular outflow tract dynamic obstruction (lvotdo) and has been associated with hypertrophic cardiomyopathy (hcm) in most instances. however, sam without left ventricular hypertrophy or with regressing hypertrophy is increasingly recognised.the echocardiographic studies of cats, diagnosed between / and / , with sam without hcm ( cats) or sam with regressing hypertrophy ( cats) were reviewed for evaluation of the anatomic and mechanic alteration contributing to lvotdo .ventriculo-aortic and aorto-septal malalignment were suggested by the significantly narrower aorto-mitral (am) and aorto-septal (as) angle compared to a group of control cats (as = versus -p = . ; am = versus -p = . ).other anatomic alterations of the lvot constituents included false tendon inserted on the septal crest ( / ), basal septal angulation ( / ), apical displacement of the postero-medial or antero-lateral papillary muscle ( / ), bifid/accessory papillary muscle ( / ), aberrant chordal insertion on the septal crest ( / ), severe papillary muscle hypertrophy ( / ), and thickened aortic cusps with sub-aortic ancillary echoes ( / ).mechanisms of lvotdo included obstructive mitral septal leaflet ( / ), protrusion of a displaced papillary muscle ( / ), impingement of an angulated basal septum into the lvot ( / ), and mechanical lvot narrowing from hypertrophic papillary muscle ( / ). basal septal impingement into the lvot was associated with apical ballooning ( / ) without obvious apical akinesis.out of cats with lvotdo without hypertrophy, had follow-up studies; only cat responded positively to atenolol treatment. all the cats with initial hypertrophy had reverse remodelling and normalised aortic ejection velocities within month to month after atenolol treatment initiation; cat developed aortic regurgitation.these data indicate that sam is not exclusively related to hcm and may be a cause, not a complication, of left ventricular hypertrophy. therapies that delay the onset of congestive heart failure (chf) in dogs with dmvd at risk of disease progression would be clinically beneficial. increases in la/ao, lveddn and serum nt-probnp and ctni concentrations are associated with decreased survival times. activation of the renin-angiotensinaldosterone system is implicated in cardiac remodeling in canine dmvd. we hypothesised that administration of spironolactone to dogs with compensated dmvd demonstrating the above risk factors would reduce the rate of cardiac remodeling associated with progressive dmvd. dogs with acvim class b dmvd were recruited to a randomized, blinded, placebo-controlled pilot study. no dogs were receiving medications for cardiac disease. all dogs demonstrated at least one of the following risk factors: echocardiographic evidence of cardiomegaly, nt-prob-np> pmol/l, ctni> . ng/ml. no dogs had evidence of other cardiac disease or renal disease, hypoadrenocorticism, hyperkalaemia, or hyponatraemia. dogs were randomized to receive spironolactone ( mg/kg orally) or placebo sid for months. comparisons between groups were made using mann-whitney tests. repeated measures linear models were constructed to compare the rate of change of variables over time. significance was set at p< . . data were analysed based on the intention to treat. twenty dogs of varying breeds were enrolled. ten dogs demonstrated risk factors, dogs risk factors and dogs risk factor. ten dogs received placebo; age range . - . years (mean ±sd, . ± . years), body weight range . - . kg ( one dog in this group died suddenly, progressed to chf and received suboptimal spironolactone dosage. nt-probnp was significantly higher in the spironolactone group at baseline. (p= . ). nt-probnp (p= . ), la/ao (p= . ) and lveddn (p= . ) increased over time in the placebo, but not in the spironolactone, group. the rates of change of nt-probnp (p= . ), la/ao (p= . ) and lveddn (p= . ) approached, but did not reach, significant differences between groups. in conclusion, treatment with spironolactone might slow the rate of increase in cardiac size in dogs with acvim class b dmvd showing risk factors for poor outcome. decreasing the rate of increase in cardiac size might delay the onset of chf. further studies are warranted to investigate these hypotheses. cardiac dysfunction is a concern in human systemic inflammatory response syndrome (sirs) patients, where increased cardiac biomarkers and decreased cardiac function have already been described. in a previous canine sirs study, an increase of cardiac biomarkers (nt-probnp, ctnt and lactate) and their prognostic value has been established. the present study evaluated the kinetics of basic echocardiographic parameters (fractional shortening (fs) and left ventricular ejection fraction (lvef), which both reflect systolic function; and the ratio of the left atrium to the aorta (la/ao), which reflects preload) in canine sirs. our hypotheses were that ( ) fs, lvef and la/ao are altered in canine sirs and ( ) that these parameters carry prognostic information.dogs with sirs, without primary cardiac disease, presenting to the emergency service were prospectively included from january until august . cardiac ultrasonography was performed by two veterinarians in a standardized fashion at initial presentation, after (t ), (t ), (t ), (t ) hours of hospitalization until discharge or death and at a control visit (t m) over one month after discharge. dogs were classified according to their underlying disease process: infection, neoplasia, trauma, gastric-dilation and volvulus (gdv), other gi diseases, and miscellaneous diseases. statistical analysis was performed with sas. univariate analysis was used to assess normal distribution. a mixed procedure and a logistic procedure was performed accordingly (p < . ).thirty seven dogs (infection, n= ; neoplasia, n= ; trauma, n= ; gdv, n= ; other gi, n= and miscellaneous diseases, n= ) were included. twenty-eight patients survived, while did not (died, n= ; euthanasia for financial reasons, n= ; euthanasia for prognostic reasons, n= ). eleven dogs had control visits, owners declined a control echocardiography, patients were lost to follow-up and died before control visit. fs and la/ao were significantly correlated with survival to discharge, however lvef was not. additionally, lvef and fs did not change significantly during hospitalization; neither compared to t m. la/ao did however increase significantly during hospitalization. la/ao at t ( . ; . - . ) differed significantly from values at t ( . ; . - . ), t ( . ; . - . ) and t m ( . ; . - . ).unexpectedly, surviving dogs had lower fs ( . %; - ) than non-survivors ( . %; - ). la/ao was associated with survival and increased rapidly after hospitalization to values similar to t m, which probably reflects the efficacy of fluid therapy in emergency cases.in this population of canine sirs patients, no echocardiographic evidence of cardiac dysfunction was demonstrated. reports from first-opinion practice of feline arterial thromboembolism (ate) are scarce. our aim was to describe and evaluate the outcome in cats with ate presenting to three first-opinion clinics.clinical records of cats presenting with ate between - were reviewed for history, clinical findings, presence of congestive heart failure (chf) and outcome. kaplan-meier and log rank analysis was performed to evaluate associations with survival.during the study period, cats presented with ate; an overall feline incidence of . %. most cats were male ( . %) and non-pedigree ( . %). signs of cardiovascular disease prior to ate included cardiomyopathy ( %), a heart murmur ( . %), a gallop sound/arrhythmia ( . %) and hyperthyroidism ( . %). most cats presented within hours of clinical signs ( . %). median age at presentation was atrioventricular block (avb) is an arrhythmia resulting from conduction abnormalities through the atrioventricular node that leads to severe signs and sudden death.the aim of this study was to evaluate long-term intrinsic rhythm variations in dogs undergone pacemaker (pm) implantation. ninety-two dogs of different breeds with rd degree avb ( avb) ( . %), advanced nd degree avb ( avb)( . %), paroxysmal avb ( %), : avb ( . %) and avb with atrial fibrillation ( . %) were retrospectively analyzed. forty-nine ( . %) were males and ( . %) females with a mean age of . + . (sd) years and a mean body weight of . kg + . . the intrinsic rhythm was evaluated the day of pm implantation (t ), after day (confidence interval % [ci %] - ) (t ), days (ci % - ) (t ), days (ci % - ) (t ), days (ci % - ) (t ). according to the avb grade at different controls, the rhythm disturbance was considered advanced, regressed or unchanged. shapiro-wilk and kolmogorov-smirnov tests were used to test normalcy, f-test to compare means in a generalized linear model and chi-squared to examine the association between categorical variables and status at each control. sixty ( . %) dogs had no intrinsic rhythm changes, ( . %) had avb progression and ( %) had avb regression. forty-eight cases of avb remained unchanged, while regressed to sinus rhythm, to : avb and to advanced avb. eight advanced second degree avbs progressed to complete avb, regressed to sinus rhythm, to : avb and remained unchanged. five paroxysmal avbs progressed to complete avb, to : avb and remained unchanged. four : avbs progressed to complete avb, regressed to sinus rhythm and remained unchanged. all avbs with atrial fibrillation remained unchanged. chi-square test showed that changes of intrinsic rhythm were associated with the type of avb (v . , p<. ) and the time of controls (v . , p<. ), while other factors were not statistically significant. regression occurred within days while progression occurred at any times. the results showed that the degree of avb at the moment of pm implantion should not be considered a definitive diagnosis since more than / of the cases could present progression or regression. because of their potential progression, pm implantation should be considered as first choice treatment also in cases of low avbs, and further studies are needed to evaluate the cause of transitory high grade avbs. tissue doppler imaging (tdi) is a complement to conventional echocardiography for assessment of myocardial function. the aims of the study were to investigate breed differences and intraobserver-variability of colour tdi variables in healthy dogs.fifty-three privately-owned male dogs were prospectively recruited. dogs were declared healthy by physical examination, blood pressure measurement, ecg, analyses of urine and blood (haematology and biochemistry), and conventional echocardiographic d and doppler examination, as part of the eu-funded lupa-project. directly following these extensive examinations, the tdi acquisition was performed by the same experienced echocardiographer using a philips hd xe with a s - (small dogs) and s - (large dogs) mhz probe. cineloops were acquired from the right short-axis view and radial colour tdi variables at the endocardium and epicardium of the left ventricular free wall were later analysed. six other healthy dogs were included in a substudy aimed at evaluating the effect of sources of variation using a hierarchial random-effects model.ten of the examined dogs were excluded due to breathing artifacts and poor quality of the curves, leaving dogs in the study; labrador retrievers ( ), cavalier king charles spaniels ( ), and dachshunds ( ), with a mean age of . ± . years (sd). a p-value < . was considered significant in the statistical analyses. kruskal-wallis one-way analysis of variance showed that labrador retrievers had significantly higher values for endocardial and epicardial systolic (s) waves, and longer time to peak for both endocardial and epicardial e-and a-waves, compared to the other two breeds. labrador retrievers also had significantly lower heart rate (hr). further analysis of the breed differences using multiple regression analysis showed major effects of body weight and hr on endocardial and epicardial s waves, while time to peak, both endocardial and epicardial, for the e-and a-waves, were primarily affected by hr. dog was the variance component having the major effect on variability of tdi variables.in conclusion, time to peak of both diastolic waves was longer in labrador retrievers compared to the small-breed dogs. furthermore, higher s-wave values in labrador retrievers might indicate a different contractility pattern in largebreed dogs, and warrants further investigation. mitral regurgitation (mr) is the most common heart disease in dogs.dogs with a more advanced stage of this disease are likely to develop pulmonary edema of heart failure. the aim of this study was to evaluate the plasma c-reactive protein (crp) concentra-tion in dogs that underwent mitral valve repair for mr.all dogs were operated between october and october . the dogs were categorized according to the international small animal cardiac health council (isachc) classification, and physical examination, thoracic radiography, and d color flow doppler echocardiography were performed before and after surgery. the plasma crp concentration and white blood cell counts were also determined before and after surgery. cardiogenic pulmonary edema was diagnosed on the basis of clinical examination and thoracic radiography.overall, dogs (mean body weight, . ± . kg and mean age, . ± . years) were enrolled; of these dogs had cardiogenic pulmonary edema. the dogs breeds were chihuahua (n = ), cavalier king charles spaniel (n = ), maltese (n = ), yorkshire terrier (n = ), shih tzu (n = ), miniature dachshund (n = ), and others (n = ). no significant difference was found for age and body weight. the vertebral heart size and la/ao ratio significantly decreased after surgery compared with the preoperative values. before the operation, crp concentration and white blood cell counts in isachc class iiib dogs ( . ± . mg/dl and ± /ll, respectively) were higher than those in class ib ( . ± . mg/dl and ± /ll, respectively), class ii ( . ± . mg/dl and ± /ll, respectively), and class iiia ( . ± . mg/dl and ± /ll, respectively) dogs. additionally, crp concentration and white blood cell counts in class iiib dogs significantly decreased after surgery compared with preoperative values. crp concentration and white blood cell counts in the dogs with cardiogenic pulmonary edema significantly increased compared with those with non-pulmonary edema. furthermore, cardiogenic pulmonary edema disappeared within months after surgery, and the crp concentrations and white blood cell counts became normal.in conclusion, crp concentration increases in dogs with mr and cardiogenic pulmonary edema. it is widely recognized that inflammatory reaction plays a key role in the development of heart failure. consequently, these data indicate the importance of strict management for pulmonary edema and inflammation. the neurotransmitter serotonin ( -hydroxytryptamine, ht) has recently been suggested to have a role in development of myxomatous mitral valve disease (mmvd) in dogs.the aim of this study was to investigate whether serum ht concentration was associated with mmvd severity in dogs, and to assess potential associations between serum ht concentrations and dog characteristics, echocardiographic variables, heart rate, systolic blood pressure, and platelet size (mean platelet volume) in the study population. client-owned dogs with naturally acquired mmvd of varying severity were prospectively recruited for the study. dogs were classified according to mmvd severity (breeds predisposed to early onset of mmvd, but without echocardiographic evidence of the disease, or mild, moderate or severe disease). serum ht concentrations were analyzed using an elisa assay.lower serum ht concentrations were shown in dogs with severe mmvd, compared with dogs predisposed to mmvd (p = . ) and dogs with mild mmvd (p = . ). unilinear and multiple regression analyses showed that serum ht concentrations decreased with increasing left atrial to aortic root ratio (la/ao), were higher in cavalier king charles spaniel (ckcs) dogs compared to dogs of other breeds, and were higher in female dogs than in male dogs. the la/ao was the variable most strongly associated with serum ht concentration.in conclusion, the finding of higher serum ht concentrations in dogs predisposed to mmvd (ckcs) and dogs with mild mmvd suggests that alterations in ht signaling might play a role in progression of early stages of mmvd. hypersomatotropism (hs) can be a common reason for development of diabetes mellitus in the cat. remission of diabetes can be achieved with an accurate diagnosis of the hs, although diagnosis is hampered by the relative complexity of confirming hs, requiring a combination of insulin growth factor- (igf- ) or feline growth hormone measurement and intracranial imaging. unfortunately, all three have limitations as diagnostic aid and when evaluating the success of therapy, particularly radiotherapy (rt). consequently, more precise markers of hs are required. the current study aimed to evaluate physiological behaviour and diagnostic potential of serum ghrelin in feline hs. as ghrelin is an endogenous ligand of the gh secretagogue receptor, it was hypothesised production of ghrelin might be suppressed in hs and subnormal serum levels could be a marker for increased gh activity.fasted (pre-insulin) serum samples were collected from normal (age matched control), uncomplicated diabetic (dm) and hypersomatotrophic diabetic (hsdm) cats. cats were categorized into the hsdm-group on the basis of elevated igf- (> ng/ml) and demonstration of a pituitary lesion on imaging. in cats additional serum samples were obtained following rt. cats were categorized into the dm-group on the basis of low igf- (< ng/ml) and normal insulin requirements (< . iu/kg). serum ghrelin was determined using a total ghrelin elisa system validated for the cat. data were tested for normality and concentrations compared between groups using unpaired t-tests and a paired t-test for the before and after rt hsdmgroups.serum total ghrelin was not different between the hsdm ( . ng/ml+/- . ) and dm-group ( . ng/ml+/- . , p= . ). a significant difference was present between the control group ( . ng/ml+/- . ) and both the hsdm (p= . ) and the dmgroup (p= . ). serum ghrelin concentrations in the hsdm cats undergoing rt (n = ) were significantly higher following completion of treatment ( . ng/ml+/- . versus . ng/ml+/- . , p= . ).the results suggest feline serum total ghrelin is suppressed to similar levels in both the diabetic and diabetic hypersomatotrophic state compared to healthy subjects. consequently, it appears serum ghrelin levels are not helpful in determining the presence of hs in the diabetic cat. however, the results do indicate treatment of hs with rt results in an increase in serum ghrelin, suggesting the presence of an independent inhibitory effect of excess gh on serum ghrelin production. this potential inhibitory effect of gh might render serum ghrelin measurement useful as an additional tool to assess hypersomatotrophic remission after rt. further studies are however indicated. to date only few studies characterized histopathological features of the endocrine and exocrine pancreas in cats with diabetes mellitus. loss of b-cells is a consistent finding but no detailed data about the presence and types of inflammatory cells are available. we recently observed that hyperglycemia increases neutrophils in the exocrine pancreas. the aims of the present study were to assess whether diabetic cats have pathological evidence of islet inflammation or pancreatitis and to define islet lesions in comparison to a well-matched control population.formalin-fixed, paraffin-embedded pancreatic samples were collected from post-mortem examination performed on diabetic and control cats died due to any disease at the clinic for small animal internal medicine, university of zurich (switzerland) between and . control cats were selected to be matched for age, sex, breed and body weight. sections were routinely stained with hematoxilin-eosin, and doublelabeled immunohistochemistry was performed for the following markers: insulin and myeloperoxidase (neutrophils), insulin and cd (t-lymphocytes), insulin and cd (b-lymphocytes), insulin and pcna (proliferation marker), and glucagon and ki- (proliferation marker). light-microscopic cell counting and morphometric analyses were performed manually and with software (image-j), respectively. data were analyzed with contingency tables and ttests.thirty-seven diabetic cats and controls were included. the mean insulin-positive cross sectional area was approximately % lower in diabetic than control cats (p< . ), that of glucagon was similar. proliferation of insulin-positive and glucagon-positive cells and the average counts of neutrophils, t-and b-lymphocytes in the islets did not differ between groups. interestingly, the presence of (t and b) lymphocytes in general tended to be more frequent in diabetic ( / = . %) than control ( / = . %) cats. in the exocrine pancreas, a trend towards increased presence of necrosis and fibrosis was observed in diabetic cats ( / = . % vs. / = %; p= . ) but inflammatory infiltration did not differ. proliferation of acinar cells was -fold increased in diabetic cats (p< . ), notably nearby islets ( -fold, p< . ).the results confirm previous observations that loss of b-cells occurs in diabetic cats. in addition, a subset of diabetic cats shows lymphocytic infiltration of the islets that might have contributed to b-cell loss. increased necrosis and fibrosis of the exocrine tissue may suggests that the diabetes leads to pancreatitis in some cats. the increased proliferation rate of acinar cells deserves further investigation. in humans this finding has been associated with chronic pancreatitis as well as transdifferentiation into islet cells. on the basis of a relatively high prevalence of hypersomatotropism (hs) amongst cats with a diagnosis of diabetes mellitus, as well as the possible subtle phenotype of these patients and the significant implications on prognosis and treatment, screening diabetic cats for hs could be advocated. for most veterinarians serum total insulin-like growth factor- (igf- ) assessment represents the most feasible and accessible means of performing screening. however, hepatic igf- production is dependent on presence of sufficient portal insulin, which can be deficient in newly diagnosed diabetic cats, resulting in false negative results. additionally, elevation of igf- has been reported in non-acromegalic diabetic cats. alternative or additional diagnostic tests for hs, as well as to evaluate the success of treatment of hs, are therefore desirable. since feline hs is associated with tissue growth, serum type iii procollagen propeptide (piiip), a peripheral indicator of collagen turnover, was hypothesised to be a useful indicator of active disease or growth hormone bioactivity. fasted, pre-insulin, serum samples were prospectively collected from uncomplicated diabetic (dm) and hypersomatotrophic diabetic (hsdm) cats. cats were categorised into the hsdm-group on the basis of elevated igf- (radioimmunoassay, > ng/ml), followed by demonstration of a pituitary lesion on intracranial imaging and into the dm-group on the basis of low igf- (< ng/ml) and modest insulin requirements (< . iu/kg). an elisa system for piiip was developed for use in the cat. data were tested for normality and concentrations compared using the mann whitney test. correlation with serum igf- was assessed by calculating a spearman's correlation coefficient (rho).dilutional parallelism using cat serum with high and low piiip-activity indicated validity of the elisa system. intra-assay coefficient of variation calculation proved adequate precision at high and low concentrations ( . % and . %) and the assay detection limit was found to be . ng/ml. median serum piiip was . ng/ml (range: < . - . ) in the hsdm group, versus . (range: < . - . ) in the dm group (p< . ). there was a significant correlation between serum igf- and piiip (rho= . , p< . ).in conclusion, serum piiip can be measured in the cat. additionally, serum piiip seems an alternative measure of growth hormone bioactivity in cats, given the significant elevation in concentration in feline hs. further evaluation of piiip in cases with hs will help determine the exact added value of evaluating this parameter in the diagnosis of hs, as well as in the assessment of treatment efficacy. glucose and galactose are transported across the brush border membrane (bbm) of enterocytes by sodium/glucose cotransporter- (sglt ), coded for by slc a . sglt is the sole route for intestinal glucose absorption and its level of expression dictates bbm transport capacity for glucose. the relevance of sglt expression in predisposition to diabetes mellitus and obesity was investigated in dogs. the aims were to assess the effect on promoter function of known snps in the ' flanking region of canine slc a , and to search for novel snps in well defined samples of dogs with varying risk for diabetes or obesity. caco- /tc cells were shown to express sglt in vitro. the kbp fragment of canine slc a ' flanking region from - to + relative to the transcription start site was cloned from canine genomic dna, ligated into pgl basic plasmids bearing firefly luciferase as a reporter gene, used for transient transfection of caco- /tc cells, and shown to drive luciferase production significantly above control (p< . ). to determine the effect of the three known snps in this region on promoter function, new promoter/reporter constructs (all possible permutations of these three snps) were created using site-directed mutagenesis. these constructs were used for transient transfection of caco- /tc cells using renilla luciferase as an internal control. no significant differences in promoter function were seen, suggesting that these three snps do not have a significant effect on the constitutive transcription of sglt mrna in dogs.a search for novel snps in this region in dogs was made in two breeds predisposed to diabetes mellitus (samoyed, cairn terrier), two breeds that rarely develop diabetes (boxer, german shepherd dog), and two breeds predisposed to obesity (labrador retriever, cocker spaniel). genomic dna from healthy individuals of each of these breeds was obtained from the uk companion animals dna archive, with kind permission. the slc a ' flanking region was amplified from each individual by high-fidelity pcr using breed-labelled primers, gel purified, mixed in equimolar amounts and sequenced by pyrosequencing ( sequencing, gs flx, roche). the sequence of the slc a ' flanking region in all individuals of all breeds tested was identical. on this evidence, variations in slc a promoter sequence between dogs do not influence the pathogenesis of diabetes or obesity in these breeds. remission of diabetes mellitus may be achieved in up to % of cats. for remission to occur, recovery of b-cell function and possi-bly of b-cell mass is required. a novel class of antidiabetic drugs that act via the incretin system increase b-cell proliferation and glucose-stimulated insulin secretion in rodents. two strategies of incretin-based therapies, glp- analogues (e.g. exenatide shortacting (ex-sa), exenatide long-acting (ex-la)), and dpp- inhibitors (e.g. sitagliptin), are successfully used in human diabetics. knowledge about the use of incretins in cats is scarce. it was demonstrated that ex-sa and a dpp- inhibitor (nvp-dpp ) increase insulin secretion after intravenous glucose stimulation in healthy cats. the effects of these drugs after meal stimulation and the use of ex-la have not been explored in cats so far.the aims of the study were to test whether ex-sa (byetta ® , q h, sc), ex-la (bydureon ® , q d, sc) and sitagliptin (januvia ® , q h, po) can be safely used in cats, and to identify the most effective drug and dose in a dose-escalation study.nine healthy cats were used. ex-sa was given to cats at . , . , and lg/kg for consecutive days each. ex-la was given to other cats at , , and lg/ kg with single injections each. sitagliptin was given to cats at , , and mg/kg for consecutive days each. a washout period of weeks was allowed between doses. on day of each treatment block, a meal response test (mrt) was performed in all cats after a h fastby feeding % of daily energy intake with subsequent blood sampling at timepoints , , , , and minutes. insulin and glucose area under the curves (auc) were calculated for each drug dose.gastrointestinal side effects of to days duration were observed in cats of each group, irrespective of the dose. well-being and appetite were otherwise conserved. ex-sa increased insulin auc by %, %, % and %, respectively, compared to insulin auc during mrt without drug administration. ex-la and sitagliptin increased insulin auc by %, %, %, % and %, %, %, %, respectively. auc for glucose was similar in all cats, irrespective of the drug and dose. we conclude that ex-sa, ex-la and sitagliptin can be safely used in healthy cats and that ex-sa increases insulin secretion more effectively than ex-la and sitagliptin. insulin detemir is a synthetic long acting insulin analogue designed to maintain basal levels of insulin in humans with diabetes mellitus (dm).pharmacokinetic studies in dogs indicate that insulin detemir has a greater effect than other types of insulin, requiring a lower dose. the objective of our study was to evaluate its efficacy and the frequency of hypoglycemia in dogs with dm treated with insulin detemir. eight dogs were included into the study. median (range) age was years ( - ), were female ( intact, spayed), and was intact male; median (range) body weight was . kg ( . - . ). dogs with relevant concurrent diseases (e.g. hypothyroidism, hypercortisolism, neoplasia, renal insufficiency) and dogs with prior administration of diabetogenic drugs were excluded. all dogs received insulin detemir bid for at least months, re-evaluations were performed after , , , and weeks and included clinical signs, blood glucose curves (bgc) and fructosamine concentrations. median (range) insulin dose was . u/kg ( . - . ) bid at admission which was not significantly different after weeks of therapy ( . u/kg, range . - . ). initially, all dogs had markedly elevated blood glucose ( . mmol/l, . - . mmol/l), and elevated fructosamine concentrations ( lmol/l, - lmol/l). mean(±sd) glucose concentrations (mmol/l) of the bgc at each re-evaluation were . ± . , . ± . , . ± . , . ± . , . ± . ; median (range) of the glucose nadir values (mmol/l) were . ( . - . ), . ( . - . ), . ( . - . ), . ( . - . ), . ( . - . ) and fructosamine concentrations (lmol/l) were ( - ), ( - ), ( - ), ( - ), ( - ), respectively. glucose concentrations (mean) were significantly (p< . ) lower after weeks therapy than before treatment. hypoglycemia (glucose nadir < mmol/l) was a consistent problem, identified in dogs ( % of the bgc of the dogs). there were episodes (in dogs) of owners reporting clinical signs (lethargy, weakness, unsteady gait) that could have been caused by hypoglycemia. based on owner opinion, clinical assessment of the veterinarian and bgc after months of therapy, good control of the disease was obtained in ( %) and moderate control in dogs ( %). according to our preliminary results insulin detemir is effective in controlling hyperglycemia in dogs with dm. it is more potent than other types of insulin which are also used bid and therefore lower doses were used. nevertheless hypoglycemia was a common finding especially in small dogs. therefore insulin detemir should be used with great caution and dilution could be considered. spontaneous hypercortisolism is due to acth-independent hypersecretion of cortisol by an adrenocortical tumor (at) in about % of the cases. although the understanding of growth and hormonal activity of ats has expanded in recent years, the pivotal factors/acts in the pathogenesis of the at remain undisclosed.the canonical wnt-pathway plays a role in cell survival and cell cycle progression and has been shown to be involved in many different tumor types, including human and mouse ats. central in this pathway is ß-catenin. cytoplasmic and/or nuclear accumulation of ß-catenin has been demonstrated in human cortisol-secreting ats. this has been partly explained by mutations in exon of ß-catenin.in our study, the activation of the canonical wnt-pathway was investigated in adrenal adenomas and carcinomas of dogs with acth-independent hypercortisolism. fifteen normal canine adrenals served as control tissue. the mrna expression was measured for wnt-ligands (wnt , wnt , wnt , wnt a, wnt b variants and , wnt , wnt a, wnt b), wnt-ligand inhibitors (dkk , wif , sfrp ) and for wnt target genes (cmyc, axin and cyclind ). in addition, the coding region of the mrna of ß-catenin was sequenced and the localization of ß-catenin was evaluated by immunohistochemistry (ihc).the results of expression analysis of wnt-ligands and wnt-ligand inhibitors demonstrated a significant downregulation of wnt and wnt b variants and . wnt target gene cyclin d was significantly downregulated in adenomas, while cmyc and axin mrna expression did not differ between ats and normal adrenals.sequence analysis of ß-catenin revealed a mutation in / of the ats: silent mutations, most probably single nucleotide polymorphisms (snps), neutral mutation (arginine to histidine), nonsense mutation resulting in a premature stop codon and deletion of basepairs combined with a neutral mutation. the amino acid change appeared not to be tumorassociated whereas the stop codon was. there were no activating ß-catenin mutations. the ihc demonstrated accumulation of ß-catenin in the cytoplasm of part of the ats, while nuclear staining for ß-catenin was rarely present.we conclude that the canonical wnt-pathway is most likely not involved in the pathogenesis of canine cortisol-secreting ats. trilostane is the treatment of choice for canine pituitary-dependent hyperadrenocorticism (pdh) however there is controversy about the ideal treatment regime. the objective of this study was to evaluate efficacy and safety of sid vs bid trilostane treatment for canine pdh.this prospective randomised study included dogs with pdh, dogs treated with trilostane sid (initial dose - mg/kg/ hrs) and with trilostane bid ( . - mg/kg/ hrs). a history, physical exam, haemogram, biochemical profile, acth stimulation test and urinary cortisol to creatinine ratios (uccr) were performed before treatment, and at one week (sid - hrs post trilostane; bid - hrs) and , , and months after treatment ( - hrs post medication)the median (±sd) daily dose throughout the study was similar in dogs receiving sid ( . ± . mg/kg/day) or bid ( . ± . mg/kg/day) trilostane. baseline cortisol concentration (mean±sd) before trilostane and at the five reevaluations in the sid group were . ± . , . ± . , . ± . , . ± . , . ± . and . ± . and in the bid group were . ± . lg/dl, . ± . , . ± . , . ± . , . ± . and . ± . . baseline cortisol was significantly higher (p= . ) in the sid group only at six-month evaluation. post acth cortisol concentrations in the sid group were . ± . , . ± . , . ± . , . ± . , . ± . and . ± . and in the bid group were . ± . , . ± . , . ± . , . ± . , . ± . and . ± . . no statistically significant differences were found at any evaluation. uccr in dogs in the sid group were ± , ± , ± , ± , ± and ± . and in the bid group ± , ± , ± , ± , ± and ± . similarly no statistically significant differences were found throughout the study. in each of the five re-evaluation times, clinical signs (polyuria, polydipsia and/or polyphagia) persisted in % ( / ), % ( / ), % ( / ), % ( / ) and in % ( / ) of dogs in the sid group. persistence of clinical signs occurred in % ( / ), % ( / ), % ( / ), % ( / ) and % ( / ) of dogs in the bid group. mild adverse effects occurred in / and in / of the dogs treated with sid and bid trilostane, respectively.based on this study, we conclude that the results of acth stimulation test and uccr in dogs with pdh treated with both protocols are similar at most re-evaluation times. however, clinical signs resolved in a greater proportion of dogs receiving trilostane bid. using trilostane twice daily will help to reduce the number of dogs that do not have a good clinical response. the effectiveness of trilostane therapy is monitored by regular acth stimulation, which is time-consuming and expensive. therefore, a monitoring system without a stimulation protocol and with less client expense would be preferable.the aim of our study was twofold: firstly, to evaluate, if baseline cortisol, endogenous acth (cacth) or the baseline cortisol to acth ratio (cortisol/acth ratio) could replace the acth stimulation test; secondly, to evaluate, if baseline cortisol provides additional information than post-acth cortisol alone or if its measurement could be abandoned.forty-one trilostane-treated dogs with pdh diagnosed between april and december were included in the study. a total of acth stimulation tests with baseline, post-acth and delta cortisol (post-acth cortisol minus baseline cortisol) and cacth results and cortisol/acth ratios were analyzed.control of adrenal gland function was classified according to the target range of post-acth cortisol concentration as: excessive (< nmol/l; group ), optimal ( - nmol/l; group ), or inadequate (> nmol/l; group ). in a second step, control of adrenal gland function was reclassified according to baseline cortisol values only and the new classification was compared with the original one.there was a significant correlation between baseline cortisol and post-acth cortisol and a significant difference of baseline cortisol between the groups, however, with a large overlap. reclassification of the adrenal gland function on the basis of baseline cortisol revealed a misclassification in / ( %) tests.endogenous acth did not correlate with baseline or post-acth cortisol and did not differ between the groups. the baseline cortisol:acth ratio differed significantly between group and and between group and , but again with a large overlap.to determine if measurement of baseline cortisol gives additional information than post-acth cortisol alone, the delta cortisol values were analysed. delta cortisol correlated significantly with post-acth cortisol but not with baseline cortisol and differed significantly between the groups, the overlap however was large.the large overlap using only single values without taking post-acth cortisol into consideration leads to an unacceptably low correct differentiation of control of adrenal gland function. therefore, the acth stimulation test and determination of post-acth cortisol cannot be replaced by baseline cortisol, endogenous acth or the cortisol/acth ratio. however, as baseline cortisol concentration does not give additional information than post-acth cortisol alone, its determination can be abandoned. in humans, recombinant human thyrotropin (rhtsh) enhances radioactive iodine uptake (raiu) in patients with differentiated thyroid cancer. this property is particularly interesting in dogs because high doses of radioiodine- ( i) are used for the treatment of this disease. no studies have been performed in veterinary medicine to optimize i treatment of thyroid cancer.the aim of this study was to evaluate the effect of rhtsh on the uptake of i in dogs with thyroid tumors.nine dogs with thyroid neoplasia were included in this prospective cross-over study. six dogs had unilateral tumors, dog had bilateral tumors and dogs had ectopic tumors. diagnosis was based on physical examination, cytology, cervical scintigraphy and, when available, histopathology. in dogs i was administered for a baseline raiu determination in week . in week (after a wash out period of weeks), these dogs received rhtsh ( lg iv) h before i injection. in patients the order of the protocol was reversed. for each scan, the dogs received mbq ( mci) of i iv and planar scintigraphy was performed h and h thereafter for tumor raiu calculation. blood samples were taken at baseline and at , , and h after rhtsh administration for measurement of serum total thyroxine (tt ) and serum thyrotropin (tsh) concentrations.rhtsh caused no statistical significant change on thyroid tumor raiu at h (p= . ) or at h (p= . ). despite the lack of overall statistical significance, after rhtsh administration the h raiu increased in tumors and the h raiu increased in tumors. when an increased raiu was observed, i uptake with rhtsh ranged . to . times baseline uptake. in patients, the post-rhtsh raiu more than doubled compared to baseline raiu. the raiu of thoracic metastases from patients could be calculated. in thoracic metastasis the raiu doubled after rhtsh; in the other the raiu decreased after rhtsh.in euthyroid patients, rhtsh induced a significant increase in tt concentrations (p= . ), confirming the biological activity of rhtsh.this study suggests that iv administration of lg rhtsh h before i has an inconsistent effect on thyroid tumor raiu, with a marked increase in uptake in some tumors and a decrease in others. further studies are necessary to determine the best dosage, route and timing of rhtsh to optimize thyroid tumor raiu.this study was partly funded by the dutch animal cancer foundation transdermal methimazole has been suggested as an alternative treatment option of hyperthyroid cats. in a previous study we could show a good clinical effectiveness of a pleuronic lecithin organogel (plo-) based product on a twice-daily basis. a reduced dose frequency is known to improve owner compliance, however no study has yet evaluated long-term treatment responses after once daily administration.objectives of the present study were to assess whether once daily administration of transdermal methimazole in its original formulation of (plo) was an effective alternative to the twice-daily treatment during a follow-up period of up to months and to evaluate t courses during a -hour period after methimazole application in selected cats.twenty client-owned cats with newly diagnosed hyperthyroidism and with available follow-up information were included in the study. methimazole was formulated in plobased vehicle and was applied to the pinna of the inner ear at a starting dose of mg/cat q hours. cats were rechecked - weeks, - weeks, - months, - months, - months, - and - months after starting therapy. additionally, in cats t concentrations were measured every hours after gel application over a hour period week after starting therapy. after - weeks, clinical improvement was observed in all animals. a change of treatment to oral medication due to erythema of the internal pinna of the ears was necessary in one cat, while none of the other animals showed any side effects during the follow-up period. significant decreases in t concentrations were determined at all rechecks compared to pre-treatment concentrations. methimazole dosage was increased in , decreased in and remained unchanged in cats. two cats with a decrease in the dose later had to be re-increased, while one cat was changed to q hours. there was no significant change in t during the -hour period and fluctuations corresponded to variations of precision in series.these results are in accordance with those of our previous study using the twicedaily regimen. we could show that once daily administration of transdermal methimazole in its original formulation of plo is an effective treatment option for long-term management of feline hyperthyroidism. further, timing of blood sampling after gel application is not important when assessing response to treatment. increase in the prevalence of large thyroid tumors, intra-thoracic thyroid thyroid scintigraphy provides valuable information regarding both thyroid anatomy and physiology and plays an integral role in the diagnosis, staging, and management of feline thyroid disease. in this study, we performed thyroid imaging on , consecutive hyperthyroid cats that were referred for radioiodine therapy between january and december . scintigraphy was performed as part of our staging protocol in which thyroid volume is estimated for i dose estimation (vet radiol ultrasound ; : ) . in each cat, the location of each area of increased radionuclide uptake (iru) was also recorded (cervical, thoracic inlet, chest). finally, each scan was evaluated for features suggesting malignancy (multiple, extensive areas of iru, heterogeneous pattern of iru with irregular, spiculated margins, extension of tumor through thoracic inlet into the thorax, and metastasis to regional lymph nodes or lung).of the , cats, most had been recently diagnosed. in cats, however, the interval between diagnosis and i treatment ranged from > to . years; almost all of these cats had received long-term antithyroid drug treatment. the , cats were divided into groups based on interval from diagnosis to i treatment: group ( - year), , cats; group (> - years), cats; group (> - years), cats; group (> - years), cats; and group (> - . years), cats. when the estimated thyroid volumes in the groups of cats were compared, a progressive, significant (p< . ) increase in median tumor volume occurred: . cm (group ); . cm (group ); . cm (group ); . cm (group ); and . cm (group ). the prevalence of cats with areas of iru within the thoracic cavity also increased progressively: . % (group ); . % (group ); . % (group ); . % (group ); and . % (group ). finally, the prevalence of suspected thyroid carcinoma ( of the , cats) also increased progressively: . % (group ); . % (group ); . % (group ); . % (group ); and . % (group ). in contrast, no increase in prevalence of ectopic thyroid tissue was found: . % (group ); . % (group ); . % (group ); . % (group ); and . % (group ). in conclusion, our results indicate that hyperfunctional thyroid tissue continues to grow and enlarge over time. thyroid carcinoma is extremely rare in cats with recently diagnosed hyperthyroidism, but the prevalence increases dramatically over time, suggesting that transformation from benign disease is common in cats controlled medically. measurement of plasma renin activity (pra) is considered the gold standard for monitoring mineralocorticoid substitution in humans with primary hypoadrenocorticism (ph). it is the most sensitive parameter to reflect insufficient as well as inappropriate high replacement. in dogs with ph mineralocorticoid substitution is currently monitored mainly by serum potassium and sodium concentrations. the role of pra for monitoring mineralocorticoid replacement has not been investigated. the aims of the study were to measure and compare pra in dogs with newly diagnosed ph and dogs with diseases mimicking ph, and to evaluate pra in dogs with ph treated with different mineralocorticoid substitution regimes.the following groups of dogs were included in the study: dogs with newly diagnosed ph (group ), dogs that were already treated for ph (group ), and dogs with diseases mimicking ph (group ). in group pra was measured before treatment and - weeks, - weeks, and - weeks after start of therapy. in group pra was measured at least twice every to months. in group pra was measured once at initial presentation. three dogs of group and dogs of group were treated with fludrocortisone (florinef ® , bristol-myers squibb). two dogs of group and dogs of group were treated with docp (percorten ® -v, novartis). pra was measured with an enzymatic assay via trapping of angiotensin i in the service d'angiologie, chuv-nes, lausanne. results were analysed by means of non-parametric methods (p < . ).pra before treatment was significantly higher in group ( . - . ng/ l/h, median . ) than in group ( . - . ng/l/h, median . ). average pra during therapy ranged from . to ng/ml/h (median ). pra did not decrease significantly in dogs treated with fludrocortisone. pra of dogs treated with docp ( . - . ng/ml/h, median ) was significantly lower compared to dogs treated with fludrocortisone ( . - . ng/ml/h, median . ). all dogs treated with docp had normal serum sodium and potassium at all re-checks, whereas dogs treated with fludrocortisone had mild to severe electrolyte abnormalities at several occasions. there was a weak correlation between pra and serum potassium.measurement of pra is a promising tool for monitoring mineralocorticoid substitution in dogs with ph. according to our preliminary results docp is superior to fludrocortisone for mineralocorticoid replacement. phenobarbital is widely used to control epilepsy in dogs. use of phenobarbital induces hepatic enzyme activity, and may decrease serum total and free thyroxine (tt and ft ), with the exact mechanisms and prevalence of this phenomenon being unknown. the aim of the present retrospective study therefore was to investigate how many dogs treated with phenobarbital show a decrease in thyroid hormones and to give insight into potential mechanisms. for this, tt , ft , and tsh were measured in canine serum samples submitted for assessment of therapeutic concentrations of phenobarbital. in a smaller subset of dogs, albumin, total protein, and transthyretin (ttr) were also measured at the diagnostic center for population and animal health, michigan, usa.according to thyroid results, dogs were classified as 'non thyroidal illness' (nti) (tt < nmol/l, ft ! pmol/l, tsh . ng/ml); 'equivocal' (e) (tt < nmol/l, ft < pmol/l, tsh . ng/ml); 'hypothyroid' (ht) (pattern of low tt and ft , tsh > . ng/ ml); and 'normal' (n) (tt ! nmol/l; ft ! pmol/l and tsh . ng/ml).forty-five dogs were classified as nti ( . %), dogs were classified as e ( . %), were classified as ht ( . %), and as n ( %); dogs did not fit in any defined category. there was no statistically significant difference in mean phenobarbital concentrations between nti, e, ht, and n ( . ± . , . ± . , . ± . , and . ± . lmol/l, respectively). twenty dogs of each group were analyzed for albumin and total protein and compared to healthy dogs that were not receiving phenobarbital. no statistically significant difference was noticed (p> . ). the attempt to measure ttr concentrations via commercially available elisa (tsz elisa, framingham, ma, usa) gave us inconsistence performance. in conclusion, % of dogs on phenobarbital treatment were classified as nti. if group e would be included, this value could be as high as %. neither phenobarbital, nor total protein or albumin concentrations had predictive value for thyroid hormone concentrations. this study was able to define the prevalence of the phenomenon that some dogs on phenobarbital therapy have low thyroid hormone concentrations, but did not give insight into the pathogenesis. ideally, demonstration of hypothyroidism in dogs receiving phenobarbital should include assays of thyroid hormones and tsh. pheochromocytoma (pheo) is a rare malignant catecholamine-secreting tumor of the adrenal medulla. catecholamines and metanephrines in plasma and in -h urine are approved biomarkers for the detection of the disease in humans, however, the question which of the tests is best is controversial. we previously demonstrated that measurement of urinary catecholamine and metanephrine to creatinine ratios is helpful for the diagnosis of pheo in dogs and that urinary normetanephrine to creatinine ratio may be the best test to discriminate between pheo and hypercortisolism (hc).knowledge on plasma catecholamines and metanephrines in dogs is scarce and no comparison between urinary and plasma parameters has been performed. the objective of the study was to measure urinary and plasma catecholamines and metanephrines in dogs with pheo, hc and in healthy dogs and to determine the test with the least overlap between the groups. six dogs with pheo, dogs with hc ( with ath, with pdh) and healthy dogs were included. urine samples were collected into hcl containing tubes to ensure a ph < , blood samples were collected on ice, centrifuged at °c and immediately snap frozen in liquid nitrogen. all samples were stored at - °c. urinary epinephrine (u-epi), norepinephrine (u-norepi), metanephrine (u-meta) and normetanephrine (u-normeta), and plasma epinephrine (p-epi), norepinephrine (p-norepi), free and total metanephrine (pf-meta and pt-meta) and free and total normetanephrine (pf-normeta and pt-meta) were analysed by hplc. urinary catecholamines and metanephrines were expressed as ratios to urine creatinine concentrations. data were analysed by non-parametric tests (p< , ).similar to our previous findings u-epi, u-norepi, u-meta and u-normeta were significantly higher in dogs with pheo and u-norepi and u-normeta were significantly higher in dogs with hc compared to healthy dogs. comparison between dogs with hc and dogs with pheo revealed significantly higher u-meta and u-normeta in the latter group. u-normeta was the only parameter with no overlap.in dogs with pheo p-norepi, pf-meta, pt-meta, pf-normeta, pt-normeta were significantly higher and in dogs with hc p-norepi, pf-normeta and pt-normeta were significantly higher than in healthy dogs. comparison between dogs with hc and dogs with pheo showed significant higher pf-meta, pt-meta, pf-normeta, pt-normeta in the pheo group. overlap was present with all parameters, but was least with pf-normeta and pt-normeta.according to our preliminary results u-normeta, pf-normeta and pt-normeta are valuable parameters for the diagnosis of pheo, so far u-normeta has performed better than the plasma parameters. chronic kidney disease (ckd) is common in geriatric cats and hypoxia might contribute to ckd progression. vascular endothelial growth factor (vegf) is a marker of hypoxia. the aim of this study was to evaluate urinary vegf as a prognostic marker in cats with ckd compared with the established progression factors, proteinuria and hyperphosphataemia. cats were recruited through geriatric clinics held at two first opinion london practices between and . diagnosis of ckd was based on concurrent findings of plasma creatinine > mg/dl and usg < . , with persistence of azotaemia for at least weeks. vegf was measured in urine samples taken from cats at diagnosis of ckd and indexed to creatinine giving vegf: creatinine ratios (uvc). survival was compared among low (< . lg/g), medium ( . - . lg/g) and high (> . lg/g) categories of uvc using the log-rank test. multivariable binary logistic regression was used to assess whether uvc was associated with an increase in plasma creatinine concentration of at least % within year of diagnosis. cats which did not demonstrate progression but were followed for < year were excluded from the study. cases which developed hyperthyroidism, received ace inhibitors, had gross haematuria, urinary tract infections, nephrotic syndrome or evidence of bladder neoplasia were also excluded. survival data are presented as median [ % confidence interval] and other descriptive data are presented as median ( th , th percentile). significance was set at p< . . cats with low uvc (n= ) had survival of [ , ] days, while those with medium (n= ) and high uvc (n= ) had survival of [ , ] days and [ , ] days respectively. there was no difference in survival time between cats with low and medium values of uvc (p= . ), but cats with both low (p< . ) and medium (p= . ) uvc had significantly longer survival than cats with high uvc. plasma creatinine concentration increased by ( , )% in cats which progressed (n= ) and ( , )% in cats with stable renal function in the year following diagnosis (n= ). uvc was also associated with progression of azotaemia (p= . ) independently of upc and plasma phosphate concentration. the progressive group had uvc of . ( . , . ) lg/g, while the stable group had uvc of . ( . , . ) lg/g. if high uvc indicates renal hypoxia, the results of this study support the hypothesis that hypoxia is associated with progression in cats with ckd. however, further studies evaluating renal hypoxia directly would be required to verify this. the term 'triaditis' is used to describe concurrent inflammation of the liver, pancreas, and small intestine, although occasionally only two of these organs may participate. the aim of this study was to investigate the frequency of coexistence of different combinations of cholangitis, pancreatitis and/or inflammatory bowel disease in cats, and describe the clinical, clinicopathological, and histopathological findings.initially cats were included in the study. thirty-nine cats had a suspicion of 'triaditis' based on clinical signs (depression, anorexia or polyphagia, vomiting, diarrhea, and/or weight loss), while cats clinically healthy, were considered as controls. each cat on presentation underwent cbc, biochemistry profile, serum total t , spec fpl ® and ftli blood examinations. cats diagnosed with intestinal parasitism, infectious disease, neoplasia, and/or hyperthyroidism were excluded from the study. biopsies from the liver, pancreas and small intestine (duodenum, jejunum, and ileum) were collected from each sick cat during laparotomy as part of the diagnostic investigation. biopsies from healthy cats were collected during laparotomy for ovariohysterectomy. all owners had signed a consent form and the study protocol was approved by the university's and state's ethics committee.of the sick cats with a suspicion of triaditis, were excluded because of neoplasia or other conditions. of the clinically healthy cats, had histopathological evidence of inflammation in their liver, pancreas, and/or intestine and were eventually grouped together with the sick cats. collectively, histopathological evaluation of the biopsies revealed cats ( with clinical signs and without) with inflammatory lesions in at least one organ. of those cats, ( . %) had histopathological evidence of ibd, ( . %) of cholangitis, and ( . %) of pancreatitis. thirteen cats ( . %) had only ibd, ( . %) of which were symptomatic. six ( . %) cats had only cholangitis, ( . %) of which were symptomatic. one ( . %) had only pancreatitis and was symptomatic. sixteen cats ( %) had concurrent ibd and cholangitis, ( . %) of which were symptomatic; ( . %) had ibd in combination with pancreatitis, ( . %) of which were symptomatic, while ( %) had ibd, cholangitis and pancreatitis and were all symptomatic. common biochemical findings were increased activities of alt ( / ), alp ( / ), and increased total bilirubin ( / ), ftli ( / ), spec fpl ( / ) concentrations.the results of our study indicate that different combinations of concurrent inflammation of the liver, pancreas, and intestine do exist in cats. it also appears that these conditions may be subclinical in many cases. several potential pathogens are found in feline faeces, ranging in significance from incidental to pathogenic. treatment recommendations are based on accompanying clinical signs. real time pcr has enabled rapid screening of small quantities of faeces for potential pathogens, with high sensitivity and specificity. co-carriage of feline faecal pathogens may reflect a symbiotic relationship or related pathogenesis, in which case identification of common cocarriage patterns may influence treatment decisions and prognosis. the primary objective of this study was to identify co-carriage of selected feline faecal pathogens. secondary objectives were to evaluate the prevalence of individual pathogens in feline faeces, and their association with pedigree status, and a history of diarrhoea. results of a commercial -way feline diarrhoea realpcr tm panel (idexx reference laboratories, uk) from june to january were evaluated. real time pcr was performed for tritrichomonas foetus, giardiaspp., cryptosporidiumspp., toxoplasmagondii, salmonellaspp., clostridium perfringens enterotoxin a gene, feline coronavirus, and feline panleukopenia virus. additional data was recorded when available, including age, gender, breed, and history of diarrhoea. weak or borderline positive results and those from pooled faecal samples were excluded. associations of the carriage of pairs of pathogens were evaluated with a chi-squared test, statistical significance set at p < . .results of pcr panels were evaluated. the prevalence of faecal pathogens was . % (tritrichomonas foetus), . % (giardia), . % (cryptosporidium), . % (toxoplasma gondii), . % (salmonella), . % (clostridium perfringens), . % (feline coronavirus), and . % (feline panleukopenia virus).salmonella was the only faecal pathogen significantly associated with a history of diarrhoea. faecal samples from pedigree cats were significantly more likely than dsh to be positive for tritrichomonas foetus, giardia, clostridium perfringens and feline coronavirus.significant co-carriage was identified for feline coronavirus with all other pathogens except salmonella. there was significant co-carriage of tritrichomonas foetus with clostridium perfringens and giardia, and also for giardia with panleukopenia virus, cryptosporidium, and clostridium perfringens. finally, there was significant co-carriage of cryptosporidium with clostridium perfringens, panleukopenia virus with cryptosporidium, and toxoplasma gondii with salmonella.in conclusion there was a moderate to high prevalence of all feline faecal pathogens tested except toxoplasma gondii and salmonella. positive pcr results were more likely in pedigree cats, and salmonella was associated with a history of diarrhoea. significant co-carriage of faecal pathogens was common, possibly reflecting common environmental risk factors, a shared pathogenesis, or a symbiotic relationship. mycoplasma spp have been identified as causative pathogen in feline lower airway disease and are not thought to colonize the lower airways of clinically healthy cats.to challenge this hypothesis, domestic shorthair cats aged - years (median, . years), without signs of respiratory disease, housed in a shelter, underwent transoral lower airway washing with sterile saline under general anaesthesia. retrieved bronchalveolar lavage fluid (balf) was subjected to microbiological and cytological analysis.during preanaesthetic clinical examination, only minor alterations were discovered: tartar in , conjunctivitis in , flea infestation in and dirty auditory canals in cats. one animal each showed dandruff, corneal ulceration and a heart murmur / .balf-cultures of animals ( . %) were positive for mycoplasma spp. . % of examined cats had mycoplasma felis in their balf (light growth in , moderate growth in and heavy growth in animals), % ureaplasma felinum/cati (light growth in , moderate growth in cases), . % mycoplasma gateae (light growth in , moderate growth in and heavy growth in cat) and . % mycoplasma feliminutum (light growth in animal). using aerobic bacterial culture, pasteurella multocida ( . %), a-haemolytic streptococci ( . %), haemolytic e. coli ( . %) and acinetobacter iwoffii ( . %) were detected, with only samples ( . %) yielding negative results.median balf-total nucleated cell count was /ll (range, - ) with a median mononucleated cell percentage of %, a median neutrophil fraction of . % and a median eosinophil proportion of . %. only samples were neutrophil-dominated ( . and . %), indicating purulent inflammation. both specimens were positive for mycoplasma felis ( light growth, heavy growth), and one showed moderate growth of pasteurella multocida. like all of the animals included in this study, the corresponding cats remained clinically healthy during a -week followup period.balf-samples showed neither cytological nor microbiological signs indicative of upper airway contamination.in contrast to earlier studies, we conclude that -at least in cats housed in shel-ters and subjected to high infection pressure -in addition to bacteria like pasteurella spp, streptococcus spp and e. coli, mycoplasma spp can occur in the feline lower airways without causing respiratory signs. inflammasomes are intracellular multi-protein complexes that coordinate the maturation of interleukin (il)- b and il- in response to pathogens and metabolic danger signals. both cytokines are vital for the maintenance of the intestinal homeostasis and have been linked to chronic intestinal inflammation in humans. both il- b and il- are produced as inactive proforms and undergo subsequent maturation through cleavage into their active forms by caspase (casp)- , which is in turn activated by the inflammasome complex. the best characterized inflammasome subtype in human inflammatory bowel disease (ibd) is nlrp , which seems to be crucial for the regulation of intestinal homeostasis. defective nlrp signaling has been suggested to contribute to ibd. additionally, il- b, il- and casp- are upregulated on the mrna and protein level in human ibd. so far, no study has investigated the role of the inflammasome and respective down-stream cytokines in canine ibd. thus, the goal of the current study was to investigate the expression of inflammasome components in duodenal tissues from dogs with ibd compared to healthy controls. rna extraction from endoscopic biopsies (ibd group n = , control group n = ) and reversetranscriptase quantitative pcr was performed in a sybrgreenbased assay using specific primers for the following canine genes: nlrp , casp- , il- b and il- . a -fold dilution of plasmid controls for each gene was used to assess assay efficiency. relative quantification of gene expression was performed using three reference genes (gapdh, sdha, tbp). comparison between groups was performed using mann whitney u tests (graph pad prism ). significance was set at p < . . eight samples ( ibd, controls) had to be excluded due to poor cdna quality (inadequate expression of reference genes). when comparing the remaining samples in both groups, casp- (p = . ) and nlrp (p < . ) expression was significantly lower in ibd dogs than controls. in contrast, il- b (p = . ) and il- (p = . ) expression was not different between groups.down-regulation of nlrp and casp- could be part of a negative feedback loop in the pro-inflammatory environment in ibd. alternatively, this could represent general disturbances in intestinal homeostasis or failure to up-regulate "danger-signaling pathways"in inflammation. final conclusions might be difficult to draw without matching protein data, as assessing il- b and il- mrna levels cannot give insight into the ratio of "pro-cytokines"to active cytokines. thus, investigating il- b and il- protein content of canine ibd tissues is warranted in the future. the trefoil factor family (tff) comprises a group of small peptides produced in goblet cells, which are crucial for epithelial restitution and maintenance of tight junction function in the gut. in humans with inflammatory bowel disease (ibd), tff expression is up-regulated, which is thought to represent an unspecific repair mechanism. however, tffs have also been shown to be involved in the local control of disease in rodent models and in humans. so far, there has been no study investigating tff expression in the canine intestine. thus, the goal of this study was to assess tff expression in gastrointestinal tissues from dogs with ibd and healthy dogs. rna was extracted from endoscopic duodenal (ibd n = , healthy controls n = ) and colonic biopsies (ibd n = , controls n = ) and cdna generated. quantitative reverse-transcription pcr was performed for canine tff and tff in a sybr-green-based assay. a -fold dilution of plasmid controls for each gene was used to assess assay efficiency. relative quantification of gene expression was performed using three reference genes (gapdh, sdha, tbp). for statistical analysis, significance was set at p < . . overall, tff expression was significantly different across groups (kruskal wallis p < . ): in control dogs, tff expression was higher in the colon than in the duodenum (mann whitney p < . ). when comparing ibd cases to controls, duodenal tff was significantly up-regulated (mann whitney p = . ). tff expression was not different across groups (kruskal wallis p = . ). however, separate analysis of the intestinal location showed significant down-regulation of tff in the colon of ibd dogs compared to controls (t-test p = . ).this study demonstrates evidence for dysregulation of tff gene expression in canine ibd. a lack of tff could contribute to defective epithelial barrier function, causing "leakiness"of tight junctions. this increased intestinal permeability could lead to an increased antigenic load from microbes or food antigens, which could perpetuate faulty immune recognition of microbe-associated molecular patterns, thus leading to increased intestinal inflammation. up-regulation of tff in the colon could in turn signify a compensatory repair-mechanism in inflammation. further investigation of tff gene or protein expression is warranted in canine ibd. canine inflammatory bowel disease (ibd) is thought to be partially caused by an aberrant immune response towards the intestinal microbiome. in humans and mice, administration of probiotics can alleviate ibd severity and/or prevent relapse by induction of a more "tolerant"microenvironment. the aim of this study was to investigate the effect of probiotic enterococcus faecium ncimb e (ef) on gene expression and protein production in canine duodenal biopsies. samples from healthy beagles and ibd dogs were cultured ex-vivo with ef ( x cfu/ml) or sterile nutrient broth/ pbs (negative control) for hours. rna extraction from biopsies and reverse-transcriptase quantitative pcr was performed in a sybrgreen-based assay using specific primers for the following canine genes: tlr , tlr , tlr , tlr , il- a, il- , il- , tgfb, il- , ifnc and tnfa; using a -fold dilution of plasmid controls for each gene to assess assay efficiency. relative quantification of gene expression was performed using three reference genes (gapdh, sdha, tbp). protein content of ifnc, il- a and tnfa was measured in culture supernatants using commercially available canine-specific elisas. a linear mixed model for each genewith disease group and treatment set as fixed parameters -was chosen for statistical analysis using spss software. significance was set at p < . .no significant interaction between disease group and treatment was observed for any gene. only expression of il- was significantly increased in ibd dogs compared to healthy dogs (p = . ) in unstimulated samples. all other significant differences were independent of disease group, but depen-dent on treatment with ef. expression of the following genes was reduced by ef treatment: tlr (p = . ), tlr (p = . ), tlr (p = . ), tgfb (p = . ), ifnc (p = . ) and tnfa (p = < . ). no significant amounts of ifnc, il- a or tnfa protein were detected in culture supernatants.this is the first study demonstrating a profound effect of ef treatment on gene expression in ex vivo cultured canine duodenal biopsies. down-regulation of several genes of innate immune receptors and pro-inflammatory cytokines was observed, with the most significant effect on suppression of tnfa expression. effects were seen both in healthy and in ibd dogs. whether this translates to a beneficial effect in a clinical situation needs further investigation. recent molecular studies have revealed a highly complex bacterial microbiota in the intestine of cats. there is mounting evidence that microbes play an important role in the pathogenesis of chronic enteropathies, as compositional changes of the intestinal bacterial ecosystem have been associated with chronic intestinal inflammation in humans and dogs. the aim of this study was to characterize the bacterial microbiota in cats with chronic enteropathies.fecal samples were obtained from healthy cats (n= ) and cats with histologically confirmed chronic enteropathies (n= ). the bacterial composition was analyzed by massive parallel s rrna gene -pyrosequencing (yielding , sequencing tags), and selected quantitative pcr assays. differences in microbial community structure between healthy and diseased cats were assessed by the phylogenetic unifrac distance method, followed by analysis of similarity (anosim) of the distance matrix. differences in bacterial groups between the disease groups were analyzed using mann-whitney u tests. the resulting p-values were corrected for multiple comparisons using the benjamini & hochberg's false discovery rate. an adjusted p< . was considered for statistical significance.the unifrac distance metric indicated no significant clustering according to disease status. however, the relative proportions of sequences belonging to bacteroides spp. were significantly decreased in the cats with chronic enteropathies compared to the healthy cats (p= . ). the employed qpcr assays confirmed the sequencing results and also showed a significant decrease in proportions of bacteroides spp. in the diseased cats. in addition, qpcr revealed also a significant decrease in turicibacter spp. in cats with chronic enteropathies compared to the healthy cats (p= . ). no significant differences in diversity indices were observed between healthy and diseased cats.in conclusion, the here used molecular approach revealed significant reductions in bacteroides and turicibacter spp. in cats with chronic enteropathies. future studies are necessary to evaluate if these microbial changes correlate with functional changes in the intestinal microbiota. campylobacter spp. represent a common cause of gastroenteritis in humans with c. jejuni and c. coli considered responsible for the majority of clinical cases. many human diagnostic laboratories use the prospect ® campylobacter microplate assay (eia) as a sole or screening test for detection of c. jejuni and c. coli as this technique is easier, faster and cheaper than campylobacter spp. culture. c. upsaliensis can cause a diarrhoeal illness similar to that caused by c. jejuni in humans but methods routinely used for c. jejuni and c. coli detection, including the eia, are not optimised or have not been evaluated for the detection of other campylobacter species. c. upsaliensis and c. helveticus have been more commonly isolated from dogs and cats than c. jejuni and c. coli in most studies. campylobacter spp. from pets are potentially zoonotic while the pathogenicity in pets remains uncertain. the significance of c. upsaliensis and c. helveticus infections may be underestimated in human medicine due to detection methods applied.the aim of this pilot study was to assess if the eia detects c. upsaliensis and c. helveticus isolated from different pets in spiked human clinical samples. in addition, the ability of eia to detect campylobacter spp. in dog and cat faeces was assessed. ten clinical human faecal samples and two healthy dog and cat faeces confirmed negative by eia were tested by a range of culture methods to exclude presence of campylobacter spp. dilutions of eight c. upsaliensis, five c. helveticus cultures, and one c. jejuni and c. coli culture, all pcr confirmed, were added to aliquots of faecal samples to obtain a range of viable bacteria from to cfu/ml. eia was performed following the manufacturer's instructions and in duplicates. fifty out of samples tested positive by eia at various dilutions: / c. upsaliensis, / c. helveticus, / of each c. jejuni and c. coli. detection limit varied between the isolates and was lower in watery than semi-solid faeces.in conclusion, eia detected c. upsaliensis and c. helveticus in spiked faeces from human clinical cases and all campylobacter spp. tested in healthy pets. eia should not be used as a sole detection method and culture methods selected following a positive eia result should enable detection of a wide range of campylobacter spp. in addition, eia may be a useful test in pets to rule out campylobacter spp. infection and in epidemiological investigations. high-resolution manometry (hrm) is the gold standard for the evaluation of functional esophageal motility disorders in humans. no information on this non-invasive technique is available in dogs. the aims of this study were to evaluate the feasibility of hrm in dogs, generate first normal values, and to examine the influence of a standard sedation on the esophageal pressure profile.the study population consisted of healthy adult beagle dogs (mean bw . kg), the study protocol was approved by the local ethics committee. hrm was performed in sitting position after a -h fast, lidocaine jelly at % was used as a lubricant. a solid-state catheter (sierra scientific, . mm diameter) with circumferential pressure sensors spaced cm apart was inserted intranasally, measurements were started after minutes adaptation time. real-time pressure imaging during catheter intubation enabled accurate placement. each manometric protocol included water and canned food bolus swallows. the procedure was repeated minutes after standard im sedation with acepromazine and buprenorphine. data were analysed using manoview software and results (awake vs. sedated) were compared using the wilcoxon test. statistical significance was set at p< . .hrm could successfully be performed in / dogs. reasons for unsuccessful examinations were: defence reactions ( ), inability to pass through the ventral meatus ( ), reverse sneezing ( ). upper esophageal sphincter (ues) characterisation comprised: baseline pressure, residual pressure (nadir of the ues relaxation during swallowing), and relaxation duration. tubular esophageal function characterisation comprised: peristaltic con-tractile integral (pci; amplitude x duration x length of the contraction wave) and the bolus transit time (btt). lower esophageal sphincter (les) characterisation comprised: baseline pressure and residual pressure (lowest continuous second mean les pressure relative to intragastric pressure during swallow induced relaxation).the median values for water/food bolus swallows in awake (sedated) dogs were calculated i) ues: baseline pressure . mmhg ( . ), residual pressure - . / . mmhg (- . / . ) and relaxation duration . / ms ( . / ) ii) tubular esophagus function: pci . / mmhg-cm-s ( . / . ) and btt . / . ms ( . / . ) iii) les: baseline pressure . mmhg ( . ), residual pressure . / . mmhg ( . / ). significant differences were found for the ues relaxation duration (water) (p= . ) and btt (food bolus) (p= . ).in conclusion, hrm is a feasible technique for the evaluation of esophageal function in dogs. patients that require sedation can still be examined, however at this point it is not clear if sedation would affect the assessment of motility disorders in dysphagic dogs. in humans ambulatory intraesophageal ph-monitoring utilizing the bravo ® capsule is the standard test for establishing pathological gastroesophageal reflux (ger). this technique not only provides information on esophageal acid exposure, but is also able to assess symptoms associated with ger. in dogs ger is poorly understood and it is not clear if ger actually represents a clinically relevant problem.the goals of this study were to examine the canine esophageal ph milieu in health and to examine esophageal ph in dogs presenting with signs commonly attributed to ger in the veterinary literature.thirteen client-owned dogs (cod) of various breeds (median bw . kg, . - ; median age y, - ) were included. clinical signs ultimately leading to ph-monitoring comprised: lipsmacking ( ), repeated swallowing motions ( ), chronic vomiting ( ), cough ( ), retching ( ), regurgitation ( ), sudden discomfort ( ), excessive surface-licking ( ), ptyalism ( ), presumed postprandial pain ( ), refusal to eat despite interest ( ), history of esophageal foreign bodies ( ), halitosis ( ) . each dog showed a median of ( - ) signs, dogs had additional diarrhea. six healthy beagles (median bw . kg, median age . y) with unremarkable gastroduodenal evaluation served as controls (c). no prior antacid or prokinetic treatment was allowed. the capsule was endoscopically placed cm above the lower esophageal sphincter, ph data were transmitted every s to a receiver attached to the dog's collar. owners were instructed to press the individually predefined symptom-buttons on the receiver whenever indicated, and not to change the daily routine. data were analysed using the rapidph ® software, reflux was defined as a single ph-measurement < . results between groups were compared using non-parametric tests.the median ph-monitoring period (cod/c) was . / . h. the following parameters (median, range for cod/c) were evaluated: number of refluxes: ( - )/ . ( - ), number of longest (> min) reflux: ( - )/ ( - ), duration of longest reflux (min): ( - )/ . , and fraction time ph < (%): . ( . - . )/ . ( - . ). there were no differences between groups. the median number of button pushes was ( - ), dogs had reflux-positive pushes ( . , , and . % of pushes). mild distal esophagitis was noted in dog. final diagnoses were: food-responsive ibd ( ), steroid-responsive ibd ( ), allergic skin disease ( ), chronic laryngotracheobronchitis ( ), muscular dystrophy ( ) .dogs presenting with historical and clinical signs interpreted as ger may not have relevant reflux episodes. considering normal values established in humans, none of the dogs would have been classified as abnormal. the aim of the present study was to evaluate the changes of some biochemical and ultrasonographic (us) parameters in a group of dogs with naturally occurring acute pancreatitis (ap) during the therapeutic follow-up.dogs with clinical signs and abdominal us findings suggestive of ap associated with increased serum canine pancreatic lipase (cpl) activity were included into the study. in these dogs, the serum concentration of c-reactive protein (crp), amylase and lipase were also measured. severity indexes were established to semi-quantitatively evaluate the severity of clinical and us findings. in particular, a clinical score ( - ) for each of the following clinical parameters was given: presence and frequency of vomiting, appetite and general condition; an us score ( =normal, =abnormal) was assigned per each of the following parameters: pancreas (echogenicity, volume, echotexture and echogenicity of the mesentery), gastrointestinal tract, biliary ducts, lymph nodes and abdominal effusion (total score - ). all dogs were treated with fluid therapy, ampicillin-sulbactam, mg/kg iv q h, buprenorphine, . mg/kg q h, and, if needed, maropitant mg/kg sc q h. the two severity scores, serum crp, amylase and lipase concentrations were measured at diagnosis (t ) and after (t ), (t ), and (t ) days, and at discharge (td) and week after discharge (td ).nine client-owned dogs were included with a median (range) age of years ( - years). median (range) clinical and us scores were ( - ) and . ( - ), respectively, at t , and ( - ) and ( - ), respectively, at td . a significant, positive correlation was found between the clinical and us score (p< . , r= . ). the median (range) serum concentration of crp (mg/dl), amylase (u/l) and lipase (u/l) was . ( . - . ), , ( - , ) and ) , respectively, at t , and . ( . - . ), ( - , ) and ( - ), respectively, at td . on admission, serum crp, amylase and lipase levels were increased in %, %, and % of dogs, respectively while they were increased in %, . % and % of dogs, respectively, at td . serum crp and amylase, but not lipase, concentrations decreased during the follow up and were significantly (p< . ) lower at td compared to t .results suggest that us findings, and crp and amylase concentrations are correlated with the recovery from the ap. further studies are warranted to evaluate the usefulness of these parameters in the follow-up of ap in a wider population of dogs. enzyme replacement therapy is the mainstay therapy for exocrine pancreatic insufficiency (epi) in dogs. 'enteric-coated' preparations have been developed to protect the enzyme from degradation in the stomach, but their efficacy has not been critically evaluated. the hypothesis of the current study was that enteric coating would have no effect on the efficacy of pancreatic enzyme supplements for dogs with epi.thirty-eight client-owned dogs with naturally occurring epi were included in this multicentre, blinded, randomised controlled trial. dogs received either an enteric-coated enzyme preparation (test group) or an identical preparation without the enteric coating (control group) over a period of days.there were no significant differences in baseline characteristics between test and control treatment groups. body weight and body condition score increased in both groups during the trial (p< . ) but the magnitude of increase was greater for the test treatment compared with the control treatment (p< . ). by day , mean body weight increase was % ( % confidence interval - %) in the treatment group and % ( % confidence interval - %) in the control group. the dose of enzyme used increased over time (p< . ) but there was no significant treatment group difference at any time point (p= . ). clinical disease severity score decreased significantly over time for both groups (p= . ) and no significant difference was noted between groups (p= . ). no significant adverse effects were reported, for either treatment, for the duration of the trial.adding an enteric coating to a pancreatic enzyme supplement conveys a therapeutic advantage, when treating dogs with epi. when liver injury is caused by toxins that inhibit hepatocyte replication, or when the proliferative potential of hepatocytes is exhausted due to the chronic condition of the disease, hepatocyte-dependent liver regeneration is strongly impaired. in this situation another cell compartment is activated to regenerate the liver: the hepatic progenitor cell compartment. bipotent hepatic progenitor cells (hpcs) can differentiate into hepatocytes or cholangiocytes. not only are hpcs activated during severe liver disease, they may well be the cells of origin for subtypes of hepatocellular carcinoma (hcc).the polycomb group protein bmi is involved in murine hpc activation and has prognostic relevance in hcc. therefore we investigated the expression of bmi in canine liver diseases including hcc. functional consequences of deregulated bmi expression are included to reveal its feasibility in liver regenerative medicine.immunohistochemistry (ihc) and laser microdissection followed by gene expression analysis were used to investigate the expression of bmi in activated hpcs and hcc. to elucidate the role of bmi in hpcs, in vitro gene silencing experiments followed by gene expression analysis, western blot analysis, and a proliferation assay (edu-incorporation) were performed in the human hpc-like cell-line heparg.ihc and gene expression showed a strong nuclear expression of bmi in activated hpcs in canine liver diseases. keratin is a marker for hpcs and cholangiocytes, and high expression is associated with poor prognosis in hccs. bmi staining was more intense in highly malignant hepatocellular carcinomas positive for keratin compared to keratin negative hccs. bmi -silencing in vitro studies revealed a significantly reduced hpc proliferation and suggested a role of bmi in differentiation (gene expression) of hpcs.these results indicate that bmi is expressed in activated hpcs in all liver diseases tested. bmi is needed for proliferation of hpcs in vitro and is potentially involved in hpc differentiation. expression of bmi in hccs suggests a role in tumour development, potentially due to a persistent activation of hpcs. therefore, enhanced activation of bmi will lead to more progenitor cells (beneficial) but most likely also to more aggressive hcc with a hpc-signature. hepatic progenitor cells (hpc) are bipotential, forming cholangiocytes or hepatocytes. as a result, they express markers of both cell types. various markers have been used to select for hpc's yet there is no specific cell marker. cd , a transmembrane glycoprotein, has been identified as a organ stem cell marker and cancer stem cell marker, and used to select hpc's in rodents and humans. canine hpc have been shown to have gene expression of cd . this study aimed to assess if cd selection could enrich for canine hpcs.a wedge of liver from a healthy dog euthanased for behavioural reasons was digested by collagenase perfusion. hepatocytes were removed by pelleting at g for mins. the remaining non-parenchymal cells (npc) were then pelleted at g for mins. magnetic assisted cell sorting (macs) for cells positive and negative for cd was performed according to the manufacturer's instructions. cd positive and negative npc were cultured on bovine type i collagen-coated plates with hepatocyte culture media (lonza). cell morphology was monitored. rna was extracted and cdna produced from cell pellets immediately after separation and also after weeks of culture. relative gene expression of cd , cholangiocyte marker keratin (k ) and hepatocytes markers k , albumin and cyp a was performed using b mg and rpl as reference genes.the cd positive fraction was % of the npcs. morphologically, cd positive cells formed more colonies of cells with a small cytoplasm to nucleus ratio, which then transformed to hepatoblastic appearance while the negative cells produced more fibroblastic cells. compared to cd negative cells, initially cd positive cells showed a -fold increase in cd , and . -fold increase in cholangiocyte marker k as well as modest increases in hepatocyte markers albumin and cyp a and a modest decrease in k . after weeks culture, positive cells expressed more albumin, cyp a , k & k compared to cd negative cultures. cd expression had reduced to a fold increase after culture.the cd positive fraction expressed cholangiocyte and hepatocytes markers compared to negative cells initially after sorting. after weeks culture, cd positive cells formed hepatocyte-like colonies, as well as continuing to have greater hepatocyte and cholangiocyte gene expression. cd expression decreased, consistent with differentiation.a large percentage of the npc were positive for cd . cd sorting also labels haematopoietic and mesenchymal stem cells therefore subsequently using facs for other markers may further enrich for specifically the hpc compartment. until now the hepatic neoplasms in dogs are classified as hepatocellular adenomas and carcinomas, cholangiocellular adenomas and carcinomas, mixed hepato-and cholangiocellular carcinomas and hepatic carcinoids. over the past decade, many advances have been made in the characterization of primary liver tumours in man. this knowledge has resulted in a proposal for a new morphological and immunohistochemical classification of primary liver tumours which facilitates the diagnosis and categorization of these tumours including their aggressiveness and prognosis. the purpose of this study was to investigate the presence and relative incidence of the various morphological types of primary hepatic neoplasms in the dog and to determine whether the new human classification also can be applied to canine hepatic neoplasms. for this study canine primary liver tumours were examined histologically and classified using several immunohistochemical markers including keratin(k) (hepatic progenitor (stem) cell/bile duct epithelium marker), heppar- (hepatocyte marker), ema (muc ; mucine producing biliary epithelium marker), pcea (canalicular, ductular, and bile duct epithelium marker), nse and chromogranin-a (neuro-endocrine markers). in addition, the tumours were graded according to cellular and nuclear pleiomorphism and mitotic index (grade: - ) and staged with respect to absence or presence of invasive growth, intrahepatic and/or distant metastases (stage: - ). of the primary liver tumours, had a hepatocellular origin ( %). these hepatocellular tumours could be subdivided in hepatocellular tumours with < % positivity for k ( %) and tumours with > % positivity for k ( %). the hepatocellular tumours with > % positivity for k were histologically poorly differentiated and often revealed lymphatic and vascular invasion in portal tracts and all showed intrahepatic and/or distant metastasis. in contrast the hepatocellular tumours with < % positivity for k were almost always well differentiated and well demarcated and did not have evidence for vascular invasion, intrahepatic and/or distant metastasis. eight of the tumours were from cholangiocellular origin ( %). all of the cholangiocellular tumours were poorly differentiated (grade and ) and showed intrahepatic and/or distant metastases. the third group of primary liver tumours had neuroendocrine characteristics, consisted of five tumours ( %) and were classified as carcinoids. they all were histologically poorly differentiated and had intrahepatic and/or distant metastases. in conclusion, the morphological types of the primary hepatic neoplasm in the dog, including their aggressiveness and prognosis, are highly comparable to the situation in man. these finding indicate that the new human classification of primary hepatic neoplasms is applicable in the dog. doberman hepatitis (dh) is a rare inflammatory liver disease characterized by female preponderance, elevated serum transaminase activity and increased hepatic copper content. immune system involvement is suggested by the presence of lymphocyte infiltration, female predisposition, abnormal expression of major histocompatibility complex (mhc) class ii antigens by hepatocytes and association of homozygosity for the mhc ii risk allele drb * of the dog leukocyte antigen system genotype.we investigated the possibility that autoantibodies are involved in dh. serum samples from subclinical and clinical dh patients and clinically healthy control dobermans were included in an elisa assay for detection of igg autoantibodies against histones (aha). the cut-off value for positivity in the anti-histone elisa was . , determined using the mean absorbance + sd of samples from healthy controls. the values for subclinical dh cases (mean ± sd: . ± . ; % confidence interval (ci) . to . ) and clinical dh cases (mean ± sd: . ± . ; %ci . to . ) were both significantly higher than values for controls (mean ± sd: . ± . ; %ci . to . ; p< . ). no seropositivity was noted in the control group.autoantibodies are the serological hallmark of autoimmune disease. normally, the immune system has an extraordinary capacity for preventing self-antigens to stimulate an inflammatory reaction. the presence of autoantibodies is therefore the consequence of a breakdown or failure of b-cell tolerance toward the corresponding autoantigens. since the appearance of aha indicates possible presence of autoimmunity, our results support the assumption that dh is an autoimmune disease. man's best friend (canis lupus familiaris) is an ideal model organism for a broad variety of naturally occurring diseases. dog breeds have a population structure suited for genetic studies of complex disorders. haploblocks within dog breeds extend up to times longer distances than in human populations. related dog breeds often share phenotypes and causative mutations, allowing for finemapping and validation. the inbreeding and bot-tlenecks of dog populations has led to an increased incidence of genetic diseases which remained incidental in the panmictic human populations.extrahepatic portosystemic shunts (ehpss) are large abnormal venous blood vessels connecting the portal vein with a major systemic vein. it results in almost complete diversion of the portal blood past the liver, leading to lack of liver function and liver growth, and hepatic encephalopathy due to brain neurotransmitter dysfunctions. there are only reported human cases, whereas the disease is widespread in dogs. shunts are more frequently diagnosed in purebred dogs than in crossbred dogs. the inheritance is complex without sex effect.a genome wide association studied with k snps was peformed on cairn terrier cases and controls of the same breed. after quality control cases and controls were analyzed in snps. allelic association was calculated with plink and grammas of the genabel package was used to correct for genetic kinship. several chromosome regions were detected with close to significant association. overlap between the two analysis methods was found on three genomic regions covering about . mb. finemapping was conducted by analyzing more snps in a larger group of cairn terriers and in cases and controls of other breeds. the finemapping resulted in two genomic regions covering and kb that were associated with significant pvalues. comparison of haplotypes of cases from different breeds confirmed the association signals. the regions of interest are analyzed by ngs of cases and controls. historically, doxycycline has been the first line drug for the treatment of canine monocytic ehrlichiosis (cme). some studies have shown that dogs may remain carriers despite doxycyxline treatment, therefore investigation for other anti-ehrlichial agents may be warranted. rifampin was suggested as a promising alternative for the treatment of cme, though its efficacy in clearing the infection has not been thoroughly evaluated. the purpose of this study was to assess the efficacy of rifampin in achieving clinicopathological recovery and clearing the ehrlichial infection from the blood and other tissues in dogs with experimentally-induced acute cme. of the purpose-bred beagle dogs that were included in the study, dogs with acute experimental cme were treated with rifampin ( mg/kg, sid, po, for weeks), infected dogs received no rifampin (infected controls) and two dogs served as uninfected controls. fourteen days after the completion of rifampin treatment, dexamethazone ( . mg/kg, iv, once) was given to the rifampin-treated dogs. clinical score, platelet counts, anti-e. canis immunofluorsecent antibody titers (ifa) and polymerase chain reaction (pcr) detection of e. canis-specific deoxyribonucleic acid in the blood, bone marrow and spleen aspirates were evaluated between the treated and untreated infected dogs on day post-inoculation (pi) (start of rifampin), on day pi (end of rifampin) and day pi (end of post-treatment monitoring). by day pi, all infected dogs became clinically ill and thrombocytopenic, seroconverted and became pcr-positive in at least one tissue. the median clinical score and ifa titers did not differ between the treated and untreated dogs at any of the three time points. Μedian platelet counts were significantly higher in the treated compared to the untreated infected dogs on day pi ( , /ll versus , , p= . ) and day pi ( , /ll versus , /ll, p= . ) (two-sample wilcoxon rank-sum [mann-whitney] test). on day pi, treated and untreated infected dogs were pcr-positive, while on day pi, treated and untreated infected dogs remained pcr positive in at least one of the tissues tested. as administered in this study, rifampin hastened hematological recovery of the infected dogs, but was inconsistent in clearing the experimentally-induced acute e. canis infection. acute phase proteins (app) are considered one of the hallmarks of the inflammatory response. among their major functions, apps seem to modulate innate immune system efficiency. in cats, serum amyloid a (saa) and a glycoprotein (agp) are two major positive apps that are increased during inflammation. this rise is presumed to be secondary to various cytokines that are involved in the innate inflammatory response.recombinant feline interferon-x (rfeifn-x) is an immune-modulator drug that is commonly used in cats naturally infected with retroviruses, namely feline immunodeficiency virus (fiv) and feline leukemia virus (felv). several studies have been performed to clarify the clinical benefits of rfeifn-x therapy in naturally infected fiv and/or felv cats. our group has previously described that c-reactive protein (crp) increased in naturally retroviralinfected cats under rfeifn-x therapy. however, the role of apps such as saa, agp and crp in the innate immuneresponse, remains unknown.the aim of this study was to evaluate saa, agp and crp serum levels in naturally retroviralinfected cats under rfeifn-x therapy.sixteen naturally retroviral infected cats ( fiv, felv and co-infected fiv/felv stray cats) housed in a lisbonanimal rescue shelter were submitted to rfeifn-x therapy. the licensed protocol was used: courses of mu/kg sc administered once daily for days, beginning on days , and . blood samples were collected for saa, agp and crp quantification before, during and after treatment (at d , , , ) . saa was quantified by elisa (phase saa, tridelta) and agp was determined by single radial immunodifusion (agp,tridelta). feline crp was quantified by elisa (kamiya biomedical company).app serum levels were compared before and after rfeifn-x therapy. a statistically significant increase of saa and agp (p= . and p= . respectively -friedman test) was observed at d in comparison to d . these findings corroborate the significant increase of crp serum levels previously described (p < . -friedman test).all the apps tested behaved similarly, showing an evident increase in their serum values after rfeifn-x therapy. these results suggest a possible immune modulation effect induced by rfeifn-x which seems to maximize the efficiency of innate immune response. further studies correlating these findings with the cytokine profile will extend our knowledge about the efficiency of rfeifn-x therapy in naturally retroviral infected cats. canine theileriosis is a tick-born disease caused by protozoan parasites of the genera theileria that has been associated with anaemia and/or thrombocytopaenia. the clinical manifestation of this disease in the dog is poorly described. this disease of emerging importance has been diagnosed in several dogs, emphasizing the need to elucidate the specific pathogenic species and characterize the clinical manifestations of the disease.this retrospective study describes the clinical characteristics, diagnostic tests, treatment and outcome of six client owned clinically ill dogs diagnosed with canine theileriosis at the onderstepoort veterinary academic hospital in south africa during - . canine theileriosis was diagnosed by polymerase chain reaction (pcr) on whole-blood followed by a reverse line blot (rlb) hybridization assay. other tickborn diseases and neoplastic conditions were excluded.the most common clinical findings were pale mucous membranes ( / dogs), lethargy ( / dogs) and oral bleeding ( / dogs). all dogs had thrombocytopaenia with a median of . x /l (range: - ) and / dogs had anaemia with a median haematocrit of % (range: - ). the anaemia was regenerative in / dogs and non-regenerative in / dogs. one dog was positive on an in saline auto-agglutination test. bone marrow cytology and core biopsies were performed in two dogs with severe non-regenerative anaemia and showed myelofibrosis. theileriasp. were detected in four dogs and theileria equi in two dogs. imidocarb dipropionate was administered in all dogs as treatment of choice for the theileriosis. five dogs that received the complete treatment achieved clinical cure. pcr post-treatment was performed in three dogs and was negative. prednisolone and azathioprine was administered in all dogs for suspected immune-mediated haematological disorders secondary to the theileriosis. one dog was euthanased one week after diagnosis.canine theileriosis should be considered a differential diagnosis for dogs with thrombocytopaenia and/or anaemia in endemic tick-born disease areas. pcr is a versatile tool for diagnosis and treatment monitoring in theileriosis. in our study, imidocarb dipropionate was effective in sterilizing the parasitic infection. the bleeding tendency seen in the theileriosis cases is most likely secondary to the thrombocytopaenia and/or concurrent thrombocytopathy as described in canine ehrlichiosis. further studies are required to determine the possible links between thrombocytopaenia, anaemia and myeloproliferative disorder observed in canine theileriosis. a.l. proksch , s. unterer , u. truyen , r.s. mueller , k. hartmann . clinic of small animal medicine, lmu university of munich, munich, germany, institute for animal hygiene and veterinary public health, university of leipzig, leipzig, germany canine parvovirosis still is a common and severe disease, especially in puppies, and there is a need for drugs that can decrease severity of symptoms and accelerate recovery. the paramunity inducer pind-orf stimulates the immune system. therefore, the aim of the study was to investigate whether pind-orf, used as an additional drug, leads to faster recovery in dogs with parvovirosis.in total, dogs with parvovirosis were randomly assigned to two groups ( dogs each). in all dogs, infection with canine parvovirus was diagnosed by fecal elisa or polymerase chain reaction (pcr). also, all dogs had clinical signs. the study was performed as prospective placebo-controlled, double-blinded trial. all dogs received either pind-orf or placebo, and additional standardized treatment for canine parvovirosis. clinical scores, complete blood count, and serum protein and albumin were evaluated daily (day - ) and at day . viral shedding was measured on day , , , and by fecal pcr and virus isolation.no significant difference could be found in clinical scores, most blood parameters, and duration of virus shedding when comparing dogs receiving pind-orf and dogs receiving placebo. the only significant difference was an increase in lymphocyte counts observed in the pind-orf group. three dogs receiving placebo did not survive, but no significant difference between groups was determined concerning survival rate.in this study, no significant influence of the paramunity inducer pind-orf on the course of parvovirosis was determined. therefore, there is no indication to recommend pind-orf therapy in canine parvovirosis. the microscopic agglutination test (mat) is currently considered as the gold standard for the diagnosis of leptospirosis in dogs. however, it is not a perfect tool to predict the infecting serogroup as cross-reactions and paradoxical reactions do exist. the objective of this study was to determine the infecting serovar in dogs with a positive polymerase chain reaction (pcr) on one or more biological sample(s) by molecular typing and to compare the results with those provided by the highest mat titer for each dog.forty-one positive pcr biological samples ( urine samples, blood samples, one kidney sample and one cerebrospinal fluid -csf-sample) from dogs with a clinical suspicion of leptospirosis between and and at least one positive pcr on blood, urine, kidney or csf (kit taqvet pathogenic leptospira, lsi, france) were submitted to molecular typing. the genomospecies were first determined by partial rpob or partial s rrna gene sequencing. the serovar was further identified by multiple loci variable number tandem repeat analysis (mlva). three variable number tandem repeat (vntr) loci were used as markers for serovar identification.the genomospecies were leptospira interrogans sensu stricto in samples belonging to dogs, leptospira borgpetersenii in three samples (three dogs) and leptospira kirschneri in two samples (two dogs). interestingly, for one dog, we found a different species in the blood and in the urine. mlva could unequivocally determine infecting serovar in samples belonging to dogs: australis in one dog, muenchen in three dogs, fugis in one dog, canicola in two dogs, autumnalis in six dogs. for only two dogs among those , the infecting serovar identified by mlva belonged to the infecting serogroup identified by mat. for samples belonging to dogs, mlva failed to identify the infecting serovar because one or more marker(s) could not be amplified. for samples belonging to dogs, amplification profile suggested coinfection or infection with a serovar that had not been previously characterized by mlva. it must be noted that for some dogs, we were able to characterize the serovar in a single kind of sample.despite lack of sensitivity of mlva applied to clinical strains in our study, this rapid method could contribute to a better knowledge of the epidemiology of canine leptospirosis with some adaptations in the choice of markers. recently, two trichomonads have been identified in diarrheal stool of puppies: pentatrichomonashominis (ph) and tritrichomonas foetus (tf) [ ] . tf infection in cats results in a chronic colitis. however in dogs, pathogenicity of tf and ph has never been studied. so the objective of this study was to evaluate the association between trichomonads' infection in puppies and the presence of diarrhea.faecal samples were collected prospectively from puppies ( to weeks of age) in french breeding kennels. for each puppy, a rectal swab was performed and the fecal consistency was evaluated using a -point numerical scale ( = liquid feces, = hard feces) [ ]. detection of trichomonads was performed by using a commercially available system "in pouchtm tf test"(biomed diagnostics, oregon usa). evaluation of this media was done as already described [ ] . observation of motile trichomonads organisms in the system culture was considered as a positive result. nine positive culture systems, from one kennel, were frozen and single-tube nested pcr assays were performed on them in order to sequence and identify the trichomonads. . % ( / ) of the cultures were positive. . % of the puppies ( / ) had gastrointestinal troubles. puppies infected by trichomonads had significantly more digestive problems than puppies not infected ( . % vs . %; p < . ). ph was systematically isolated in the positives cultures.ph was the only trichomonads isolated in the nine culture system tested by single-tube nested pcr assay. these results show the poor specificity of the medium to distinguish tf from ph. this observation underlines the necessity to use pcr for precise identification of type of trichomonads. trichomonads are significantly associated to the presence of digestive disorders in puppies. some etiological studies on the subject (by considering the co-infection with fly snapping, fly-biting or jaw snapping are names given to a syndrome in which dogs appear to be watching something then suddenly leaping and snapping at it. fly-biting dogs are generally referred to neurologists or behaviourists because the abnormalities are often interpreted as focal seizures or as obsessive compulsive disorder (ocd). there is one published case report of fly biting presumably caused by dietary intolerance in a cavalier king charles spaniel.the aims of this case series were ) to characterize fly biting, ) perform a complete medical evaluation of dogs presented with fly biting, and ) evaluate the outcome of this behaviour following appropriate treatment of the underlying medical condition.seven dogs presented for fly-biting behaviour (fb) were assessed. all dogs underwent a complete medical and behavioural history as well as physical and neurological examinations. further investigation was performed if an abnormality was found on examination or if the history was suggestive of an underlying problem. based on clinical presentation, physical examination, neurologic examination, and laboratory test results, a diagnosis was made and a specific treatment recommended. response to treatment was monitored and evaluated following phone conversations with owners at day , and from onset of treatment. many gastrointestinal disorders were found in fb dogs which included eosinophilic and lymphoplasmacytic infiltration of the stomach and small bowel, delayed gastric emptying and gastroeosophageal reflux. complete resolution of the fb was observed in / dogs diagnosed and specifically treated for the underlying gastrointestinal (gi) disease. one dog was diagnosed with chiari malformation and responded temporarily to pain management.in conclusion, this prospective case series indicates that fly biting behaviour may be caused by an underlying medical disorder, gi disease being the most common. resolution of this behaviour is possible following specific treatment of the underlying medical condition. although several diagnostic tests have been developed to diagnose fip, there are still difficulties to differentiate between fip and diseases with similar clinical appearence in vivo. fip is an inflammatory disease, therefore, as in other inflammatory conditions, the concentrations of apps are expected to be increased. the aim of this study was to evaluate the ability of the apps to distinguish fip from other diseases.serum samples from cats presented with effusion were obtained for measurement of three apps. the diagnostic work-up was performed with respect to fip (jvim ; : ) and further diagnostic procedures were performed depending on the medical condition. cats were diagnosed as having fip and cats had another disease (cardiac , tumor , other ). serum amyloid a (saa) and haptoglobin (hp) were measured by automated analyser using feline validated assays (eiken and tridelta, respectively), alpha- -acid glycoprotein (agp) was measured using a manual method based on single radial immunodiffusion (tridelta).the median concentrations of saa, hp and agp were significantly (p< . ) higher in cats with fip compared to cats without fip (saa: . ; range . - . lg/ml; . ; range . - . lg/ml,), (hp: . ; range . - . mg/ml; . ; range . - . mg/ml,), (agp: ; range - lg/ml; , range - lg/ml).all major feline apps seem to be useful diagnostic tools to help in differentiating fip from other diseases. however, the concentration of apps in some diseases (septic processes, disseminated neoplasias) was as high as in fip. protothecosis is an uncommon disease of people and animals caused by prototheca spp., an unicellular aerobic algae. to date, about cases have been described in dogs in north america and australia. in europe, only cases have been documented in the last decades (poland, italy, greece and spain). affected dogs show signs referable to the gastro-intestinal tract, particularly the colon, but ocular and neurologic signs are also reported. the disease has an insidious onset, a slow progression and fatal course. the aim of the present study is to describe clinical and laboratory findings in cases of canine protothecosis from the north of italy, diagnosed between - . medical records were retrieved and information pertaining history, clinical and instrumental data, as well as follow-up, were collected.the median age of the dogs was years (range: - ), of them were female and were boxers. major complaints were chronic large bowel diarrhea with hematochezia and weight loss observed since a median time of months (range: to ). previous treatment with gastrointestinal diets, antiparasitic drugs and antibiotics yielded no improvement. additionally, dogs developed uveitis during the disease course. in all dogs a complete blood count, a serum biochemical profile, including protein electrophoresis, and abdominal ultrasound were performed; serum ctli, folate and cobalamin were available in dogs and urinalysis in one. the results of the above laboratory tests were normal. ultrasonography was unremarkable in dogs and showed increased colon wall thickness in the other . definitive diagnosis was obtained from endoscopic biopsies of the colon and/or rectal scrapings in dogs and from biopsies of the colon at necropsy in one. in each case spheroid, ovoid or irregularly-shaped organisms suggestive of prototheca spp. were observed. different treatments were attempted without benefit in dogs. in one dog transient improvement was obtained with itraconazole. the median survival time was months (range: - ).the present work indicates that protothecosis should be included in the list of differential diagnosis in dogs with large bowel diarrhea, especially in those with chronic refractory colitis or developing ocular signs. dogs infected with prototheca spp. have a guarded prognosis. diagnosing protothecosis in dogs over a -year period may suggest that the disease is emerging in some southern european countries. idiopathic pulmonary fibrosis (ipf) is an interstitial fibrotic pulmonary disease, mainly described in the west highland white terrier (whwt). the diagnosis is challenging and ultimately relies on lung histopathology. identification of biomarkers specific for the disease would be very helpful. ccl (mcp- ) is a chemotactic cytokine for monocytes. it is a known biomarker in human ipf. in dogs with ipf, increased ccl gene expression has been described in lung tissue. the aim of the present study was to compare serum ccl concentration in dogs with ipf versus healthy dogs and dogs with other chronic pulmonary diseases.thirteen dogs with ipf (ten whwts, two scottish terriers, one yorkshire) mean age years, range - ), nine dogs with eosinophilic bronchopneumopathy (ebp) (various breeds, years, - ), ten dogs with chronic bronchitis (cb) (various breeds, years, - ) and ten healthy whwts ( years, - ) entered the study. diagnosis was established after clinical, radiographical, bronchoscopic (and balf analysis) examinations, as well as, in dogs with ipf, either lung high resolution computed tomography (six dogs) or histopathology (three dogs) or both (four dogs). ccl concentration in serum was determined by elisa (canine cll /mcp- quantikine ® , r&d sytems). results in the different groups were then compared using non-parametric test (mann-whitney rank sum test).serum ccl concentration was elevated in dogs with ipf (median; interquartile range = . pg/ml; . - . ) compared to healthy whwts ( . ; . - . ), (p< . ). serum ccl value in ipf dogs was higher than in ebp dogs ( . ; . - . ) (p= . ) and than in cb dogs ( . ; . - . ) (p= . ).the present study shows that ( ) idiopathic pulmonary fibrosis (ipf) is a progressive interstitial fibrotic disease, described in humans and in dogs. etiology and pathogenesis of ipf are poorly known in both species, even if a genetic basis is suspected in dogs because of the predisposition of the west highland white terrier (whwt). serum transforming growth factor beta (tgfb ) concentration is elevated in both healthy whwts and whwts with ipf, as compared to healthy dogs of various breeds. in human ipf, pathways involving tgfb , a cytokine with profibrotic properties, seem to be central in the pathogenesis and are considered as potential therapeutic targets. tgfb is produced as a pro-protein and usually stored as a latent complex. activation of the latent complex is an important step that regulates tgfb function. multiple activation mechanisms have been identified including binding to integrins and thrombospondin (thbs ).the aim of the present study was to quantify tgfb expression, as well as expression of proteins involved in tgfb activation, by quantitative rt-pcr, in lung tissue from dogs with ipf versus control dogs.total rna was extracted from lung tissues from dogs with ipf ( whwts, scottish terrier, lhassa apso) and control dogs (various breeds). ipf was confirmed by histopathology on all samples. expression of tgfb , integrins (itgb and itgb ) and thbs was measured by qrt-pcr. for each gene, a relative copy number was calculated for each sample and results were normalised using two stably expressed housekeeper genes (rps and tbp). statistically significant differences between the groups were assessed using a student t-test or a mann-whitney rank sum test with significance defined as a p < . .expression of tgfb and itgb was not statistically different between the two groups. expression of itgb was significantly lower (p< . ) while thbs expression was significantly higher (p= . ) in the ipf group relative to controls.results of the present study could not confirm that increased gene expression of tgfb by lung tissue is the source of the high circulating tgfb level in ipf. this study highlights different activating pathways of tgfb in ipf lungs compared to control lungs with a shift toward an increased activation via thbs in canine ipf. obesity is the most common nutritional problem in dogs, and its detrimental effect on basal lung function parameters has been recently shown using whole-body barometric plethysmography. the -minute walk test ( mwt) has been recently demonstrated to be a non-invasive easy-to-perform test in clinical settings, able to discriminate between healthy dogs and dogs with pulmonary disease.the aim of this study was to investigate the effect of body weight loss (bwl) on pulmonary function assessed by mwt and arterial blood gas values.six experimental beagles and privately-owned dogs, all obese but otherwise healthy, were enrolled in a diet-induced bwl program. physical examination, bw and body condition score (bcs) assessment, arterial blood gas analysis and mwt were performed when dogs were obese (bcs - / ), and repeated with animals in the middle of their bwl program (overweight, bcs - / ) and at the end of it (lean, bcs / ). for the mwt, dogs were walked for minutes, along an inside m-long hallway. heart rate (hr) and oxygen saturation (spo ) were measured by pulse oximetry before the test (pre-test value), after three minutes of walk (mid-test value) and at , , , and minutes post-test.all dogs concluded the bwl program (initial bw: , ± , kg; final bw: , ± , , means±se, p , ). bwl caused a significant increase in the walked distance (lean: , ± , m; overweight: , ± , m; obese: , ± , m; means±se, p , ) and a decrease in pretest respiratory rate (rr) (lean: ± , /min; overweight: ± , / min; obese: ± , /min; means±se, p , ). resting arterial blood gas results were not influenced by bwl and neither did the pre-test hr and spo values measured by pulse oximetry. obese dogs showed significant higher hr mid-test values compared to overweight and lean dogs (lean: , ± , /min; overweight: , ± , /min; obese: , ± , /min; means±se, p , ). moreover, spo values recorded at and minute post-test were significantly higher in overweight and lean dogs, compared to obese dogs. also, hr values registered at , , and minutes post-test were all lower in overweight and lean dogs.in conclusion, obesity negatively affects the blood oxygenation level during and shortly after physical exercise in dogs, with subsequent hr increase. bwl induces a significant decrease in resting rr and it improves pulmonary function during exercise, even before achieving the targeted ideal bw. the mwt, but not pre-test arterial blood gas values, is an efficient tool to demonstrate the efficacy of bwl. there are few diagnostic laboratory methods available for evaluation of platelet function and contribution to thrombotic events in the clinical setting. the impedance whole blood platelet aggregometer multiplate ® has recently become available. although it is being marketed for monitoring effect of antiplatelet therapy, it can also be used for assessment of platelet aggregation in response to various agonists, reflecting platelet function, activity and reactivity in response to disease. the purpose of this study was therefore to investigate multiplate ® as a diagnostic tool for detection of variations in platelet aggregation in dogs with diseases known to predispose to hypercoagulability and thrombosis and to evaluate whether there is a correlation between multiplate aggregation response and the maximal amplitude (ma) measured by thromboelastography (teg).twenty clinically healthy dogs and eighteen diseased dogs with neoplasia, generalized inflammation or protein losing enteropathy ornephropathy admitted to the university hospital for companion animals, university of copenhagen, were included in the study. citrated and heparinised blood samples were collected. multiplate ® aggregations were performed on diluted heparinised whole blood for minutes using adp, collagen (col) and arachidonic acid (aa) as agonists and nacl as buffer control. results were recorded as area under the curve (auc). dilute ( : ) tissue factor teg analyses were performed on citrated whole blood.diseased dogs had significantly increased auc compared to healthy dogs for nacl buffer control (p= . ), adp (p< . ) and col (p= . ) whereas no significant difference was obtained for aa as agonist (p= . ). teg-ma was significantly higher (p= . ) in diseased dogs compared to healthy dogs. a significant correlation was not found between teg-ma and multiplate auc using adp (p= . , r=- . ), col (p= . , r=- . ) or aa (p= . , r=- . ) .these results demonstrate that multiplate ® aggregation responses are significantly increased in a population of diseased dogs with diseases known to predispose to hypercoagulability and thrombosis, but results are not significantly correlated to teg-ma. this suggests that the multiplate method can be used to detect increased platelet reactivity in dogs with diseases known to predispose for hypercoagulability and thrombosis and that multiplate provides additional information on platelet function than teg alone in this patient group. further studies are needed to determine how multiplate and teg-ma results correlate to thrombosis and whether there may be an added benefit of using them in combination. hypoalbuminaemia is a commonly identified biochemical dyscrasia. the clinical impact of this can be far reaching, particularly in severely affected animals. problems recognised to ensue include altered colloid osmotic pressure and cavity effusions, clotting abnormalities, altered carriage of drugs, hormones and electrolytes, along with acid-base disturbances.the aim of the study was to determine the incidence of side effects encountered when administering % intravenous human albumin to dogs suffering with hypoalbuminaemia. animals were presented to a specialist referral centre in hampshire, uk for various conditions resulting in hypoalbuminaemia. hypoalbuminaeamia was considered to be present when the serum albumin concentration was < g/l. a total of albumin infusions were given to dogs presenting with a serum albumin concentration of g/l or lower. albumin was given as an intravenous infusion of mg ( ml)/kg following premedication with chlorphenamine. the duration of administration was between minutes and hours. the dogs were monitored for potential adverse reactions during the administration of albumin and in the post-infusion period. potential adverse reactions included hypotension, hyperthermia, tachycardia, tachypnea, peripheral oedema, agitation/restlessness and collapse. none of the animals showed adverse reactions during the administration of albumin or in the post-infusion period. one animal demonstrated mild hyperaesthesia that resolved prior to completion of the infusion.of the animals presented, survived to discharge, animals were euthanased and died. each dog received an average of . (sd . ) albumin infusions during the period of hospitalisation. there was no significant difference in the starting albumin concentrations between the survivors and nonsurvivors. there was no significant difference in the increase in albumin concentration post-infusion between survivors and non-survivors. there was no significant difference in the number of albumin infusions between the survivors and non-survivors. the concentration of albumin pre-infusion did not negatively impact on survival and discharge from the hospital. the results of this study demonstrate no significant complications during or following administration of % human albumin solution in dogs. there was no association between the administration of albumin, number of albumin infusions or amount of albumin administered in any patient and outcome. albumin infusion may improve the chances of severely hypoalbuminaemic dogs surviving to be discharged from the hospital. the use of human albumin for this purpose enables the limited supplies of canine plasma to be reserved for dogs needing plasma transfusion for other reasons. recently, metabolic syndrome has gained attention in human medicine given its associations with development of diabetes mellitus and cardiovascular diseases. canine obesity is associated with the development of insulin resistance, altered lipid profiles, and mild hypertension, but the authors are not aware of any existing studies examining the existence of ms in obese dogs. thirty-five obese dogs were assessed before and after weight loss. the guidelines of the international diabetes federation were modified in order to produce a definition for canine metabolic syndrome (cms), which included a measure of adiposity (using a -point body condition score [bcs]), systolic blood pressure, plasma cholesterol, plasma triglyceride, plasma glucose, and urine protein:creatinine ratio (upcr). by way of comparison, total and regional body fat mass were assessed by dual-energy x-ray absorptiometry, whilst adiponectin, insulin, and high-sensitivity c-reactive protein (hscrp) were also assessed with validated assays.systolic blood pressure (p= . ), cholesterol (p= . ), triglyceride (p= . ), insulin (p< . ), and upcr (p= . ) all decreased after weight loss, whilst plasma adiponectin increased (p= . ). however, hscrp did not change with weight loss. prior to weight loss, dogs were defined as having cms. there was no difference in total or regional body fat mass between these dogs and those who did not fit the definition of cms. however, plasma adiponectin concentration was less (p= . ), and plasma insulin concentration was greater (p= . ) in cms dogs.in this study, up to a third of obese dogs suffer from cms, and this is characterized by hypoadiponectinaemia and hyperinsulinaemia. these studies can form the basis of further investigations to determine pathogenetic mechanisms and the health significance for dogs, in terms of disease associations and outcomes of weight loss. cystourethroscopy has greatly enhanced the diagnosis and treatment of numerous conditions in veterinary medicine. rigid telescopes used in female cytoscopy provide well-illuminated highly detailed images using rod lens system technology. the flexible ureteroscope has been used for male urethrocytoscopy but is limited in image quality and procedural abilities due to the fiberoptic system, diminished illumination, and smaller working channel. the purpose of this study was to describe the technique, efficacy, and complications using a novel percutaneous perineal approach to the male urethra in order to gain access for rigid cystoscopy.the perineal approach was performed ten times in nine dogs for ectopic ureter laser ablation of idiopathic renal hematuria sclerotherapy. the dogs were placed in dorsal recumbency. using fluoroscopic guidance, an gauge renal access needle was advanced trans-perineally into the pelvic urethra. guidewire access was obtained and the access site dilated to accept a fr peel-away sheath. a rigid cystoscope was then placed through the sheath to perform the procedures. urinary catheters were placed following three of ten procedures for three to eighteen hours. the only identified peri-operative minor complication included urination form the perineal site approximately six hours post-operatively once in a single dog. no signs of stranguria or pollakiuria or incisional complications were identified in any of the dogs post-operatively at follow-up examination or contact (range to days).the percutaneous perineal approach in male dogs for rigid cystoscopy appears to be a safe and effective means of facilitating endoscopic procedures. irh results in chronic upper urinary tract bleeding. in humans, ruptured renal pelvic hemangiomas/angiomas are typically the cause. although benign, anemia, ureteral and urethral obstruction(s) can ensue. with the advent of endourology renalsparing therapies like ureteropyeloscopic-guided electrocautery or sclerotherapy has replaced ureteronephrectomy. the objective is to describe the use of endoscopic-fluoroscopic-guided sclerotherapy for the treatment of irh in dogs and report the first clinical outcomes.each uvj was identified cystoscopically. once the bleeding was confirmed a retrograde ureteropyelogram was performed. a ureteropelvic junction balloon was used for ureteral occlusion and pelvis filling volumes were recorded. four dwells were performed ( % povidone iodine mixture; and sterile liquid . - % silver nitrate). a double-pigtail ureteral stent was placed.seven dogs had sclerotherapy. five unilateral, bilateral, and developed contralateral bleeding (n= units). five were right and left-sided. there were males and female. the median age and weight was years and . kg, respectively. median procedure time was minutes. there was complication of severe renal discomfort and pyelectasia in an unstented dog.cessation of hematuria occurred in / renal units (median hours). two had recurrence within weeks; both resorted to intermittent mild hematuria. two failed treatment. median follow-up time was months (range, . - ) .overall, topical sclerotherapy for irh can be safe and effective. this is the first report of local sclerotherapy for irh in dogs and could be considered a valuable endoscopic-guided therapy prior to ureteronephrectomy. further investigation is required. percutaneous nephrolithotomy (pcnl) is considered the standard -of-care for removal of nephroliths > . cm in people, minimizing morbidity and preserving renal function. success rates are reported to be - %. most veterinary nephroliths remain clinically silent and removal is only recommended for complicated stones. morbidity of nephrotomy can be severe. the objective is to describe endoscopic-guided nephrolithotomy (enl) in canine and feline patients and report clinical outcomes, hypothesizing it is safe and effective. patients that had either pcnl or surgicallyassisted endoscopic nephrolithotomy (senl) were retrospectively evaluated. a renal puncture needle and balloon-dilation-sheath combination was used for tract formation. a nephroscope provided visualization for intracorporeal lithotripsy. stone fragments were removed and a ureteral stent was placed. nine dogs and cat ( renal units) were included. four had pcnl and senl. indications included recurrent utis ( ), worsening azotemia ( ), and ureteral-outflow obstructions ( ). median weight was . kg ( . - . ). stone composition was calcium oxalate ( ), mixed struvite ( ), urate ( ), and cystine ( ). median stone size was cm ( . - ). median pre-and month post-operative creatinine was . ( . - . ) and . mg/dl ( . - . ), respectively. the median procedure time was minutes. successful removal of all stones were documented in / ( . %). procedure-related complications occurred in units, all were easily managed. median follow-up time was days ( - ). four patients are still alive. no patient died from the procedure. overall, enl can be safely performed in dogs and cats, yielding similar success rates to people. advanced endourologic experience is recommended. fgf- is a key regulator of plasma phosphate concentration. it is elevated in cats with naturally occurring ckd and increases as renal function declines. elevated fgf- concentrations are an independent predictor of survival time in human haemodialysis patients, but this association has not previously been examined in cats with ckd. this study investigated if fgf- was independently associated with survival time (all cause mortality) in cats with newly diagnosed ckd.cats diagnosed with ckd at two london-based first opinion practices between and were identified. ckd was defined as plasma creatinine concentration > lmol/l with concurrent urine specific gravity (usg) < . or plasma creatinine concentration > lmol/l on two consecutive visits. cats were excluded if no residual edta plasma was available to measure plasma fgf- concentration within days of the date of diagnosis of ckd. fgf- concentrations were measured using a previously validated human intact fgf- elisa. plasma fgf- , creatinine, phosphate and total calcium concentrations, packed cell volume (pcv), systolic blood pressure (sbp), usg and age were entered into univariable cox regression models of survival time. fgf- concentrations were logarithmically transformed due to a highly skewed distribution. statistical significance was defined as p< . . variables with p< . were carried forwards into a backwards, stepwise multivariable cox regression analysis of survival time. ninety-one cats were included in the study. at the end of the follow-up period (february ), cats had been euthanased or died (median (range) survival time ( - ) days) and cats were alive or lost to follow-up (median (range) survival time ( - ) days). median (range) age at diagnosis (n= ) was . ( . - . ) years. univariable cox regression analysis indicated that plasma creatinine (p< . ), phosphate (p< . ) and log-fgf (p< . ) concentrations were negatively associated with survival time, and that pcv (p< . ) and usg (p< . ) were positively associated with survival time. in the multivariable model (n= ), plasma logfgf (hazard ratio (hr)= . , % confidence interval (ci) for hr= . - . ; p= . ) and creatinine concentration (hr= . , % ci for hr= . - . ; p< . ) were negatively associated with survival time, and pcv (hr= . , % ci for hr= . - . ; p= . ) was positively associated with survival time.plasma fgf- concentration is a novel prognostic indicator in feline ckd, independent of other factors including plasma creatinine concentration and pcv. future studies should investigate whether belgium ragdoll breeder organizations often forewarn ragdoll cat owners that renal problems may develop due to polycystic kidney disease (pkd), chronic interstitial nephritis, familial renal dysplasia or nephrocalcinosis. in several european countries, screening of ragdoll cats for kidney disease is already performed for years, without scientific evidence. therefore, we aimed to investigate if ragdoll cats are predisposed for kidney disease based on the laboratory parameters, one ragdoll cat was diagnosed with iris stage chronic kidney disease (ckd) one of these six cats was the ragdoll cat with iris stage ckd. in one ragdoll cat, pkd could not be excluded on ultrasonography because one cyst was detected in one kidney. however, none of the ragdoll cats was genetically positive for pkd.based on this study, pkd and ckd appear to be uncommon in ragdoll cats residing in belgium and the netherlands. however, renal infarcts were seen more commonly in ragdoll cats compared to an age-matched control group spain urine markers are advocated to early detect kidney damage in the clinical practice, nevertheless histology remains the gold standard. the aim of this study was to evaluate quali-quantitative proteinuria and possible renal damage using different non-invasive tests in dogs affected by leishmaniasis.based on clinical signs and serology/cytology, affected dogs (leish) were included. fifteen healthy, non-proteinuric dogs were selected as control. upon admission, all dogs underwent to physical examination, systolic blood pressure (sbp) measurement, clinicopathological evaluation urine high resolution agarose and silver staining sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis (hre; sds-page) were performed. a cut-off of kda was selected to classify bands in high or low molecular weight (hmw; lmw). data were analyzed with non-parametric statistics and roc curve analysis (roc). a difference was considered significant for p< . leish dogs presented significantly higher upc (mean , median . ) and uac (mean . , median . ). rri values were significantly higher in leish (mean . ) than control (mean . ). rri was significantly correlated to wbc (r= . ), hemoglobin (r= . ) and albumin concentrations (r= . ), usg (r= . ) and upc (r= . ). hre and sds-page protein patterns allow to distinguish p from np and control dogs. sds-page revealed a significantly higher number of bands in leish dogs ( - ) than in control ( - ). np and blp dogs presented a significantly lower number of lmw bands than p. number of bands was significantly correlated to upc (r= . ) and uac (r= . ) in humans, diabetes mellitus (dm) is an important cause of renal damage. main lesions include thickening of the glomerular basement membrane and mesangial expansion, whereas tubular atrophy and vascular hypertrophy are less frequent. in cats, although diabetes is a common endocrinopathy, it is yet unknown whether dm causes renal damage. the aim of the present study was to compare renal histopathological features and clinical parameters of kidney function in diabetic cats against a well-matched control population.formalin-fixed, paraffin-embedded kidney samples were retrieved from diabetic and control cats that died between and due to any disease at the clinic for small animal internal medicine, university of zurich (switzerland), and in which a post-mortem examination was performed. control cats were selected to be matched for age, sex, breed and body weight. serum creatinine and urea levels were analyzed if they had been measured within days before death. kidney sections were stained with haematoxilin-eosin, periodic acid-schiff (pas), masson's trichrome, acid fuchsine orange-g (afog), and periodic acid methenamine silver (pams). with optical microscopy glomerular, tubulointerstitial and vascular parameters were identified and scored using a grading scale. data were analyzed with contingency tables and t-tests.thirty-two diabetic cats and matched controls were included. with optical microscopy, scores of glomerular lesions (i.e., sclerotic glomeruli, mesangial or endocapillary hypercellularity, increased mesangial matrix, immunodeposits, glomerular basement membrane thickening, mesangial interposition), tubulointerstitial lesions (i.e., inflammation, fibrosis, tubular atrophy, necrosis and lipidosis, intratubular mineralizations) and vascular lesions (i.e., small or large artery hypertrophy) did not differ between the groups. overall, glomerular, tubulointestitial and vascular lesions were observed in . %, . % and . % of diabetic cats and in . %, . % and . % of the controls. similarly, serum creatinine and urea levels were not different between groups (creatinine: ± vs. ± lmol/l, reference: - lmol/l; urea: . ± . vs. . ± . mmol/l, reference: . - . mmol/ l).the results suggest that dm in cats does not lead to microscopically detectable renal lesions or clinically relevant renal dysfunction when compared to a well-matched control group. we hypothesize that the short life expectancy of diabetic cats and the low prevalence of hypertension are main reasons for the difference to human diabetics.early diagnosis of aki and differentiation from non-renal disease or ckd remains challenging in veterinary medicine. in human medicine ngal is used as a real time indicator of aki but few data exist in veterinary medicine. in this study plasma and urine ngal was measured in healthy dogs with normal gfr (plasma inulin clearance) and dogs with renal azotemia (creatinine > . mg/dl and/or urea > mg/dl persisting at least hours after correction of prerenal factors). based on history, clinical course, laboratory and ultrasonographic findings, azotemic dogs were diagnosed with aki (n= ) or ckd (n= ). urine and plasma ngal was measured with a dog ngal elisa kit (bioporto ® diagnostics a/s, gentofte, denmark). intra-assay variability for plasma and urine ngal was . % and . %, respectively. azotemic dogs had significantly higher plasma ngal concentrations and urine ngal-creatinine ratios compared to healthy dogs (p< . , mann-whitney u-test). median (min-max) plasma ngal concentration in healthy dogs, dogs with aki and ckd was . ( . - . ) ng/ml, . ( . - . ) ng/ml and . ( . - . ) ng/ml, respectively. using a multiple linear regression model in the azotemic dogs with ngal as dependent and age, weight, sex, aki vs. ckd, dialysis and survival as independent variables revealed a significant differ-ence only for aki vs. ckd (p = . ). in conclusion, ngal can be measured successfully in plasma and urine of healthy dogs and dogs with kidney disease. dogs with aki had significantly higher plasma ngal concentration compared to dogs with ckd. xanthine urolithiasis is a rare condition accounting for . % of all canine urolithiasis in one study. this pathology has been reported as a primary disorder in dogs, most notably in cavalier king charles spaniels (ckcs). xanthine is an intermediate product of purine metabolism, which is converted from hypoxanthine by xanthine oxidase. xanthine is only slightly soluble in urine and therefore hyperxanthinuria may lead to urolith formation. it has been speculated that some ckcs have an inherited mutation in the xanthine oxidase gene. in humans, isolated deficiency of xanthine oxidase occurs rarely and approximately % of individuals are asymptomatic, despite having significant xanthinuria. therefore we hypothesised that asymptomatic xanthinuria may be commonplace in the uk population of ckcs. in support of this, a previous case report of a symptomatic ckcs reported significant xanthinuria occurring in an asymptomatic sibling. in order to examine the prevalence of xanthinuria in ckcs, urine concentrations of hypoxanthine and xanthine metabolites as well as creatinine were measured in clientowned cavalier king charles spaniel dogs and dogs of other breeds from three first-opinion veterinary practices in the uk. urine samples were collected by free catch and purine metabolites were measured by high-performance liquid chromatography. ratios of xanthine/creatinine and hypoxanthine/creatinine from the two populations were compared by mann whitney u test and were found not to be significantly different (p= . and p= . respectively). in the control population, the xanthine/creatinine ratio ranged from . to . (median . ), while in the ckcs population it ranged from . to . (median . ). these results are markedly lower than the previously reported case of xanthine urolithiasis in a uk ckcs dog, which utilised the same reference laboratory (xanthine/creatinine ratio . ). these data suggest that asymptomatic xanthinuria is not prevalent in the uk ckcs population. key: cord- -kbqh beq authors: behzad, catherine; taheri, hassan; kashifard, mehrdad title: establishing safe out-of-hospital infusion centers may improve the quality of care in patients with ibd during the covid- pandemic date: - - journal: inflamm bowel dis doi: . /ibd/izaa sha: doc_id: cord_uid: kbqh beq nan we read with great interest the article by occhipinti and pastorelli about the challenges in the care of patients with inflammatory bowel disease (ibd) during the covid- pandemic. we experienced a similar situation at rouhani university hospital, which is the largest medical center in northern iran. with intensification of the sars-cov- epidemic in february , our hospital was transformed into a covid- referral center and outpatient clinics, including the ibd clinic, were closed. the majority of the hospital wards became exclusive covid- -operating units. however, medical wards were maintained covid- -free to provide care for patients with urgent situations and essential services. to accommodate the care of our patients with ibd, we expanded on our existing telemedicine service, which included phone access to a trained individual at any time to answer questions. during the outbreak, we began using this service to provide necessary information about the symptoms and prevention of covid- by phone message. in addition, we gave special consideration to patients with ibd who were on immunosuppressive therapy and decided to continue biologic infusions in the hospital's covid- -free wards. our patients' conditions were regularly followed by phone. however, the news about the rapid spread of the virus caused high rates of anxiety among our patients and most of them developed an overwhelming fear of admission to the hospital, even for essential purposes. in our experience, % of the patients whose scheduled biologic infusion dates were during the peak of the outbreak in early february through march refused to come to the hospital to receive their infusions. all of our patients emphasized that they were afraid of viral contamination, and % of these patients admitted that despite knowing that the biologic infusion ward was located in the covid- -free area of the hospital, they were truly terrified of coming to the epicenter of covid- in our region. the patients noted that if the infusion ward had not been located in the hospital, then they would have come to receive their infusions. to date, there is no definite cure or vaccine for covid- , , and there are rising concerns of a second wave of infections in many countries. establishing safe biologic infusion centers outside the hospital may relieve the anxiety of patients with ibd who require infusions during the covid- outbreak and in turn diminish the risk of ibd relapse in these difficult times. challenges in the care of ibd patients during the covid- pandemic: report from a "red zone" area in northern italy pharmacologic treatments for coronavirus disease (covid- ): a review current status of multiple drug molecules, and vaccines: an update in sars-cov- therapeutics offline: the second wave key: cord- -mz i qp authors: furfaro, federica; vuitton, lucine; fiorino, gionata; koch, stephane; allocca, mariangela; gilardi, daniela; zilli, alessandra; d’amico, ferdinando; radice, simona; chevaux, jean-baptiste; schaefer, marion; chaussade, stanislas; danese, silvio; peyrin-biroulet, laurent title: sfed recommendations for ibd endoscopy during covid- pandemic: italian and french experience date: - - journal: nat rev gastroenterol hepatol doi: . /s - - - sha: doc_id: cord_uid: mz i qp the current coronavirus disease (covid- ) pandemic caused by severe acute respiratory syndrome coronavirus (sars-cov- ) infection has required a complete change in the management of patients with inflammatory bowel disease (ibd) who need to undergo endoscopic procedures. several preventive measures must be taken to avoid the spread of infection among health-care professionals and patients with ibd, including the use of personal protective equipment, greater attention to endoscopic room hygiene and rescheduling of non-urgent procedures. this perspective aims to provide a guide based on the italian and french experience to better face the difficulties encountered by endoscopists during this global health emergency. in particular, recommendations regarding the use of personal protective equipment to prevent covid- transmission, both for patients and health-care professionals, are proposed and different scenarios in endoscopic ibd management are evaluated to suggest when endoscopy could be rescheduled and replaced by alternative biomarkers. the researchers collected samples from the bathroom of a patient with the confirmed presence of sars-cov- in stool by reverse transcriptase pcr (rt-pcr) and no diarrhoea. samples from the surface of the toilet bowl and sink were positive and post-cleaning samples were negative, suggesting that stool could be a potential route of transmission . sars-cov- likely infects the gastrointestinal epithelium, producing virions secreted in the stool by the infected gastrointestinal cells . importantly, lamers et al. demonstrated , using an experimental model of human small intestinal organoids and confocal and transmission electron microscopy, that sars-cov- infects and replicates within enterocytes in vitro and that the intestinal epithelium supports virus replication . however, whether the viral concentration of sars-cov- in stool is sufficient for the transmission of infection remains currently unclear. furthermore, the presence of sars-cov rna in stool specimens can be independent of the presence of diarrhoea or other gastrointestinal symptoms (anorexia, nausea, vomiting, abdominal pain) . nevertheless, there are cases of patients with symptoms and lung imaging compatible with covid- but with negative pharyngeal swab and positive stool real-time rt-pcr for sars-cov- (ref. ). thus, both routes, airborne transmission and faecal-oral transmission, should be considered in understanding and reducing the risk of infection during endoscopic procedures. according to a study in the united states, the rate of infection in endoscopy units normally ranges from . for screening colonoscopies to . for oesophagogastroduodenoscopy for every , procedures . the goal of protective measures in the endoscopy setting should be to maintain the risk below these numbers during the covid- pandemic. currently, it is not clear whether endoscopic procedures (particularly colonoscopy) are of high risk for covid- transmission. considering that the available evidence is quite slim, we suggest the use of all disposable protective measures to prevent infection transmission. in this perspective, we provide some guidance from the humanitas research hospital (rozzano, milan, italy) and the société française d'endoscopie digestive (sfed) for performing endoscopy in patients with ibd during the covid- pandemic based on the italian and french experience. recommendations regarding the use of ppe to prevent covid- transmission are proposed and different scenarios in endoscopic ibd management are evaluated. the recommendations discussed are based on the available evidence and expertise as there are limited data with respect to covid- in patients with ibd with or without gastrointestinal symptoms. in the past few months, different societies and experts have already proposed advice or recommendations to manage endoscopy and/or patients with ibd during the covid- pandemic, including the american gastroenterological association (aga; recommendations for gastrointestinal procedures) , the british society of gastroenterology , the international organization for the study of ibd (recommendations for patients with ibd) , the european society of gastrointestinal endoscopy and european society of gastroenterology and endoscopy nurses and associates (position statement on gastrointestinal endoscopy) , the chinese society of ibd (guidance for patients with pre-existing digestive diseases) , the asian pacific society for digestive endoscopy , a panel of experts in the united states and a panel of international experts . currently, the available evidence on covid- in the context of ibd, as well as in relation to reported gastrointestinal symptoms, is very limited. accordingly, the recommendations proposed presuppose that everyone (both patient and hcps) are at high risk -the recommendations will probably evolve as the pandemic progresses. the data about the spread of covid- in countries that applied strict containment measures suggest that the risk of being infected is higher at the community level than in hospitals. thus, the first protection measure is to avoid having potentially infected individuals admitted to the hospital and endoscopy rooms. furthermore, the infection of patients with ibd by hcps must be avoided. although covid- can be transmitted by the - % of the affected individuals who are asymptomatic , symptomatic patients remain the main source of infection. patients with ibd who need endoscopy should be checked for typical symptoms of covid- such as fever, cough, dysgeusia, dysosmia and dyspnoea. the most challenging covid- symptoms might be gastrointestinal symptoms, including abdominal pain, nausea, vomiting and/or diarrhoea, occurring in a reported - % of individuals , , which can be present in patients with ibd but are rarely the only symptoms in patients with covid- (ref. ). the who report from china indicates that, within a median time of - days after infection (range - days), these patients can develop fever and respiratory symptoms (usually mild) . at humanitas research hospital and in french centres following guidance from the sfed, endoscopic procedures were reduced, limiting access to the centre to patients with ibd for whom endoscopy is necessary and not deferrable. to postpone elective surgeries and endoscopies at this time is also suggested in the international organization for the study of ibd recommendations . in particular, endoscopy is performed for patients with a new clinical flare both in ulcerative colitis and crohn's disease, when delay is not recommended (that is, to rule out superinfections in severe flares refractory to steroids), or in patients with ibd with dysplastic lesions or polyps for whom endoscopic resection was already planned. a nasal swab, as with screening procedures prior to endoscopic examina tions, could be an option, although fast-track sars-cov- tests are not generally validated and false-negative tests (up to %) might provide false reassurance. at humanitas research hospital, patients who have a scheduled endoscopy are contacted by phone the week before and again - days before the procedure to identify patients at risk of having covid- prior to the commencement of bowel cleansing and before their arrival at the hospital. if a patient refers to specific symptoms, such as fever (> . °c), cough, dysgeusia, dysosmia and dyspnoea, or if a patient has been in contact with a confirmed case of covid- (confirmed by testing) or with individuals highly suggestive of covid- infection, hcps can decide to postpone the endoscopic examination and reschedule it, according to clinical condition. furthermore, patients can be advised to perform specific tests, such as a nasal swab, or to contact the green number for a covid- emergency (a specific telephone covid- emergency service in italy). for patients accessing the endoscopy service ( fig. ), a checkpoint is available at every public entrance of the hospital (in italy) and at the entrance of the endoscopy unit (in france) to assess body temperature and current and/or previous symptoms of covid- . in italy, the endoscopist or the endoscopy nurse must double-check the same information before the patient enters the endoscopy room. at the checkpoints, hcps are also evaluated, assessing body www.nature.com/nrgastro temperature and covid- -related symptoms, and dedicated staff provide everyone with a simple surgical mask and an alcoholic solution to clean hands, on the assumption that there could be asymptomatic carriage of the infection. in both italy and france, patient relatives and caregivers are strictly forbidden from entering the hospital before the endoscopic procedure and are stopped at the checkpoints unless the patient requires specific assistance such as for patients in a wheelchair or relatives of patients under years of age. medical and nurse students are not allowed in the endoscopy units during the pandemic crises. additionally, appointments for endoscopy procedures must be organized to avoid crowding in the waiting room and to maintain a reasonable distance between patients; if possible, only one patient will be in the waiting room at a given time. in the endoscopy room, both the patient with ibd and hcps must be protected. potentially, there is a risk of transmission from the patient to the endoscopist and from the endoscopist or other hcps to the patient. the use of adequate ppe, in particular masks, protects from the risk of being infected by respiratory viruses, with no notable differences between surgical masks and n masks (ffp in europe) in a randomized clinical trial in the context of seasonal human coronaviruses (but not specifically sars-cov- ), influenza viruses and rhinoviruses , . in march , the aga and the sfed recommended the use of n (ffp ) masks, instead of surgical masks, to protect hcps during upper and lower gastrointestinal procedures, regardless of the covid- status of the patient (with low to moderate certainty of evidence). a meta-analysis of retrospective studies demonstrated an increased risk of covid- transmission during aerosolgenerating procedures (upper endoscopy), but the panel of experts extended this recommendation to lower gastrointestinal procedures . the panel of experts con sidered possible aerosolization during colonoscopy, in particular during the insertion and removal of instruments through the biopsy channel and the presence of the virus in the stool and advised on the use of n masks for lower gastrointestinal procedures as a precautionary measure to protect the endoscopist from the risk of possible covid- transmission from the patient if infected by sars-cov- (ref. ). unfortunately, in clinical practice, it could be difficult to have these kinds of ppe for every procedure; in this instance, the aga suggests the use of the same n mask all day long and changing a surgical mask covering the n mask . the sfed suggests changing the mask once at midday and practicing endoscopy with the minimal mandatory staff (doctor and nurse) . the usual procedures of disinfection and decontamination by neutral detergent and viricidal disinfectant, . % sodium hypochlorite or % ethanol of surfaces and devices are effective in clearing the virus , . the major issue is the possibility that the virus can remain alive and contagious in aerosols, as van doremalen et al. demonstrated that sars-cov- remained viable in aerosols for at least h, with a reduction in infectious titre from . to . tcid (median tissue culture infectious dose) per litre of air . to avoid this risk, at humanitas, patients and hcps wear masks during the entire procedure and during the entire time spent at the hospital. in the endoscopy room, entry is restricted to the endoscopist, the nurse and the patient. considering that the endoscopy staff are at higher risk of infection during the procedures and could spread the virus, regular patient care (such as infusion management) is separated from the endoscopy unit, with other separate, dedicated staff, to decrease risk of viral transmission. during the procedure in the endoscopy room, the patient dresses in a cotton gown, a hairnet and a surgical mask ( fig. ). hcps must remove contact lenses, if present, and must dress following this specific order: a hairnet, a long water-resistant gown (in blue in fig. ) with back closure, a n (or ffp ) mask, goggles for eye protection and over-sleeve gloves over the gown (the surgical gown, not because of sterility, but because they are longer than others and can cover the skin up to the wrist). to avoid becoming infected and/or passing on potential infection to different patients, before a patient's entrance into the endoscopy room, hcps must wear, over the other layers, a single-use gown (in white in fig. ) and a pair of nitrile gloves (in blue in fig. , double gloves are also recommended in the aga recommendations ). hcps change the white disposable gown and the blue gloves for each procedure, as is normal in clinical practice outside of the covid- era. the sfed recommends long waterresistant gowns only for hcps caring for patients who are covid- -positive and disposable plastic gowns when treating other patients . before and after all patient interaction and before putting on and removing the ppe, hand washing (keeping the surgical gloves on, as if they were hcp's own skin) with water and soap or alcohol-based hand solution is mandatory. when anaesthesiology is mandatory for sedation, the sfed advises orotracheal intubation to protect the endoscopy team from covid- aerosols . nevertheless, because of the pandemic, it is very likely that anaesthesiologists would not be available for deep sedation because they are attending to patients with covid- in the intensive care unit. thus, conscious sedation remains the most feasible option and can be provided and managed even though the patient is wearing a mask. at humanitas, in the covid- inpatient department, we have negativepressure rooms (airborne isolation rooms) to prevent generated aerosols from diffusing outside the room. unfortunately, we do not have a negative-pressure room in the endoscopy department and so emptying the endoscopy room of all non-used equipment is essential to facilitate room cleaning and disinfection. the use of air renewal in the operative room is the most protective tool . the timing to open the endoscopy room for air renewal depends on the adequacy of ppe worn by patients and hcps. on the other hand, in several centres, scarce availability of and access to ppe have been reported; centres using surgical masks for ppe should open the endoscopy room for more time to allow air renewal. in the ibd endoscopy room, we do not perform room disinfection after each procedure for covid- -negative patients, rather dedicated staff perform a standard room disinfection using : dilution of household bleach and water once a day as a standard decontamination process. after each procedure, all endoscopes and reusable accessories are reprocessed with a standardized and uniform reprocessing procedure. kampf et al. demonstrated that human coronaviruses can be efficiently inactivated by surface disinfection procedures with specific agents . we could assume that these methods would work for sars-cov- , indicating that current endoscope disinfection techniques could be sufficient to prevent covid- infection . beds are also cleaned with specific disinfection products and bed sheets are changed for each patient. finally, avoiding viral dissemination to patients with ibd who are potentially immuno compromised because of therapies (steroids, immunosuppressants or immunomodulators) is particularly important. given that nurses and physicians are at particular risk of contamination and could also be asymptomatic carriers of sars-cov- , maintaining physical distance wherever possible, hand cleaning and the wearing of surgical masks outside the endoscopy units should always be the rule at any time of the day at the hospital for all hcps. for patients with ibd with known or presumptive covid- with non-deferrable endoscopic procedures we follow the same precautions used for non-ibd patients with covid- (ref. ). the procedure should be performed in a dedicated room and the exams must be concentrated in the last part of the day to enable the correct room cleaning and disinfection protocol after the endoscopic procedure. the patient has to be transferred to the endoscopy room at a specific time, without encountering obstacles. once the patient is in the endoscopy room the door will be closed until the end of the procedure . there is a specific protocol for wearing and removing ppe, with a specific order as noted earlier. the ppe has to be donned in this order: hairnet, gowns, filtering face-piece (n or ffp mask), goggles for eye protection, pairs of gloves. at the end of the endoscopy procedure, the gloves and gown should be the first to be removed and a new pair of nitrile gloves should be worn to prevent self-contamination. subsequently, the ppe has to be removed in this order: goggles, respiratory protection, hairnet and, finally, the new pair of gloves worn. at the end of the examination, the removal of the ppe from patients with covid- is crucial to protect hcps from contamination. in late february , the who reported the development of covid- symptoms after a median time of - days post-infection of sars-cov- (range - days) . in a cohort of admitted hospital patients in wuhan, huang et al. reported a median time from illness onset to dyspnoea of days in % of patients . patients undergoing endoscopy could therefore develop symptoms after the procedure in instances in which they have contracted the sars-cov- infection at the community level just before endoscopy. therefore, ibd nurses perform a follow-up call at least week after the procedure to identify potential new covid- cases and to verify whether the protective measures have worked in avoiding contamination and dissemination in the endoscopy environment; of note, the patient can contract the disease elsewhere once outside the hospital setting. patients with ibd normally undergoing planned endoscopies to monitor endoscopic response to therapy or to evaluate endoscopic disease activity could be managed instead by checking clinical activity with patient-reported outcomes (pros; for instance, number of bowel movements and abdominal pain for crohn's disease or bowel movements and rectal bleeding for ulcerative colitis) and non-invasive tests such as stool and blood exams and cross-sectional imaging. indeed, all protective measures already described can limit, but not exclude, the risk of infection for patients during endoscopy: patients can be at risk of sars-cov- infection whilst travelling to the hospital (that is, by public transportation or potentially by use of public toilets). whether alternative non-invasive tools can replace endoscopy, at least for the monitoring of patients under therapy, has been a topic of debate since before the covid- pandemic. cross-sectional imaging, such as bowel ultrasonography or magnetic resonance enterography (mre), are valid alternatives; however, they also require hospital attendance, with similar challenges as with endoscopy in protecting patients and hcps from sars-cov- . the use of blood tests and faecal biomarkers could play a key role in avoiding endoscopy when this procedure is not urgent or necessary (fig. ) . the calm trial demonstrated that treatment escalation and tight control based on levels of faecal calprotectin and serum c-reactive protein (crp), compared with treatment based on clinical management, was associated with a statistically significant higher proportion of patients with crohn's disease achieving endoscopic remission at week ( % versus %, % ci . - . ; p = . ). in patients with ulcerative colitis, faecal calprotectin levels had a statistically significant positive correlation with endoscopic extent, mucosal healing and histological activity , . in this context, the use of home tests for faecal calprotectin levels to avoid manipulation of stools and virus in the laboratory is of particular interest. home faecal calprotectin tests, where available, together with remote monitoring of symptoms and pros, could be a safe alternative to monitor patients by avoiding endoscopies. patients with ibd with onset of gastrointestinal symptoms during covid- pandemic, such as abdominal pain and diarrhoea, will need to undergo specific examinations to identify the correct diagnosis. first, the diagnosis of covid- will be considered, as sars-cov- infection can occur with gastrointestinal symptoms. in cases of gastrointestinal symptoms in covid- , the median duration of symptoms is days (range - days) and thus shorter than an ibd flare, which typically does not resolve without therapy. evidence of concomitant fever, cough, dysosmia and fatigue can help in the clinical diagnosis and should prompt nasal swab and pcr testing for sars-cov- . the second step is to exclude usual infectious diseases, such as salmonella, shigella, campylobacter, clostridioides difficile (formerly known as clostridium difficile) or parasitological infection, by performing microbiological analysis of stool samples. finally, faecal calprotectin can help discriminate between ibd and irritable bowel syndrome, keeping in mind that it could be elevated in cases of covid- infection, as reported in a letter published in gut by effenberger et al. . if a new diagnosis of ibd is highly suspected (and supported by cross-sectional imaging for crohn's disease), colonoscopy or proctosigmoidoscopy with biopsies are indicated to confirm the diagnosis, assess endoscopic activity and extension of disease, and initiate the appropriate therapy. in patients with mild symptoms, the endoscopic procedures should be postponed, as treatment might also be delayed. in patients with suspected crohn's disease and isolated small bowel manifestations, without obstructive symptoms or bleeding, enteroscopy should be postponed. if necessary to assess a diagnosis and commence treatment, capsule enteroscopy could be an option , considering that the patient will have to attend the hospital twice with this approach, first for administration of the patency capsule and a second time for capsule endoscopy. in that scenario, all the adequate safety measures will be respected. acute severe ulcerative colitis (asuc) is a medical emergency occurring in about - % of patients with ulcerative colitis during their lifetime . multiple definitions of asuc exist; the first was proposed by truelove and witts in (ref. ), who suggested a list of criteria to assess patients on initial admission to hospital and to predict the risk of colectomy. the criteria were stool frequency (≥ per day), blood in the stool, heart rate (> bpm), temperature (> . °c), haemoglobin levels (< g/l) and crp levels (> mg/l). the risk of colectomy was directly correlated with the number of variables present at , and days and was % when three or more criteria were present . in , travis et al. proposed the oxford score, evaluating only a few parameters to re-assess after days of treatment in patients treated with intravenous corticosteroid as first-line therapy . in this case, to determine the risk of colectomy, the researchers recommended assessment of only stool frequency and serum crp levels (> stools per h or - stools per h and crp levels > mg/l). in the covid- era, if a patient with ulcerative colitis is admitted to hospital with bloody diarrhoea, fever and high serum crp level, it is probably asuc, yet these symptoms could be similar to the gastro intestinal manifestations of covid- ; notably, rectal bleeding has been described in % ( of ) and . % ( of ) of patients with covid- (ref. ). in some cases of covid- , gastrointestinal symptoms are associated with respiratory symptoms, usually after an initial period of - days with isolated gastrointestinal symptoms . at the humanitas clinical centre emergency room, every patient, regardless of respiratory symptoms, has a nasal swab taken and tested to exclude covid- before hospitalization. differential diagnosis with gastrointestinal infections, such as c. difficile infection, is crucial. if the suspicion of asuc is high, endoscopy is useful to confirm an ulcerative colitis flare, to exclude other diagnoses and to perform concomitant biopsies for cytomegalovirus (cmv) infection. cmv-positive patients with ibd have a higher risk (nearly double) of steroid resistance than cmv-negative patients with ibd as well as a worse prognosis . several methods for analysing cmv infection in tissue are available; the gold standard for the diagnosis of cmv infection is related to colonic biopsies (haematoxylin and eosin staining via detection of inclusion bodies, so-called owl's eyes, and/or immunohistochemistry assay on histology) rather than serology (antigen or virus detection in blood) . in , pcr-based cmv detection was also applied to evaluate cmv infection in tissue from patients with ibd. this test was highly sensitive ( - %) but had limited specificity ( - %) , . the sfed advise to keep performing flexible proctosigmoidoscopy in this setting, even in the context of the covid- pandemic . at humanitas, proctosigmoidoscopies for asuc are also still performed. after surgery, both in crohn's disease and ulcerative colitis, there is an indication to perform endoscopy to assess endo scopic disease activity. in patients with crohn's disease after ileocaecal resection, endoscopic recurrence could precede clinical mani festations; indeed, ileocolonoscopy is recommended at - months from surgery , . endoscopy also plays an important part in the postoperative management of patients with ulcerative colitis. in these patients, proctocolectomy with ileal pouch-anal anastomosis represents the procedure of choice in cases of colitis refractory to treatment or high-grade dysplasia on random biopsy samples according to the european crohn's and colitis organisation guidelines . endoscopic surveillance of the pouch after surgery is a controversial issue; however, a so-called screening pouchoscopy with biopsies at year from surgery is recommended to assess pouch inflammation and histological activity . after first endoscopy, patients are stratified according to several risk factors such as dysplasia or cancer identified before or at operation time and concomitant diagnosis of primary sclerosing cholangitis (psc) . in the covid- era, endoscopy to check postoperative recurrence could be delayed. the possible alternative is to check inflammation activity by performing non-invasive tests, such as a faecal calprotectin home test. in symptomatic patients with crohn's disease, an accurate differential diagnosis is the first step (gastrointestinal infection, covid- , irritable bowel syndrome symptoms) using faecal and haematological tests and pro measures (by specific questionnaires filled in by the patient). if there is an indication to start therapy, the alternative to endoscopy could be performing bowel ultrasonography to assess disease activity, localization and extension of disease . compared with endoscopy and mre, bowel ultrasonography needs less time to be performed ( min versus - min; patient spends less time at the hospital), no preparation (bowel preparation, causing diarrhoea and/or vomiting, could increase the risk of spread ing covid- ) and the patient can attend hospital alone (for colonoscopy with sedation it is necessary that a caregiver takes the patient home). for colorectal surveillance and screening of dysplasia, endoscopies planned according to the schedule recommended by the european crohn's and colitis organisation guidelines and postponed during the lockdown in patients without alarming signs will be rescheduled in the next months. indeed, in may , lockdown restrictions are starting to be lifted. during the lockdown we could not use laboratory markers for colorectal cancer surveillance because they are not currently available and standard faecal tests (that is, immunological tests such as enzyme-linked immunosorbent assay (elisa)) for screening of colorectal cancer used for the general population cannot be used in patients with ibd ; indeed, patients with ibd are excluded from national screening programmes. thus, faecal tests cannot be an alternative for dysplasia screening in patients with ibd during the pandemic and the procedures will have to be rescheduled once it ends. however, ten hove et al. demonstrated, in a large surveillance cohort of patients with ibd with colonic disease and no additional high-risk features, that two consecutive negative colonoscopies predict a very low risk of colorectal cancer occurrence on follow-up . the interval www.nature.com/nrgastro between the first colonoscopy and the subsequent surveillance examination was . years and the duration of follow-up after the first surveillance procedure was . years. once the covid- emergency is over and endoscopic departments reopen for patients with ibd, there will be a high number of procedures to recover; thus, a change in surveillance intervals could be useful to better manage the endoscopy department's new agenda in prioritizing patients . indeed, during the covid- pandemic, a new stratification of patients could be an option in a selected population and might be safe, excluding high-risk patients such as those with psc or patients with previous dysplastic lesions. if the pandemic lasts more than months we suggest not to further delay surveillance colonoscopy for patients with ibd and concomitant psc or with prior dysplasia because a time frame longer than months would not be acceptable given the risk of colorectal cancer development. in that case, these patients will be considered as a priority for endoscopic examination. obstructive symptoms, such as vomiting, nausea and abdominal pain, require a rapid intervention. in patients with crohn's disease with a known stricture, management is generally conservative: gastrointestinal nasogastric decompression, bowel rest, intravenous fluids and electrolyte replacement, guided by test results . in cases of repeated obstructive episodes, if the stricture is < cm, endoscopic dilatation is a therapeutic option. if severe and disabling obstructive symptoms are present, a planned endoscopic dilatation should be performed to avoid intestinal occlusion and subsequent admission to the emergency room. in the covid- era, symptomatic patients might have to attend a hospital that is not dedicated to ibd because of movement restrictions, with the risk that the acute obstructive situation could suggest an indication to surgery to a physician not experienced in ibd. thus, instead of surgery, which requires an anaesthesiologist and surgical teams available and several days for recovery and discharge, endoscopic dilatation is the best management option. the suggestion is to limit upper gastrointestinal procedures, if possible, because of the higher risk of virus transmission in cases of asymptomatic covid- -positive patients. oesophagogastroduodenoscopies are indicated in cases of acute gastrointestinal bleeding or for dilatation of symptomatic stenosis in the upper gastrointestinal tract in patients with crohn's disease . for colonoscopy, if the endoscopic procedure is not deferrable, proctosigmoidoscopy instead of full colonoscopy might be preferred, depending on the clinical situation. proctosigmoidoscopy can reduce the procedure time, the need for sedation and the need for oral bowel preparation. in many trials, the recruitment of new patients and screening visits have been discontinued by their sponsors. the status of monitoring colonoscopies for participants already recruited in clinical trials should be discussed at the local level and with trial sponsors. if the execution of endoscopy is mandatory to retain patients in the clinical trial, endoscopy might be performed. the investigational product could be the only therapeutic option for the patient and the alternative, like surgery, could be more challenging in the current pandemic. however, the benefits of avoiding surgery and/or corticosteroids by receiving trial medication must be balanced against the risk of the unknown effects of the investigational product on the course of covid- . planned trial visits, whenever possible, should occur virtually and investigation tools could be shipped to patients' homes with specific protocol amendments , . based on the italian and french experience, we propose some recommendations for every indication of endoscopy in patients with ibd (table ). in brief, we propose the performance of endoscopic examinations only for those patients for whom endoscopy is required for the commencement of appropriate treatment. the justification is that appropriate and timely treatment will decrease the time of hospitalization, or even the need for hospitalization, whilst avoiding disease complications associated with ibd. proctosigmoidoscopies for asuc, severe ulcerative colitis flares, and ulcerative colitis and crohn's disease first diagnosis (excluding mild cases) should be performed, if possible, in the centre. for moderate to severe crohn's disease flares, alternative faecal calprotectin tests and mri or ultrasonography should be discussed (table ). therapeutic endoscopies for bowel dilatation will also be maintained in symptomatic occlusive patients. telemonitoring and faecal calprotectin tests at home. a combination of pros with faecal calprotectin home test could accurately predict the presence and severity of endoscopic lesions in patients with ibd. however, all non-emergency outpatient visits for patients with ibd should be delayed or cancelled during the covid- pandemic. furthermore, standard faecal calprotectin tests require a technician to manipulate the stools to perform the test and to collect the stools from the patient; in french university hospitals, it is recommended not to collect stool samples during the pandemic . telemonitoring, using specific questionnaires and a faecal calprotectin home test, has been studied prior to the covid- era to manage patients with ibd at home and seems safe as conventional follow-up in both paediatric (teenagers) and adult populations . in a paediatric population study, children and teenagers aged - years with ibd in clinical remission at baseline were randomly assigned to follow-up by conventional visit or telemonitoring for year; no statistically significant difference was observed between the two groups in terms of flare or change in quality of life . in an adult population study, patients with ibd were randomly assigned into either a home monitoring group (patients performed a faecal calprotectin home test and completed a symptom questionnaire) or a control group (patients had their follow-up visit in the ibd centre); the study duration was months and the disease course between the two groups was similar. in the home monitoring group, patients with a higher disease burden were more adherent than those with a better health-related quality of life . the specific questionnaires used measured pro, which are important for symptom management. previously, a pro measure (monitor ibd at home questionnaire or miah) was proposed to predict endoscopic inflammation in patients with ibd . the miah combined with a faecal calprotectin home test compared with endoscopy showed an excellent diagnostic accuracy (sensitivity of . % and specificity of . % for patients with crohn's disease, and sensitivity of . % and specificity of . % for patients with ulcerative colitis). in summary, telemonitoring seems safe, effective and accurate. in the covid- era, telemonitoring could be an optimal instrument to monitor disease activity, delaying endoscopy when possible. head-to-head comparison of three different faecal calprotectin home tests, compared with measurement of calprotectin concentration with the elisa method by two experienced laboratory technicians, agreed sufficiently . the disadvantages of a faecal calprotectin home test are related to stool manipulation because stool handling is required to add a sample to the test kit. in french university hospitals it is recommended to no longer collect stool samples because of the risk of contagious sars-cov- transmission. moreover, telemonitoring could be misleading as an alternative to endoscopy because high faecal calprotectin levels could be unrelated to ibd activity and symptoms; for instance, abdominal pain or diarrhoea could be related to irritable bowel syndrome or other clinical conditions. cross-sectional imaging. bowel ultrasonography and mre are non-invasive and well-tolerated techniques shown to have the same level of accuracy in assessing and monitoring crohn's disease and ulcerative colitis disease activity compared with ileocolonoscopy . ct and ct enterography are also accurate, in particular in assessing crohn's disease complications , such as fistulae, strictures and abscesses, or in first crohn's disease diagnosis during the covid- pandemic. ct is fast and readily available, but it exposes individuals to ionizing radiation; therefore, current guidelines recommend limiting its use. in patients with ulcerative colitis, bowel ultrasonography is useful for monitoring the disease course, for assessing short-term treatment response after or months (is it possible for the exam to be performed by gastroenterologists during the follow-up visit) and it has the potential to predict the therapeutic response . during the covid- pandemic, bowel ultrasonography could become an excellent ally to assess crohn's disease activity . however, patients still have to attend to hospital to undergo bowel ultrasonography, as with endoscopy, although bowel preparation or sedation are not necessary and fasting h before the imaging exam is sufficient. furthermore, bowel ultrasonography has the advantage of a rapid assessment of disease activity (localization, extension, grade of inflammation, presence of complications such as fistula, strictures or intrabdominal abscesses). unfortunately, bowel ultrasonography is not a widespread technique worldwide; therefore, where available, mre can give a good assessment of disease activity and is also non-invasive. in all cases using cross-sectional imaging, the disadvantage is the impossibility of performing biopsies and observing the mucosa directly. these types of only imaging allow assessment of indirect signs of inflammation, such as bowel wall thickening and vascularization, mesenteric inflammation, and the presence of enlarged lymph nodes, or complications related to the disease (obstruction, fistula, abscesses). as recommended by the european association of cardiovascular imaging, to decrease the risk of peri-procedural transmission of sars-cov- between patient and health-care staff, the indication for any imaging test should be carefully considered and only tests that are 'essential' to a patient and could change patient management or care should be performed . ultrasonography, mre and ct have the potential for contamination of hospital personnel and patients by direct contact, by the contamination of equipment and facilities (all equipment has the potential to carry droplets), by patient transportation or when the procedure is time consuming (higher for mre). to clean the instrument, including the probes, warm water, a mild detergent and water-soluble disinfectant are suggested; alternatively, dedicated disinfectant wipes could be an option. the suggested ppe is the same as used in endoscopy both for hcps and for patients. to reduce contamination, for known covid- -positive patients, a good strategy is to reserve a dedicated mre, ct or ultrasonography scanner . the protection of patients with ibd requiring endoscopy is a key point to consider in their daily ibd management. measures of prevention should be planned and adopted in all 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on precautions, indications, prioritization, and protection for patients and healthcare personnel janssen and takeda; consulting fees from abbvie, ferring and gilead; and research grants from msd a. received consultancy fees from nikkiso europe and lecture fees from janssen, abbvie and pfizer. d.g. received consultancy fees from nikkiso, sofar spa, boiler spa, j&j, pfizer, takeda and roche. s.d. has served as a speaker, consultant and advisory board member for schering-plough the authors contributed equally to all aspects of the article. the authors disclose the following: f.f. received consultancy fees from msd, abbvie, janssen and amgen. l.v. has key: cord- -o k r yo authors: simpson, kenneth w. title: the role of the microbiota in feline inflammatory bowel disease date: - - journal: august's consultations in feline internal medicine, volume doi: . /b - - - - . - sha: doc_id: cord_uid: o k r yo nan the role of the microbiota in feline inflammatory bowel disease inflammatory bowel disease (ibd) is the collective term applied to a group of chronic enteropathies characterized by persistent or recurrent gastrointestinal (gi) signs and inflammation of the gi tract. it is widely accepted that ibd involves a complex interplay among host genetics, the intestinal microenvironment (principally bacteria and dietary constituents), the immune system, and environmental "triggers" of intestinal inflammation. however, the specific steps that lead to ibd and the basis for phenotypic variation and unpredictable responses to treatment are not known. recent advances in microbiology have enabled unforeseen insights into the composition and spatial distribution of intestinal bacteria, fungi, viruses, and protozoa (collectively the microbiota) in health and disease across species. - this chapter summarizes current knowledge of the role of the microbiota in feline ibd with a focus on bacteria. until recently, our knowledge of the bacterial composition of the feline gi tract was based on the culture of duodenal/ jejunal juice or mucosa, colonic contents, and feces. - these studies found that bacteroides spp., clostridium spp., enterococcus spp., streptococcus spp., fusobacteria spp., and eubacteria spp. are the most common bacteria cultured from the feline gi tract. in general, the number and type of bacteria vary according to the intestinal region, with the number and proportion of strictly anaerobic bacteria increasing from the duodenum to the colon, peaking at colony-forming unit (cfu)/g of feces. although the small intestine contains fewer bacteria than the colon or feces, bacterial numbers in the small intestines of healthy cats vary widely, from less than to greater than cfu/ml, and frequently exceed the cfu/ml reported as the upper limit of normal in healthy people. this has important implications for clinicians seeking to make diagnosis of small intestinal bacterial overgrowth (sibo). the advent of molecular microbiology has enabled the identification of bacteria using their genetic signatures without the need to grow them. culture-independent analyses have revealed that culture-based methods dramatically underestimate the diversity of the enteric flora, typically identifying only % of the bacteria recognized by their s rna signatures. in other words, culture-based methods only identify bacteria that you know how to grow. the principal cultureindependent methods used to identify bacteria are polymerase chain reaction (pcr), s ribosomal ribonucleic acid (rrna) sequencing, and fluorescence in situ hybridization (fish; box - ). the s sequencing is frequently employed as a first step to create an inventory of bacteria that are present and their relative proportions. - bacteria of interest can then be targeted for precise enumeration by pcr (primers with or without probes against a bacterial target) or fish with oligonucleotide probes directed against bacterial or s rrna. fish can also be used to gain insight into the spatial distribution of bacteria in formalin fixed biopsies. for example, are they luminal, adherent, crypt associated, or invasive? s-based studies in cats indicate that firmicutes (most are gram-positive bacteria) is the dominant bacterial phylum throughout the gi tract, with clostridiales (predominately clostridium cluster xiva) and lactobacillales (includes the enterococcaceae, lactobacillaceae, and streptococcaceae families) being the predominant orders. , , these findings broadly parallel the results of culture-based studies where clostridium spp. were identified in duodenal aspirates of more than % of cats. - enterococcus spp., streptococcus spp., and lactobacillus spp. were the dominant species from the jejunum, and enterococcus spp. and lactobacillus were the dominant species from the colon and feces. the s-based analyses to date have indicated that the microbiota tends to be more similar within an individual than when comparing the same intestinal region among different cats. fishbased studies of duodenal mucosa-associated bacteria in healthy cats have shown that the majority of bacteria are present in free and adherent mucus, with a median of low-grade, t-cell, alimentary lymphoma in cats means that careful consideration has to be given to the number and regional distribution of mucosa-associated lymphocytes (e.g., clusters of intraepithelial lymphocytes) to enable distinction between lymphoplasmacytic enteritis and lymphoma. , it is common practice for pathologists to emphasize mucosal cellularity as the dominant histopathologic feature, with scant information provided about mucosal architecture. however, the finding that abnormalities in mucosal architecture, such as villous atrophy and fusion, correlate with proinflammatory cytokine upregulation and the severity of clinical signs in cats with ibd indicates the importance of reporting architecture and cellularity. there is a paucity of studies that have evaluated the microbiota in cats with gi disease. studies to date have included client-owned cats with lymphocytic plasmacytic enteritis, less commonly neutrophilic or granulomatous ileitis or colitis; foster kittens with ill thrift; and colony-housed cats with signs of gi disease. the only culture-independent study of the duodenal mucosal bacteria in cats to date employed fish analysis to evaluate the numbers and types of bacteria associated with the duodenal mucosa and their relationship to clinical signs, histopathology, and mucosal cytokines. cats with signs of gi disease had more mucosal enterobacteriaceae than healthy cats (figure - ) . the total number of mucosal bacteria was strongly associated with changes in mucosal architecture and the density of cellular infiltrates, particularly macrophages and t cells. a subset of bacteria comprising enterobacteriaceae, escherichia coli, and clostridium spp. correlated with abnormalities in mucosal architecture (principally atrophy and fusion), proinflammatory cytokine upregulation (interleukin [il]- , - , and - ), and the number of clinical signs exhibited by the affected cats ( figure - ). this study shows that changes in the number and type of mucosa-associated bacteria are related to the presence and severity of ibd in cats and raises the possibility that abnormal mucosal flora are involved in the etiopathogenesis of feline ibd. these changes in the microbiota closely parallel the microbial shifts, termed dysbiosis, observed in people, dogs, and murine models of ibd. - , from a comparative standpoint, the histopathologic findings, microbial shifts, and cytokine profiles in cats with moderate to severe lymphoplasmacytic ibd resemble those associated with active celiac disease in people. [ ] [ ] [ ] the potential for ibd-associated dysbiosis to effect change outside of the gut is increasingly recognized. studies in cats with inflammatory liver disease and severe pancreatitis (k.w. simpson and d.c. twedt, unpublished observations) have revealed the presence of intrahepatic and intrapancreatic bacteria, notably e.coli and enterococcus spp., and raise the possibility that the inflamed, dysbiotic gut may be the source for these bacteria (figure - ). , ( - ) bacteria/mm of mucosa. in healthy cats, the total numbers of bacteria hybridizing to probes against clostridium spp., bacteroides spp., streptococcus spp., and enterobacteriaceae represented only % of bacteria hybridizing to the eub- probe. when considering the role of bacteria in ibd, it is important to understand that ibd is a term applied to a diverse group of chronic enteropathies that are characterized by persistent or recurrent gi signs and inflammation of the gi tract. ibd is often subcategorized on the basis of clinical signs (e.g., vomiting, diarrhea, and/or weight loss), clinicopathologic abnormalities (e.g., low cobalamin and/or low albumin), ultrasonographic findings (e.g., muscularis hypertrophy and/ or regional lymphadenopathy), histopathology (cellular infiltrates and mucosal architecture), the anatomic region(s) involved (e.g., proximal small intestine, ileum, and/or colon), and response to therapy (e.g., diet-responsive, antibioticresponsive, steroid-responsive, or unresponsive). , [ ] [ ] [ ] [ ] [ ] feline ibd is also considered in the context of concurrent inflammation of other organs, such as the liver and pancreas ("triaditis") and kidneys. the most commonly diagnosed form of feline ibd is lymphocytic-plasmacytic inflammation of the proximal small intestine. unfortunately, this nomenclature is somewhat misleading as the numbers of lymphocytes and plasma cells in the small intestinal mucosa of cats with and without gi signs are broadly similar. thus the term lymphoplasmacytic enteritis is often more helpful at distinguishing this group of patients from those with neutrophilic, granulomatous, or eosinophilic infiltrates, than identifying cats with increased numbers of lymphocytes and plasma cells. there is growing evidence to implicate bacteria and other infectious agents in the development of granulomatous and neutrophilic intestinal inflammation across species. nonbacterial infections associated with this type of intestinal inflammation in cats include feline infectious peritonitis, fungi, and tritrichomonas fetus. , bacterial pathogens such as salmonella, campylobacter, and yersinia have been associated with neutrophilic enteritis, whereas mycobacteria have been associated with granulomatous inflammation and regional lymphadenopathy. the discovery of invasive e. coli in dogs with granulomatous colitis has changed our perception of this disease as an idiopathic immune-mediated condition to a bacterially driven disease in a susceptible host. to date, there is only a single report of granulomatous/histiocytic ulcerative colitis in cats with invasive bacteria that responded to antibiotics. ileocolitis associated with spiral bacteria identified as an anaerobiospirillum spp. has been described in six cats. four cats had clinical signs related to the gi tract, whereas two did not. the most significant and consistent histologic change was present in the colon and consisted of marked multifocal to diffuse dilations of the crypt lumina that were filled with large numbers of spiral bacteria, often accompanied by necrotic epithelial cells and degenerate leukocytes or associated with crypt abscesses consisting of independent evaluation using pcr, s sequencing, or fish analysis. , , eubacterial fish analysis of formalin-fixed tissues can be performed on the same tissue block used for histopathology. it seems a reasonable first step for screening for the presence of bacteria and their regional distribution within the gut (i.e., luminal, adherent, or invasive). for example, fish analysis enabled the identification of invasive spiral bacteria in the ileum, colon, and regional lymph nodes of a cat with pyogranulomatous ileocolitis associated with recurrent episodes of fever and leukocytosis whose inflammatory process was considered sterile on the basis of histopathology (see figure - c). fish analysis of colonic mucosa from a cat with dilated crypts and neutrophilic infiltrates enabled the detection of bacteria in and around degenerating glands (see figure - d) that responded to tylosin but not metronidazole or enrofloxacin. approximately % of foster kittens die before weeks of age, with most of these kittens demonstrating clinical signs or postmortem evidence of enteritis. , lesions observed via light microscopy in the gi tract of kittens were largely nonspecific as to etiology and characterized in many cases as necrosis of the crypt epithelium and accumulation of polymorphonuclear neutrophils. changes in the surface epithelium ranged from focal sloughing to multifocal erosions, particularly over submucosal lymphoid aggregates, to diffuse epithelial necrosis and mucosal ulceration. inflammatory cells were usually relatively sparse and consisted mainly of lymphocytes, with fewer histiocytes and polymorphonuclear neutrophils. the presence of an anaerobiospirillum spp. was demonstrated by genus-specific s pcr and electron microscopy. nucleotide sequencing of three affected cats showed a close relationship to anaerobiospirillum succiniciproducens. the colons of three clinically healthy cats without lesions and one cat with mild colitis not associated with spiral bacteria were negative for anaerobiospirillum spp. in the same assay. another study has also correlated the presence of bacteria with spiral morphology with enterocolitis in cats, but the identity of these bacteria was not determined. it is becoming apparent that routine histopathology is an insensitive means of detecting infectious agents in tissue samples with evidence of neutrophilic and granulomatous inflammation. although histochemical stains for fungi, argyrophilic and acid-fast bacteria, coronavirus, and culture may improve detection (figure - a) , it is these cases that are likely to benefit most from additional culture- consisting of mild inflammatory infiltrates and crypt abscesses. ghosh and colleagues used culture and fish analysis to characterize the ileum mucosa-associated enterococcal community of apparently healthy and terminally ill foster kittens. in healthy kittens, enterococcus hirae was the most common species of ileum mucosa-associated enterococci and was often observed to adhere extensively to the small intestinal epithelium (see figure - b). e. faecalis, with numerous virulence traits and multiple antimicrobial resistances, was more commonly isolated from the ileum mucosa of kittens with terminal illness. in addition, attachment of e. coli to the intestinal epithelium was significantly associated with terminal illness and was not observed in any kitten with adherent e. hirae. fish analysis was performed to investigate the microbiota in a colony of cats that developed severe gi disease while undergoing an experimental trial. this colony of cats, which underwent initial clinical investigations at cornell university, had an ill-defined virus-like enteropathy that was associated with multisystemic and sometimes fatal disease that led to closure of the colony and its relocation to a research facility. these cats are described by inness and colleagues as having ibd, but they clearly had an unexplained severe enteropathy that is far removed from what is considered ibd in clientowned cats. fish analysis showed total bacteria, bifidobacterium spp., and bacteroides spp. counts were higher in healthy cats when compared with affected cats, whereas desulfovibrio spp. (producers of toxic sulphides) numbers were found to be significantly higher in affected cats. the fecal microbiota of colony cats with chronic diarrhea was evaluated using massive parallel s sequencing before and after dietary modification to determine the impact of diet change and related improvement in diarrhea. alterations in the intestinal microbiota were associated with improvement in diarrhea, but their relationship to disease was unclear. from the limited clinical studies performed to date, it is clear that changes in the microbiota (i.e., dysbiosis) can accompany a variety of gi disorders in cats. it remains to be determined if dysbiosis is a cause or a consequence of gi disease in cats. recent research indicates that acute, nonspecific intestinal inflammation can induce a consistent shift in the microbiome from firmicutes to proteobacteria, accompanied by a reduction in microbial diversity and proliferation of e. coli that recapitulates the dysbiosis of ibd. there appears to be an interdependence of inflammation and dysbiosis, with inflammation promoting dysbiosis, and dysbiosis promoting inflammation. genetic susceptibility may impact the threshold for dysbiosis in response to an external trigger and may also influence the ability of an individual to resolve the selfperpetuating cycle of dysbiosis and inflammation generated by an acute insult. from a therapeutic standpoint, it is tempting to equate dysbiosis with a need for antimicrobial therapy. however, this may not be required in patients that lack evidence of invasive bacteria (e.g., neutrophilic or granulomatous inflammation, intramucosal bacteria) or enteric translocation (e.g., fever, neutrophilia, regional lymphadenopathy). because intestinal dysbiosis is an endpoint of many adverse stimuli, simply removing the initiating inflammatory stimulus, without recourse to antimicrobial intervention, may effect clinical resolution. from a mechanistic standpoint, bacteria and diet are frequently separated, but it is important to consider that they are not mutually exclusive, and the ability of diet to alter microbial populations in healthy cats is well established. [ ] [ ] [ ] [ ] changes in the microbiome are present in diet-responsive enteropathies such as celiac disease, , and responses to diet, without recourse to immunomodulatory drugs, have also been observed in people with crohn's disease. clinical signs in dogs with lymphoplasmacytic enteritis and concurrent dysbiosis can also resolve in response to a controlled diet without recourse to antimicrobial therapy. with this in mind, it is noteworthy that % of cats with chronic gi disease, which would typically be defined as low-grade lymphoplasmacytic enteritis, responded to an antigen-restricted diet without recourse to antimicrobial or immunosuppressive therapy. similar responses have also been reported in cats fed hydrolyzed diets. thus it seems prudent that cats with signs of chronic gi disease (that have undergone a thorough workup to exclude infectious or parasitic agents, non-gi disorders, exocrine pancreatic insufficiency, and intestinal structural abnormalities requiring surgery) with biopsy findings that are considered normal, minimal change, or "lymphoplasmacytic enteritis" are treated in a sequential, stepwise manner with progression to more aggressive therapeutics dictated by a lack of response. for example, dietary modification (e.g., antigen-restricted or hydrolyzed diet) and cobalamin supplementation for weeks could be followed by the addition of an antimicrobial (e.g., tylosin) for weeks, then by the addition of an immunosuppressive agent (e.g., prednisolone). this stepwise approach, which has been very effective in dogs with chronic enteropathies, will hopefully identify subsets of cats that are diet-responsive, antibiotic-responsive, prednisoloneresponsive, or unresponsive, and provide a phenotype that is important in ongoing efforts to inform understanding of the pathogenesis, diagnosis, and treatment of ibd. in cats with more severe lymphoplasmacytic enteritis, simultaneous therapy with diet, vitamins, antibiotics, and immunosuppressive agents may be prescribed, with treatments sequentially withdrawn if remission is achieved. in cats with neutrophilic or granulomatous inflammation, infectious agents must be strongly suspected. an aggressive search for organisms in the intestinal mucosa or regional lymph nodes (culture, histopathology and special stains, pcr, and fish analysis) and potential systemic dissemination is of paramount importance to enable specific therapy. for proven and suspected bacterial infections, antimicrobials are the mainstay of therapy, and choice of treatment should take into consideration the spatial distribution of bacteria (i.e., antimicrobial sensitivities determined in vitro have to be reconciled with the ability of an antimicrobial to penetrate tissues and cells harboring the bacteria). immunosuppression of these patients should be a last resort. despite great expectations that probiotic bacteria given alone or in combination with prebiotics will alleviate or prevent the dysbiosis associated with ibd or chronic enteropathies, studies that show a positive effect are lacking. some formulations have been evaluated in healthy cays and cats with chronic diarrhea, , but clinical trials in cats with ibd have not been reported to date. stepwise approach to treating cats with lymphocytic plasmacytic enteritis with diet, then antimicrobials, and then immunosuppression as the default approach in most cases. in cats with evidence of neutrophilic or granulomatous intestinal inflammation, the onus is very much on the clinician to search aggressively for infectious agents before defaulting to immunosuppression of an idiopathic disease. much remains to be learned about the complex interplay among host genetics, the intestinal microenvironment (principally bacteria and dietary constituents), the immune system, and environmental triggers of intestinal inflammation that lead to the development of ibd in cats. recent advances in microbiology provide new insights into the composition and spatial distribution of intestinal bacteria, fungi, viruses, and protozoa in health and disease. a picture is emerging that correlates changes in the relative proportions of resident enteric bacterial populations-"dysbiosis"-with clinical signs and mucosal inflammation. the role of dysbiosis in the disease process remains to be elucidated. is it a cause or a consequence, or a cause and a consequence, of intestinal disease? from a clinical perspective, it seems wise to adopt a characterization of fecal microbiota in cats using universal s rrna gene and group-specific primers for lactobacillus and bifidobacterium spp assessment of microbial diversity along the feline intestinal tract using s rrna gene analysis pitfalls and progress in the diagnosis and management of canine inflammatory bowel disease food sensitivity in cats with chronic idiopathic gastrointestinal problems subnormal concentrations of serum cobalamin (vitamin b ) in cats with gastrointestinal disease feline idiopathic inflammatory bowel disease: what we know and what remains to be unraveled ultrasonographic thickening of the muscularis propria in feline small intestinal small cell t-cell lymphoma and inflammatory bowel disease pancreatitis and triaditis in cats: causes and treatment the distribution of leucocyte subsets in the small intestine of healthy cats outcome of cats with low-grade lymphocytic lymphoma: cases feline gastrointestinal lymphoma: mucosal architecture, immunophenotype, and molecular clonality inflammation drives dysbiosis and bacterial invasion in murine models of ileal crohn's disease unraveling the ties between celiac disease and intestinal microbiota intestinal t-cell responses in celiac diseaseimpact of celiac disease associated bacteria the hla-dq genotype selects for early intestinal microbiota composition in infants at high risk of developing coeliac disease evaluation of fluorescence in situ hybridization for the detection of bacteria in feline inflammatory liver disease the classification of feline colitis fluorescence in situ hybridization for identification of tritrichomonas foetus in formalin-fixed and paraffin-embedded histological specimens of intestinal trichomoniasis first description of mycobacterium heckeshornense infection in a feline immunodeficiency viruspositive cat feline histiocytic colitis: a case report with electron microscopy ileocolitis associated with anaerobiospirillum in cats chronic gastroenterocolitis in nine cats in situ molecular diagnosis and histopathological characterization of enteroadherent references enterococcus hirae infection in pre-weaningage kittens mortality in kittens is associated with a shift in ileum mucosa-associated enterococci from enterococcus hirae to biofilm-forming enterococcus faecalis and adherent escherichia coli molecular characterisation of the gut microflora of healthy and inflammatory bowel disease cats using fluorescence in situ hybridisation with special reference to desulfovibrio spp fecal microbiota of cats with naturally occurring chronic diarrhea assessed using s rrna gene -pyrosequencing before and after dietary treatment bacterial flora in the duodenum of healthy cats, and effect of dietary supplementation with fructo-oligosaccharides dietary protein concentration affects intestinal microbiota of adult cats: a study using dgge and qpcr to evaluate differences in microbial populations in the feline gastrointestinal tract effects of dietary fiber on the feline gastrointestinal metagenome the gut microbiome of kittens is affected by dietary protein:carbohydrate ratio and associated with blood metabolite and hormone concentrations efficacy of a commercial hydrolysate diet in eight cats suffering from inflammatory bowel disease or adverse reaction to food the relationship of serum cobalamin to methylmalonic acid concentrations and clinical variables in cats open-label trial of a multi-strain synbiotic in cats with chronic diarrhea effect of feeding a selected combination of galactooligosaccharides and a strain of bifidobacterium pseudocatenulatum on the intestinal microbiota of cats key: cord- - mu tdr authors: zingone, fabiana; buda, andrea; savarino, edoardo vincenzo title: screening for active covid- infection and immunization status prior to biologic therapy in ibd patients at the time of the pandemic outbreak date: - - journal: dig liver dis doi: . /j.dld. . . sha: doc_id: cord_uid: mu tdr coronavirus disease has been recently classified as pandemic infection by the world health organization. patients with inflammatory bowel disease (ibd) are invited to follow the national recommendations as any other person. it is unclear whether a more aggressive clinical course might develop in asymptomatic covid- infected subjects during biological therapy and current evidence does not support treatment suspension. however, during pandemic, the start of treatment with immunosuppressive drugs and biologics should be postponed whenever possible and based on an individual risk assessment. when clinical conditions and the disease activity do not allow a treatment delay, before starting a biological therapy, screening of ibd patients for covid- active infection by rt-pcr should be advisable, even in absence of clinical suspicion. serum antibody testing, when available, could provide evidence of infection as well as identify patients already immune to the disease. organization. patients with inflammatory bowel disease (ibd) are invited to follow the national recommendations as any other person. it is unclear whether a more aggressive clinical course might develop in asymptomatic covid- infected subjects during biological therapy and current evidence does not support treatment suspension. however, during pandemic, the start of treatment with immunosuppressive drugs and biologics should be postponed whenever possible and based on an individual risk assessment. when clinical conditions and the disease activity do not allow a treatment delay, before starting a biological therapy, screening of ibd patients for covid- active infection by rt-pcr should be advisable, even in absence of clinical suspicion. serum antibody testing, when available, could provide evidence of infection as well as identify patients already immune to the disease. coronavirus disease has been recently classified as a pandemic infection by the world health organization and it can present with different clinical manifestations: as an asymptomatic carrier state, acute respiratory disease, and pneumonia. adults represent the population with the highest infection rate, with severe clinical courses and deaths being more likely in older patients with underlying comorbidities compared to mild cases ( ) . also, male gender seems to be at higher risk of morbidity and mortality. due to the rapid spread of the disease, governments and the medical community are taking measures to prevent transmission, from common sense recommendations to radical quarantine measures ( ) . accumulating evidence shows that patients with covid- infection may also experience gastrointestinal symptoms, including diarrhea, nausea, vomiting and abdominal discomfort prior to the common respiratory symptoms current recommendation suggests postponing the start of treatment with immunosuppressive drugs and biologics, whenever possible, based on an individual risk assessment during the covid pandemic ( - ). however, since postponing the drug start is not always possible depending on the clinical activity of the patients, particular measures should be considered in order to reduce covid- -related risks. a viral screen is commonly suggested before starting biologics ( ) due to the higher risk of serious and opportunistic infections in ibd patients ( ) ( ) ( ) , that becomes particularly high in patients older than years ( , ). since it is expected that a same higher risk might occur in sars-cov- , we believe that, at least temporarily, there is an urgent need to update the current recommendations for prebiological screening ( ) . knowledge of virus dynamics and host response is crucial to fully evaluate the impact of immunosuppressive therapies on the clinical course of covid- in ibd patients and to provide management guidance to healthcare professionals. in conclusion, before starting biological therapy, until new data will be available, using a pragmatic approach, the physician should screen all patients for active covid- by rt-pcr, even without clinical suspicion of infection. thus, we suggest updating the common screening recommended prior to biological therapy in ibd patients ( we declare no competing interests. asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus (sars-cov- ): facts and myths taking the right measures to control covid- covid- : gastrointestinal manifestations and potential fecaloral transmission evidence for gastrointestinal infection of sars-cov- . st interview covid- ecco taskforce. ecco crisis task force nd interview covid- ecco taskforce what should gastroenterologists and patients know about covid- ? british society of gastroenterology consensus guidelines on the management of inflammatory bowel disease in adults opportunistic infections with anti-tumor necrosis factoralpha therapy in inflammatory bowel disease: meta-analysis of randomized controlled trials crohn's disease activity and concomitant immunosuppressants affect the risk of serious and opportunistic infections in patients treated with adalimumab first-and second-line pharmacotherapies for patients with moderate to severely active ulcerative colitis: an updated network meta-analysis risk factors for opportunistic infections in patients with inflammatory bowel disease viral screening before initiation of biologics in patients with inflammatory bowel disease during the covid- outbreak covid- : consider cytokine storm syndromes and immunosuppression immunosuppression for hyperinflammation in covid- : a double-edged sword? temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study key: cord- -hhd f authors: fiorino, gionata; allocca, mariangela; furfaro, federica; gilardi, daniela; zilli, alessandra; radice, simona; spinelli, antonino; danese, silvio title: inflammatory bowel disease care in the covid- pandemic era: the humanitas, milan, experience date: - - journal: j crohns colitis doi: . /ecco-jcc/jjaa sha: doc_id: cord_uid: hhd f the outbreak of the covid- caused by coronavirus sars-cov , is rapidly spreading worldwide. this is the first pandemic caused by a coronavirus in history. more than confirmed cases worldwide are reported involving the sars-cov , with more than covid- -related deaths on march , . fever, chills, cough, shortness of breath, generalised myalgia, malaise, drowsiness, diarrhoea, confusion, dyspnoea, and bilateral interstitial pneumonia are the common symptoms. no therapies are available, and the only way to contain the virus spread is to regularly and thoroughly clean one’s hands with an alcohol-based hand rub or wash them with soap and water, to maintain at least m [ feet] distance from anyone who is coughing or sneezing, to avoid touching eyes, nose, and mouth, and to stay home if one feels unwell. no data are available on the risk of covid- and outcomes in inflammatory bowel disease [ibd] patients. outbreak restrictions can impact on the ibd care. we aim to give a viewpoint on how operationally to manage ibd patients and ensure quality of care in the current pandemic era. the outbreak of the new coronavirus [sars-cov ] officially named sars-cov- , which causes covid- , is rapidly spreading worldwide. this is the third serious coronavirus outbreak in less than years, following sars in - and mers in , and the first pandemic caused by a coronavirus in history. the outbreak was first reported in december in china, but it has rapidly spread to other asian countries and, since february , to italy and europe, with increasing incidence in all european countries, and now in all continents. currently [as of march , ] , more than confirmed cases worldwide are reported by the sars-cov , with more than covid- -related deaths. currently, italy is the most affected country in europe [more than cases], accounting for % of confirmed cases requiring hospitalisation for covid- , % of patients being admitted to intensive care units, and % mortality. compared with mers-cov and sars-cov, sars-cov appears to be less fatal but more contagious. the virus shares . % of its genome with the sars-cov, and is able to use all ace proteins, except for mouse ace , as an entry receptor to enter ace -expressing cells, but not cells that do not express this receptor. the sars-cov does not use other coronavirus receptors, such as aminopeptidase n [apn] and dipeptidyl peptidase [dpp ]. the main route of contamination appears to be by small virus-laden droplets displaced by airflows. however, there is increasing evidence that ace protein, which has been proven to be a cell receptor for sars-cov- , is abundantly expressed in the glandular cells of gastric, duodenal, and rectal epithelia, supporting the entry of sars-cov- into the host cells. the continuous positive detection of the viral rna from faeces suggests that the infectious virions are secreted from the virusinfected gastrointestinal cells, and therefore the faecal-oral route should be considered. , pooled analysis of confirmed covid- cases reported between january , and february , from provinces, regions, and countries outside wuhan, hubei province, china, estimates that the median incubation period is . days ( % confidence interval [ci], . to . days), and . % develops symptoms within . days [ci, . to . days] of infection. under conservative assumptions, out of every cases [ th percentile, ] will develop symptoms after days of active monitoring or quarantine. the major clinical manifestations in coronavirus infection, are fever, chills, cough, shortness of breath, generalised myalgia, malaise, drowsiness, diarrhoea, confusion, dyspnoea, and bilateral interstitial pneumonia. covid- pneumonia manifests with chest ct imaging abnormalities, even in asymptomatic patients, with rapid evolution from focal unilateral to diffuse bilateral ground-glass opacities that progress to or co-exist with consolidations within - weeks. at the moment, infection by sars-cov is diagnosed by a sars-cov nucleic acid amplification test from an oropharyngeal swab; however, the combination of amplification test and ct may improve the diagnosis of covid- , , as % of patients can have lung involvement with no symptoms, and amplification tests on biological samples might be negative in almost % of patients with infection. moreover, patients may have viral rna present in faeces and, at smaller rate, in urine for - days after the oropharyngeal swab returns to negative. , a small study on patients with different grades of severity of covid- pneumonia showed clinical characteristics of common viral pneumonias. in this study, there were statistically significant differences in the expression levels of interleukin- receptor [il- r] and il- in the serum of the three groups [p < . ], among which the critical group was higher than the severe group and the severe group was higher than the mild group. no statistically significant differences in serum levels of tumour necrosis factor-alpha [tnf-α], il- , il- , il- , hs-crp, lymphocyte count, and ldh were found among the three groups [p > . ]. low cd + t cells in blood are associated with longer virus clearance time and more severe course of the disease, resulting in a longer time when the virus may be present in stools during the rehabilitation phase. advice at the moment, no reports on ibd patients have been published and no specific recommendation can be given to ibd patients based on direct evidence. because the sars-cov infection is not an opportunistic infection and is extremely contagious, patients with ibd should follow the same recommendations given by the world health organization [who] to the general population as follows. -regularly and thoroughly clean hands with an alcohol-based hand rub or wash them with soap and water. our ibd unit follows more than patients coming from all regions of italy [almost % of patients come from other regions], and it is part of one of the largest university hospitals in lombardy. since the outbreak has started, our hospital has been massively involved in the management of covid- patients, requiring many clinicians to be reassigned to dedicated covid- infected inpatients. the overload of hospitalisations and the distraction of doctors and nurses to this emergency situation, together with the limitations in travel even within the same area, has led to the need of a rapid change in the management of ibd patients at any level. ibd patients are severely worried about the impact of their disease and medications on the risk and the prognosis of covid- , and many of them are forced to come to hospital because of active disease, complications and drug administration. in order to maintain the quality standard of care, our unit has adopted several strategies [ figure ]. in our unit, two physicians have been assigned to the inpatient care. three physicians have been assigned to the infusion/clinical trial unit and endoscopy and two physicians to the remote monitoring. since the multidisciplinary team [mdt] meetings can increase the risk of contacts among people, these have been converted to virtual clinics. any elective surgery has been postponed, and only urgent cases are admitted and managed, both in the gastroenterology and the surgical units. one checkpoint to investigate suspected symptoms and signs of covid- , to test body temperature, and to provide personal protective equipment [ppe] has been posted at every public entrance of the hospital. all workers and patients must be checked before entering the hospital. nurses have rescheduled all patients coming for infusions in order to avoid crowding in the waiting area, and have moved infusion seats to a safe distance from each other. the access to our unit has been restricted to only patients needing infusions or clinical trial procedures, and no caregivers are allowed to stay in the area. the entire team wears ppe and strictly follows the who recommendations to prevent any contamination. our daily activity in the infusion unit has been limited to intravenous drugs for patients coming from the surrounding area. our hospital pharmacy has arranged home delivery and adequate drug supplies [at least months] to all patients receiving subcutaneous drugs. patients living in other regions who need infusions are referred to the closest ibd unit temporarily within the italian ibd network, and this also happens in case of mild-to-moderate ibd flares. patients scheduled for a follow-up visit are required to stay at home and to complete a questionnaire about ibd symptoms and quality of life, together with their routine laboratory tests, to the nurse and the dedicated doctor, who give recommendations and information about therapy and follow-up procedures. in order to limit the access to the hospital for invasive procedures, decisions are taken based on patients' reported outcomes [pro], c-reactive protein [crp] , and faecal calprotectin levels in asymptomatic patients. based on the assumption that the risk of coronavirus infection is not different between the general population and ibd patients, but that ibd flares are difficult to manage in this situation, we advise all patients to continue their therapies, especially if in remission. specific web pages are available to give patients updated information on covid- for ibd patients. the use of steroids during covid- is controversial, but it seems that low-dose and short-term steroids are not associated with worse prognosis even in patients with critical covid- pneumonia, therefore they can be used to treat ibd flares in case of need. thiopurines and jak inhibitors can decrease the number of activated t-cells; however, we are not advising to stop these treatments in patients in remission with these agents, who strictly follow the preventive recommendations. since there are no data in for or against monoclonal antibodies [although antiil- agents appear to be promising for covid- pneumonia ], patients continue their treatment; but we are postponing the start of new therapies if the patients has no symptoms [i.e., prevention of postoperative recurrence in patients with low-moderate risk of recurrence]. patients strongly need to be reassured in this situation. our nurses advise patients to strictly follow the who and the italian ministry of health recommendations. patients are invited to find any general information on the national ibd society [ig-ibd], and on the patients' association [amici] websites. additional information is given by email and telephone, case by case. our hospital sends newsletters by email every day to all patients recorded in our general database. this covid- is heavily impacting on everybody's daily life, including health care professionals [hcps] and patients. ibd teams need to support political decision making to rapidly adapt priorities, and they should also adapt current ibd strategies to guarantee a minimum standard level of quality of care. collaboration and communication between hcps and patients is fundamental. the role of patients' associations together with ibd scientific societies is also crucial. therapies should not be stopped, but alternative and safer ways of administration and remote monitoring should be considered. patients should avoid leaving home especially because, beside the common risk of contracting sars-cov from air droplets, the additional risk of infection in public toilets cannot be excluded. data on the incidence and prognosis of covid- in ibd patients are strongly needed. the deadly coronaviruses: the sars pandemic and the novel coronavirus epidemic in china coronavirus covid- global cases novel coronavirus -ncov: prevalence, biological and clinical characteristics comparison with sars-cov and mers-cov a pneumonia outbreak associated with a new coronavirus of probable bat origin evidence for gastrointestinal infection of sars-cov- characteristics of pediatric sars-cov- infection and potential evidence for persistent fecal viral shedding detection of sars-cov- in different types of clinical specimens the incubation period of coronavirus disease [covid- ] from publicly reported confirmed cases: estimation and application radiological findings from patients with covid- pneumonia in wuhan, china: a descriptive study comparison of different samples for novel coronavirus detection by nucleic acid amplification tests persistence and clearance of viral rna in novel coronavirus disease rehabilitation patients implications of covid- for patients with pre-existing digestive diseases q&a on coronaviruses quality of care standards in inflammatory bowel diseases: a european crohn's and colitis organisation [ecco] position paper international organization for the study of inflammatory bowel disease [ioibd]. ioibd update on covid for patients with crohn's disease and ulcerative colitis on the use of corticosteroids for -ncov pneumonia potential benefits of precise corticosteroids therapy for severe -ncov pneumonia for the zhongnan hospital of wuhan university novel coronavirus management and research team, evidence-based medicine chapter of china international exchange and promotive association for medical and health care [cpam]. a rapid advice guideline for the diagnosis and treatment of gf and sd drafted the manuscript; ma, ff, dg, az, sr, and as reviewed the manuscript; all the authors approved the final version of the manuscript. key: cord- -gu tq n authors: martin arranz, eduardo; suarez ferrer, cristina; garcía ramírez, laura; rueda garcía, jose luis; sánchez-azofra, maría; poza cordón, joaquín; noci, jesus; zabana, yamile; barreiro-de acosta, manuel; martín-arranz, maría dolores title: management of covid- pandemic in spanish inflammatory bowel disease units: results from a national survey date: - - journal: inflamm bowel dis doi: . /ibd/izaa sha: doc_id: cord_uid: gu tq n background: the outbreak of covid- has rapidly evolved into a pandemic that has represented a challenge to health systems worldwide. inflammatory bowel disease (ibd) units have been forced to change their practices to address the disease and to ensure the quality of care. methods: we conducted a national survey among ibd gastroenterologist members of the spanish working group on crohn’s disease and colitis regarding changes of practice, ibd treatments, and diagnosis and treatment of covid- . results: we received answers from spanish hospitals. one hundred percent of the ibd units rescheduled onsite visits to telematic consultation, and elective endoscopic and surgical procedures were delayed. protective measures were also taken in the infusion units ( % of health centers) and hospital pharmacies, with . % sending subcutaneous medications to patients. no switching between intravenous and subcutaneous anti-tumor necrosis factor drugs were made. we also found that . % of ibd units advised their patients to maintain treatment if they were asymptomatic for covid- . for patients with covid- symptoms, . % of ibd units referred them to primary care or the emergency department. in addition, . % of ibd units made a covid- diagnosis through polymerase chain reaction and/or chest x-ray. modifications in ibd treatment and treatment recommended for covid- are also discussed. conclusions: we report a representative national survey of changes made in the structure, diagnosis of covid- , and modifications in ibd treatments within ibd units. in december , an alert was issued from the chinese population of wuhan warning about a severe acute respiratory syndrome caused by a new coronavirus type (sars-cov- ). this disease, which has been named coronavirus disease (covid- ), exponentially spread across all continents in just a few weeks. on march , , when approximately , people around the world were affected by covid- , the world health organization officially declared the disease a pandemic. this pandemic has affected more than . million people and caused more than , deaths as of april , . in spain, the first imported case of covid- was detected on january , , the first official death was registered on february , and months later, approximately , cases and , deaths have been documented. on march , when the pandemic reached confirmed cases in the country, the government of spain declared a state of alarm with lockdown and social distancing measures. clinical manifestations of covid- range from asymptomatic carriers or mild respiratory symptoms to cases of sars and death. several risk factors have been linked to covid- mortality, but predictors of pneumonia or severe complications of the disease are yet to be determined. the course of covid- in patients with immunemediated diseases such as inflammatory bowel disease (ibd), and especially in patients undergoing immunosuppressant or biologic therapies, is still unknown. ibd units with a high covid- burden have been forced to rapidly adapt to this new situation. progressively national and international recommendations have been issued, [ ] [ ] [ ] [ ] including changing in-person appointments to telematic consultation, either by phone or e-mail; cancellation of all elective endoscopic procedures and management of immunosuppressants and biologics; and postponement of elective surgery if possible. the goal of this survey was to compile information about organizational changes and protocols created for covid- management among the different units and sites treating ibd in spain. we designed a survey for ibd-specialized gastroenterologists, members of the spanish working group on crohn's disease and ulcerative colitis (geteccu) working in different ibd units in spain. the survey included items in sections asking about the structure of ibd units, structural changes adopted because of the current pandemic, recommendations regarding immunosuppressive and biologic treatments during the sars-cov- pandemic, adopted measures regarding suspected covid- in patients with ibd, and management of patients with ibd with confirmed covid- . the survey was scientifically approved by geteccu and sent on its behalf via e-mail to all of its members. answers were collected using google forms llc (mountain view, ca). google forms allows survey organizers to draw results from a survey and export them directly to spreadsheets. answers were obtained from march to april , . the deadline for submission was april . results from the survey are presented through descriptive analysis. continuous variables are displayed as medians and interquartile range. categorical variables are reported as percentages (%) with % confidence intervals. comparison between variables with normal distribution was done using the student t test for quantitative variables and the χ test was used for qualitative variables. normal distribution was confirmed using the shapiro-wilk test. if nonnormal distribution was detected, we used the corresponding nonparametric test. statistical analysis was done using stata for mac os (stata . ; november , stata corp llc). we considered statistical significance to be a p value of < . . we received answered surveys from geteccu members across different hospitals in spain (fig. ) . most of the respondents ( doctors, . %) are part of an ibd unit of which have received the geteccu's quality of care certification for comprehensive care ibd units. thirteen doctors ( %) attend patients in monographic ibd clinics, and the rest of the surveyed doctors ( , . %) usually attend ibd patients but only in general consultation (fig. ). the mean of ibd-dedicated gastroenterologists was . per hospital, attending ibd consultation an average . days per week (sd = . ). in addition, % of sites ( ) have a specialized ibd nurse. during the covid- crisis, % of hospitals surveyed have cancelled elective onsite consultation and changed to phone assistance, % have also attended patients via e-mail, and % have used social media to inform patients about the latest updates regarding ibd and sars-cov- . the median date that hospitals applied these measures was march (march -march ). we found that % ( hospitals) canceled outpatient clinics before the state of alarm was declared, and % ( sites) suspended them on the first working day after the state of alarm was declared (fig. ) . forty-seven percent of hospitals canceled elective endoscopic procedures on march (march -march ), and % also suspended nonemergent surgeries on march (march -march ). self-isolation recommendations for nonimmunosuppressed ibd patients were the same as those that the spanish ministry of health issued for the general population in % of the doctors surveyed. in cases ( . %), patients were also advised to wear a face mask when leaving their homes, hospitals ( . %) recommended teleworking and/ or sick leave, and respondents ( . %) applied all mentioned measures to their patients. regarding immunosuppressed ibd patients, only . % of the surveyed doctors ( respondents) did not apply additional measures in addition to general recommendations for their patients. we found that hospitals ( . %) recommended teleworking and/or sick leave, in cases ( . %) patients were advised to wear a mask in public spaces, and the remaining participants ( . %) recommended all aforementioned measures. we found that . % of sites recommended that asymptomatic patients remain on immunosuppressants to continue with the same regime, and . % ( sites) lowered the immunosuppressant dose. all surveyed doctors ( %) advised patients on biologic therapies without suspected covid- to continue treatment. all ( %) respondents also applied protective measures in the day-care unit facility for intravenous biologics: % increased the distance between seats and supplied surgical masks and % also provided a checklist regarding covid- symptoms to be filled out by patients before treatment administration. one site ( . %) used the checklist as the only preventive measure. none of the respondents recommended changing intravenous biologic treatment to equivalent subcutaneous biologic treatment. when asked about patients on subcutaneous biologics, doctors working at % of the sites noted that they had changed their usual practice with or more measures. for example, . % of respondents ( doctors) advised patients to send someone else to collect treatment if possible, . % of respondents ( ) recommended gloves and a surgical mask if patients had to go to the hospital pharmacy, and at . % of sites ( ) treatment was delivered to a patient's home by the hospital pharmacy for some or all patients. thirteen percent of sites continued their usual practice. regarding the management of ibd patients contacting their physicians with mild symptoms suggesting covid- , . % of surveyed ibd units referred patients to their primary care physician or the emergency department; of these, % ( sites) referred all immunosuppressed patients to the emergency department and % ( sites) referred all ibd patients regardless of their current treatment. at sites ( . %), the diagnosis of covid- was made directly at the ibd unit via reverse transcription polymerase chain reaction and/or chest x-ray: sites performed these tests only in immunocompromised ibd patients, and hospital performed these tests in all ibd patients with suspected sars-cov- infection. no significant differences were found regarding the management of ibd patients with suspected covid- between sites with monographic ibd consultation or an ibd unit and those attending ibd patients in general consultation. differences were also nonsignificant regarding the number of ibd-dedicated gastroenterologists per site ( . vs . ; p = . ) and the presence of an ibd nurse (p = . ). regarding ibd patients diagnosed with covid- who did not need hospitalization, . % of respondents referred patients to the local emergency department or infectious diseases unit for the prescription of experimental covid- treatments. we found that . % also referred patients to their primary care physician. only . % of respondents prescribed specific covid- treatment without referral. preferred treatments for mild cases of covid- in nonhospitalized ibd patients are summarized in fig. . these regimes have likely changed over time as more information regarding covid- and its experimental treatments has become available during these past weeks. answers from respondents related to the management of ibd therapies in patients with suspected or confirmed sars-cov- infection are shown in table . no statistically significant differences were found between respondent sites in any of the proposed clinical situations regarding covid- and ibd. the covid- pandemic has severely affected spain, with , cases confirmed by polymerase chain reaction and , deaths registered up to april , , and has forced all hospitals to change their clinical practice, suspending all ambulatory procedures to minimize the risk of infection and relocating doctors and nurses to attend covid- patients. evidence about the clinical course and prognosis of covid- in ibd patients is lacking. though preliminary data [ ] [ ] [ ] suggest that covid- has the same course as in the general population, strict surveillance of ibd patients is advised because of the high proportion of immunosuppressive therapies and increased risk of opportunistic infections. in addition, covid- can also cause gastrointestinal symptoms mimicking an ibd flare, such as diarrhea, nausea, or vomiting. because of these considerations, specialized ibd units have been required to make changes beyond general population measures. some ibd units have published their experience during this pandemic, , , and national and international associations have issued recommendations regarding sars-cov- and ibd. [ ] [ ] [ ] [ ] this survey reflects the early adjustment of spanish ibd units to this exceptional situation. in most cases, changes were adopted before official recommendations were published. the main measures adopted were the fast change to telematic assistance, cancelling ambulatory elective endoscopic and surgical procedures, and making structural changes in outpatient facilities and hospital pharmacies regarding administration and delivery of biologics. differences in adoption dates for these measures can be explained because of geographic variability in the arrival of the epidemic. even if the peak of the epidemic is contained, many of these changes will have to remain active to minimize the risk of infection among patients during the following months. regarding the diagnosis of covid- in ibd, most ibd units referred patients to other departments, such as primary care, emergency, or infectious diseases departments. only hospitals carried out diagnostic procedures directly at the ibd consultation; all of these have daily onsite ibd assistance. there is also great variability concerning the management of ibd therapies depending on the clinical scenario, probably because of the lack of scientific evidence, the variation in local protocols, and the different official recommendations published during the pandemic. the main strength of this study is the large number of respondents, with more than ibd units from most regions in spain, so the sample provides very good representativeness of the situation in spanish ibd units during the epidemic. this survey has some limitations. first, most of the answers come from ibd units in tertiary hospitals; this may constitute a potential bias regarding available resources for patient management. the epidemiological variability between regions can also affect answers, as the different incidences of covid- also imply different burdens on the health care system. finally, experimental options to treat sars-cov- infection have continuously changed during the pandemic, so items on the survey regarding treatment would have different answers depending on the reply date. the worldwide pandemic caused by covid- has forced ibd units across spain to abruptly change their daily clinical practice, mainly by adopting telematic assistance as their routine form of consultation during these weeks. spanish ibd centers have developed a fast, widespread, uniform response to this pandemic. there is variability regarding the management of treatments in ibd patients affected by covid- . more evidence is needed to define protocols for this matter. maps and trends. coronavirus resource center clinical characteristics of coronavirus disease in china clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study challenges in the care of ibd patients during the covid- pandemic: report from a "red zone" area in northern italy resumption of activity in gastroenterology departments. recommendations by sepd, aeeh, geteccu and aeg on behalf of the international organization for the study of inflammatory bowel disease. management of patients with crohn's disease and ulcerative colitis during the covid- pandemic: results of an international meeting british society of gastroenterology guidance for management of inflammatory bowel disease during the covid- pandemic aga clinical practice update on management of inflammatory bowel disease during the covid- pandemic: expert commentary current data characteristics and prognosis of patients with inflammatory bowel disease during the sars-cov- pandemic in the basque country outcomes of covid- in patients with ibd in italy: an ig-ibd study evidence for gastrointestinal infection of sars-cov- inflammatory bowel disease care in the covid- pandemic era: the humanitas, milan experience the authors acknowledge tamara verges and belen poladura for investigational support, all survey respondents, and geteccu. key: cord- -tjqpiyp authors: day, alice s.; wood, jessica a.; halmos, emma p.; bryant, robert v. title: practical guidance for dietary management of patients with inflammatory bowel disease during the sars-cov pandemic date: - - journal: j acad nutr diet doi: . /j.jand. . . sha: doc_id: cord_uid: tjqpiyp abstract the recent outbreak of the severe acute respiratory syndrome coronavirus (sars-cov ) pandemic has affected almost every nation worldwide. coronavirus disease (covid- ) has resulted in considerable morbidity and mortality along with major disruption to global financial markets and social function with mandated social isolation precautions in many countries. severe covid- have been reported in older people and those with medical comorbidities, raising concern for those with pre-existing gastrointestinal illness. resulted in considerable morbidity and mortality along with major disruption to global financial markets and social function with mandated social isolation precautions in many countries. severe covid- have been reported in older people and those with medical comorbidities, raising concern for those with pre-existing gastrointestinal illness. health organization and there are > million reported cases worldwide ( th july ). although the majority of patients will experience a mild disease course, around % of symptomatic patients will require hospitalization or intensive care treatment, with a mortality rate of around % overall. the clinical manifestations of covid- include fever, chills, despite theoretical risks, current evidence does not suggest that patients with ibd have an increased risk of developing the international secure-ibd registry reported on cases of covid- in patients with ibd (on th july ), with marginally higher rates in those with crohn's disease (cd) ( / , %). most patients had mild disease and were managed as outpatients ( %). sixty deaths ( %) amongst this cohort were reported, fourteen of whom were receiving biologic therapy, which is comparable with non- ibd populations. current guidelines therefore advocate for minimizing exposure to covid- infection while continuing therapy for their underlying ibd to prevent flares and complications, including escalation of immunosuppression as clinically indicated. , it must be acknowledged that it is unlikely any high quality evidence will allow timely production of guidelines specific for ibd patients during the covid- pandemic, so observational data and anecdotal lessons learned from the first countries affected by the novel coronavirus are providing direction for the rest of the world. despite reassuring registry data and published guidelines, many patients with ibd and their treating clinicians alike may be reticent to persist with immunosuppressive therapy in the setting of the covid- pandemic. anxiety as to therapy-related vulnerability may lead to widespread and often inappropriate cessation of medical therapy, as well as exploration of non-immunosuppressive alternatives for management of ibd. dietary strategies represent an appealing option for those determined to avoid immunosuppression and for those in whom escalation of medical therapy is indicated for a disease flare. exclusive enteral nutrition therapy (een), involving consumption of only nutritional formula without intake of food for a defined period, has a strong evidence base for remission induction in cd when barriers to adherence are overcome. moreover, een has been shown to delay or avoid ibd surgery, which is critical at a time when hospitalization increases risk of covid- exposure as well as in the setting of stretched health care resources. , beyond dietary management of ibd, nutritional optimization, and evaluation and treatment of both malnutrition and obesity- related illness are important to best equip patients to face the need for dietary management of functional gut symptoms, already common in patients with ibd, is likely to increase during this period of enormous psychological stress. there is not a diet to prevent virus transmission or to reduce the severity of respiratory illness, however, there is evidence to suggest that malnutrition worsens outcomes for critically ill patients or those with respiratory disease, whilst obesity increases risk of more severe disease, and increases risk of hospitalization in younger patients. - therefore, both conversely, overweight and obesity is also highly prevalent in ibd populations, with - % reported as overweight or obese. , isolation restrictions are reported to broadly influence unhealthy dietary habits including less fresh food, increased snacking on ultra- processed foods, increased alcohol consumption and less physical activity, therefore weight gain and over nutrition in individuals with ibd is of concern during this pandemic. , , obesity is associated with persistent disease activity, has been shown to negatively impact biologic therapy and surgical outcomes, and is associated with poorer mental health (anxiety and depression). , routine nutrition screening will also assist in early identification of table ). , it must be acknowledged that these guidelines do not take into account the with a view to protracted use during the pandemic without consideration of these practical points of difference, as the duration of this pandemic is uncertain but likely to be prolonged. rather, een is best used to avoid corticosteroid therapy for remission induction, acting as a bridge to a suitable maintenance therapy. a major barrier to een is adherence, particularly in adults, although remission rates of - the global pandemic has resulted in a huge change in almost all of our activities of daily living, including work, studies, social interactions, physical activity, and even access to essential shopping items and services. it is foreseeable that this pandemic may increase exacerbations of mental health disorders and associated conditions. indeed, survey data on in many countries, safety measures have been put in place to limit community transmission of coronavirus, including use of telehealth for medical services and restrictions on use of endoscopy for ibd disease activity assessment. fortunately, dietitians have the advantage of not needing to physically examine patients, so a switch to telehealth is generally straightforward, aside from an inability to assess nutritional status by physical examination. cost and/or waiting lists may be a barrier to this new format of health care delivery, . consider telehealth dietetic consultation for ibd patients to ensure safe and equitable access to dietetic services during this pandemic, the cost of which may be mitigated by government supported healthcare schemes. in these unprecedented times, established paradigms for delivery of care must be re-evaluated and a pragmatic and practical approach to dietary management must be taken. existing dietary recommendations must be adapted to current social, financial and health service disruptions with a specific focus on safe, adequate nutrition, and optimization of nutritional status. access to an experienced ibd dietitian is an essential service for ibd patients who may need their nutrition care plans adapted during this global health crisis. management of ibd during the covid- outbreak thank you to christopher p. filosi who kindly designed the graphic published as figure . key: cord- -aqfgl cm authors: clough, jennie n; hill, katie l; duff, alexa; sharma, esha; ray, shuvra; mawdsley, joel e; anderson, simon; irving, peter m; samaan, mark a title: managing an ibd infusion unit during the covid- pandemic: service modifications and the patient perspective date: - - journal: inflamm bowel dis doi: . /ibd/izaa sha: doc_id: cord_uid: aqfgl cm nan the covid- pandemic has posed significant challenges to the provision of inflammatory bowel disease (ibd) unit infusion services in terms of the redeployment of specialist staff and reduced capacity because of social distancing. given the recommendation for patients to remain on their usual biologic medication wherever possible, we modified our service to ensure that our patients were adequately protected on the unit and surveyed attenders regarding their views and concerns. one hundred seven ( . %) of patients who attended for an infusion between may and , , responded to our survey. during this period, patients cancelled or did not attend their infusion. patients were telephoned hours before their infusion to screen for covid- symptoms and were advised to attend the unit unaccompanied. the number of chairs in the unit was reduced to permit social distancing, and staff wore personal protective equipment for all interactions. results showed that . % of patients found it "quite easy" or "very easy" to access information about their ibd care during the pandemic, with . % accessing information available from crohn's and colitis uk, . % using the ibd registry self-evaluation tool (https://ibdregistry.org. uk/), and . % contacting our e-mail ibd helpline for advice. in addition, . % of patients felt "very" or "somewhat uncomfortable" at the prospect of attending hospital for their usual infusion, but . % felt on attending that the measures taken to reduce the risk of contact with covid- were "completely adequate." we agreed upon changes to our standard protocols to reduce the nurse contact time with each patient, including removing the requirement to wait minutes after the infusion and performing observations only at the start of the infusion unless the patient felt unwell or was having an induction infusion. according to the survey, . % and . % of patients, respectively, felt that they would want these changes to remain in place postpandemic. no adverse events were observed as a consequence of these changes. overall, only . % of patients reported being "quite" or "very concerned" about access to ibd services over the next - months, but levels of anxiety were significantly higher about the impact of the pandemic in general (fig. ) . of the patients who had had a telephone appointment, . % found it completely acceptable as a method of follow-up. although these responses represent a self-selecting group of patients, we are encouraged that the majority felt prepared to attend for their usual infusion with appropriate infection prevention precautions. changes made to infusion procedure did not result in an increased risk of adverse reaction. british society of gastroenterology guidance for management of inflammatory bowel disease during the covid- pandemic figure . levels of concern about ibd treatment during covid- . opa, outpatient appointment key: cord- - gxpeaf authors: piciucchi, matteo; sbrozzi-vanni, andrea; rossi, alice; satriano, alissa; dell’amico, iginio; francesco, vincenzo de; zullo, angelo; manta, raffaele title: preliminary considerations regarding the risk of covid- and disease severity in chronic gastrointestinal conditions date: - - journal: ann gastroenterol doi: . /aog. . sha: doc_id: cord_uid: gxpeaf the novel rna betacoronavirus sars-cov- is driving great efforts in clinical and basic research and several studies of the epidemiology, risk factors, clinical and virological features of this infection are already available. however covid- is a totally new pathological entity, and many gray areas regarding associated diseases still need to be elucidated, especially in the group of patients who suffer from preexistent gastrointestinal disease. the aim of this review is to summarize the published data on the correlation between chronic gastrointestinal disorders and covid- . the high number of infected and deceased patients during the global pandemic of the novel rna betacoronavirus, also called sars-cov- , is driving great efforts in clinical and basic research [ ] . since the discovery of sars-cov- at the end of , several studies of the epidemiology, risk factors, clinical findings, therapy and virological features of this infection have become available and are totally free to clinicians via a pubmed search [ , ] . in the great italian epidemic area, the fight against this infection is showing the first signs of improvement due to the rapid spread of knowledge and the more severe social restrictions adopted. during the first days of april , a better understanding of the mechanisms of diffusion and pathogenesis of sars-cov- infection led to a decrease in newly infected subjects and in patients' hospitalization, in both regular wards and icu recovery, and a progressive flattening in the daily number of deaths [ ] . however covid- is a totally new pathological entity and many gray areas regarding associated diseases still need to be elucidated. in particular, several risk factors that are involved in acquiring the infection and that affect the clinical course and severity of covid- related diseases have been identified (figure ), but conclusive data are still lacking [ , ] . there is growing evidence, recently summarized in a systematic review [ ] , that cardiovascular diseases, particularly arterial hypertension, diabetes and advanced age are the major negative prognostic factors for both contagion and disease severity [ ] . similarly, chronic pneumopathy, chronic kidney diseases, immunosuppression, and oncological conditions were found to be highly prevalent in covid- patients, especially in those who progress to sars and require intensive care [ ] [ ] [ ] . in contrast, there remain very few published reports about sars-cov- infection in the setting of chronic gastrointestinal (gi) disorders (table ) . a retrospective analysis of data from hospitalized covid- patients in epicentral and nonepicentral areas of china did not show a high prevalence of preexistent chronic gi comorbidities in these patients [ ] . preliminary data from the usa's centers for disease control and prevention regarding covid- patients demonstrated a negligible ( cases; . %) prevalence of underlying gi diseases [ ] . current epidemiological data from deceased patients in italy also did not report a significant rate of gi comorbidity in these patients [ ] . however, data from a multicenter chinese study found that gi diseases were the third most common comorbidity, being present in . % of hospitalized covid- patients [ ] . a higher rate ( %) of preexistent gi disorders was also reported for covid- patients in wuhan [ ] . unfortunately, neither of these studies specified which conditions were reported as chronic gi disorders. only one study specifically described a % rate of chronic gastritis and gastric ulcer in covid- patients in wuhan [ ] . moreover, none of the abovementioned studies evaluated a possible correlation between these comorbidities and the course of covid- -related diseases. as far as inflammatory bowel disease (ibd) is concerned, there are no data to suggest a higher risk of sars-cov- infection or a higher severity of related diseases in these patients, compared to the general population. to date, only one of the ibd patients in the largest ibd referral centers of china has tested positive for covid- [ , ] . current data from the secure ibd database show that worldwide barely cases of around , , identified covid- patients had underlying ibd [ ] . notably, in vitro studies suggest that a soluble form of angiotensin converting enzyme (ace) , over-expressed in both inflamed intestine and blood of ibd patients, may be a competitive ligand for sars-cov- , reducing full-length virus binding to cell surface and possibly limiting infection spread in these subjects [ ] . however, a fatal case of covid- pneumonia has recently been reported in an -year-old italian female patient, admitted during recovery from left-side ulcerative colitis reactivation treated with systemic steroid therapy [ ] . world health organization recommendations discourage steroid use in covid- , because of the risk of viral spreading, but rapid steroid tapering or discontinuation is notoriously dangerous in ibd patients with active, severe disease. therefore, in the absence of clear sars-cov- infection symptoms, it is reasonable to maintain the scheduled steroid treatment until its ending [ , ] . with regard to pediatric ibd patients, only children have had covid- globally, all with mild infection and no need for hospitalization, despite therapy with immunomodulators and/or biologics. no cases have been reported in china and south korea, but in pediatric patients biological treatment was preventively delayed, and ( %) of them showed ibd reactivation [ ] . at present, there is no evidence that would justify stopping biological and immunosuppressive treatments in epidemic areas, taking into account the high risk of ibd reactivation and hospitalization, especially in a pediatric setting. however, starting a new treatment with these drugs is not recommended in epidemic areas [ ] . based on these considerations, accurate surveillance is required in ibd patients receiving steroid, immunosuppressive or biological therapy, including strict social isolation [ , ] . as suggested by chinese and european ibd associations, patients need to be remotely managed with telephone contacts and minimal access to the hospital structure, whenever possible [ , ] . regarding the liver, it has been observed that chronic liver diseases (cirrhosis, nonalcoholic fatty liver disease, etc.) italian covid- surveillance group [ ] no gi comorbidities reported (among , deceased patients) usa centers for disease control and prevention [ ] . % (of patients) liang et al [ ] no gi comorbidities reported (among hospitalized patients) zhang et al [ ] % (of patients) chen et al [ ] % (of patients) wu et al [ ] . % (of patients) gi, gastrointestinal were present in - % of patients with covid- . on the other hand, alteration of liver enzymes, albumin reduction and increased bilirubin may occur in as many as - % of patients, more frequently in the severe form of the disease. therefore, liver function should be properly monitored during the disease so as to promptly detect any worsening. it is unclear whether the alterations of liver function depend on the viral infection or the hepatotoxicity of drugs used [ ] . mild acute pancreatitis was reported in % of patients during covid- pneumonia, and it was supposed that high ace expression in pancreatic β-cells may be a possible viral target [ , ] . β-cell injury causes an alteration of glycemic control and a worsening of preexisting diabetes in these patients. it is thus conceivable that patients affected by underlying chronic pancreatitis may have a more severe course regarding these covid- -related complications during the infection. therefore, patients with chronic pancreatitis should be appropriately informed by clinicians about this risk. at present, no data are available on sars-cov- infection in patients with celiac disease or other chronic gi diseases. in conclusion, during the new covid- pandemic emergency, the efforts of gastroenterologists should be aimed towards a better understanding of covid- -related diseases in the setting of chronic gi disorders. more specifically, prospective studies to analyze the risk of the infection and its possible course in patients with ibd, pancreatitis and liver diseases are needed in order to improve care for these patients. identification of a novel coronavirus causing severe pneumonia in human: a descriptive study epidemiological and clinical predictors of covid- italian covid- surveillance group. characteristics of covid- patients dying in italy. report based on available data on april th prediction models for diagnosis and prognosis of covid- infection: systematic review and critical appraisal prevalence of underlying diseases in hospitalized patients with covid- : a systematic review and meta-analysis prevalence of comorbidities and its effects in patients infected with sars-cov- : a systematic review and meta-analysis clinical characteristics and outcomes of hospitalised patients with covid- treated in hubei (epicenter) and outside hubei (non-epicenter): a nationwide analysis of china covid- response team. preliminary estimates of the prevalence of selected underlying health conditions among patients with coronavirus disease -united states clinical characteristics of imported cases of covid- in jiangsu province: a multicenter descriptive study epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of patients infected with sars-cov- in wuhan chinese society of ibd, chinese elite ibd union chinese ibd quality care evaluation center committee. implications of covid- for patients with pre-existing digestive diseases are patients with inflammatory bowel disease at increased risk for covid- infection? a fatal case of covid- pneumonia occurring in a patient with severe acute ulcerative colitis international organization for the study of inflammatory bowel disease. management of patients with crohn's disease and ulcerative colitis during the covid- pandemic: results of an international meeting paediatric ibd porto group of espghan. covid- and paediatric inflammatory bowel diseases: global experience and provisional guidance ioibd update on covid for patients with crohn's disease and ulcerative colitis liver injury in covid- : management and challenges pancreatic injury patterns in patients with covid- pneumonia covid- and diabetes mellitus: unveiling the interaction of two pandemics key: cord- -bukm m q authors: song, woo-jin; li, qiang; ryu, min-ok; nam, aryung; an, ju-hyun; jung, yun chan; ahn, jin-ok; youn, hwa-young title: canine adipose tissue-derived mesenchymal stem cells pre-treated with tnf-alpha enhance immunomodulatory effects in inflammatory bowel disease in mice date: - - journal: research in veterinary science doi: . /j.rvsc. . . sha: doc_id: cord_uid: bukm m q abstract canine inflammatory bowel disease (ibd) is an intractable autoimmune disorder that results in various gastrointestinal and systemic symptoms. mesenchymal stem cells (mscs), which release immunomodulatory factors such as tumor necrosis factor-α (tnf-α)-induced gene/protein (tsg- ) and prostaglandin e (pge ), have been suggested as an alternative therapeutic option for ibd treatment in veterinary medicine. furthermore, although it is known that mscs pre-treated with pro-inflammatory cytokines show enhanced anti-inflammatory properties via the secretion of soluble factors, the underlying mechanisms of ibd remain unclear. the aim of this study was to demonstrate the therapeutic effects and corresponding mechanisms of canine adipose tissue-derived (cat)-mscs stimulated with tnf-α in mouse models of ibd. mice with dextran sulfate sodium (dss)- or dinitrobenzene sulfonic acid (dnbs)-induced colitis were injected intraperitoneally with cat-mscs pre-treated with tnf-α. colitis severity was assessed and colon tissues were collected for histopathological, enzyme-linked immunosorbent assay, and flow cytometry analysis. cat-mscs stimulated with tnf-α secreted higher concentrations of immunomodulatory factors such as tsg- and pge , which play a key role in inducing phenotypic alterations in macrophages. consequently, tnf-α-pre-treated cat-mscs further regulated colonic inflammatory cytokines such as interleukin (il)- β, il- , and il- , and ameliorated dss- or dnbs-induced colitis in mice. additionally, we demonstrated that m macrophages (f / +/inos+ cells) were decreased in colon tissues from mice treated with tnf-α-pre-treated cat-mscs, whereas m macrophages (f / +/cd + cells) were increased. these results may suggest a new cell-based therapeutic option for treating ibd. canine inflammatory bowel disease (ibd), which leads to gastrointestinal or systemic clinical signs, is diagnosed by ruling out the possibility of other diseases such as infection or tumor and performing histopathological assessment (cerquetella et al., ; craven et al., ) . ibd is an intractable autoimmune disease and immunosuppressive drugs are used to reduce inflammation (allenspach et al., ; dossin and lavoue, ) . however, no alternative treatments exist for dogs with ibd that do not respond to the conventional therapies. therefore, mesenchymal stem cells (mscs) that can effectively modulate inflammation might be an alternative therapeutic option (iyer and rojas, ) . recent studies have revealed that soluble factors released by mscs such as prostaglandin e (pge ), hepatocyte growth factor, indoleamine , -dioxygenase, and tnf-stimulated gene/protein (tsg- ) contribute to immunomodulation (bassi et al., ; montemurro et al., ; teng et al., ) . therefore, mscs exert strong anti-inflammatory effects, although injected mscs did not migrate into inflamed tissue in a previous study (sala et al., ) . kang et al. and chae et al. also demonstrated that canine and feline mscs secrete soluble immunomodulatory factors (chae et al., ; kang et al., ) . in addition, our previous studies have shown that tsg- released from human and canine mscs ameliorates colitis in mice song et al., b) . our previous study demonstrated that canine mscs pre-treated with tumor necrosis factor (tnf)-α and interferon (ifn)-γ exerted enhanced anti-inflammatory effects in vitro by releasing higher concentrations of pge , an immunomodulatory factor (yang et al., ) . fan et al. also revealed that human mscs stimulated with interleukin (il)- β showed enhanced efficacy in mice with colitis (fan et al., ) . in addition, recent studies have demonstrated that mscs pre-treated with pro-inflammatory cytokines showed enhanced secretory abilities (broekman et al., ; heo et al., ) . however, few studies have assessed the therapeutic effects of pro-inflammatory cytokine-stimulated canine mscs. therefore, in this study, we used canine adipose tissue (cat)-mscs stimulated with tnf-α, and revealed the therapeutic effects and their mechanisms in two mouse models of ibd. canine adipose tissues were obtained from healthy -year-old dogs using protocols approved by the institutional animal care and use committee (iacuc) of seoul national university performed in accordance with approved guidelines. the dogs were negative for canine parvovirus, canine coronavirus, and canine distemper virus infections. mscs were isolated and cultured as previously described . briefly, adipose tissue samples were washed five times in dulbecco's phosphate buffered saline (dpbs; pan-biotech, aidenbach, germany) containing % penicillin-streptomycin (ps; pan-biotech), and cut into small pieces in a petri dish. the samples were digested with collagenase type ia ( . %, gibco/life technologies, carlsbad, ca, usa) for min at °c. the samples were neutralized with dulbecco's modified eagle's medium (dmem; pan-biotech) containing % fetal bovine serum (fbs; pan-biotech). after centrifuging the adipose tissue mixture at ×g for min, the pellet containing mscs was passed through a -μm cell strainer (thermo fisher scientific, rockford, il, usa) to remove undigested debris. cells were resuspended in dmem containing % fbs and % ps, seeded onto a cell culture dish at a density of cells/cm , and incubated at °c and % co . after days, cultures were washed with dpbs to remove non-adherent cells and incubated with fresh medium. the culture medium was changed every - days until cells reached - % confluence. the cells were then subcultured and seeded at a density of , cells/cm in culture dishes. before experimentation, the cells were characterized using flow cytometry to evaluate the expression of several stem cell markers. cells were suspended in dpbs and monoclonal antibodies against the following proteins: cluster of differentiation (cd) -fluorescein isothiocyanate (fitc), cd -fitc, cd -phycoerythrin (pe), cd -pe (bd biosciences, san diego, ca, usa), cd -fitc, and cd -allophycocyanin (ebiosciences, san diego, ca, usa). cell fluorescence was analyzed with a facs aria ii system (bd biosciences). additionally, the cells' differentiation abilities were evaluated using stempro adipogenesis, osteogenesis, and chondrogenesis differentiation kits (gibco, grand island, ny, usa) according to the manufacturer's instructions. the differentiated cells were stained with oil red o, alizarin red, and alcian blue. cat-mscs at approximately - % confluence were stimulated with canine recombinant tnf-α ( ng/ml; prospec protein specialists, nj, usa) for h. the cells were used for tnf-α-stimulated cat-msc groups. c bl/ j mice (male, -week-old) were purchased from nara biotech (seoul, korea) and housed under standard conditions (controlled temperature, humidity, and light cycle). all procedures involving mice were approved by the institutional animal care and use committee of seoul national university (protocol no. snu- - ), and the protocols were performed in accordance with approved guidelines. two different mouse models for ibd were used for this study (dextran sulfate sodium (dss)-, and dinitrobenzene sulfonic acid (dnbs)-induced colitis models), and each colitis model was made as previously described (kim et al., ; martín et al., ; morampudi et al., ; solomon et al., ) . for the first experiment, colitis was induced by % dss ( - kda; mp biomedical, solon, oh, usa) in the drinking water from day to day , whereas mice offered normal water were used as the naive group. the following experiments were performed on day : cat-mscs ( × ) stimulated with tnf-α in μl pbs; cat-mscs ( × ) in μl pbs; or an identical volume of pbs was injected intraperitoneally into the dss-induced colitis mice. for this experiment, mice were randomly divided to the following four groups: naïve (n = ), dss + pbs (n = ), dss + cat-msc (n = ), dss+ tnf-α-cat-msc (n = ). for the second experiment, dnbs (sigma-aldrich, st. louis, mo, usa) colitis was induced by rectal administration of dnbs ( mg/mouse in % ethanol) into mice. six hours after dnbs infusion, cat-mscs were administered intraperitoneally as described above. for this experiment, mice were divided into five groups: naïve (n = ), ethanol (sham; n = ), dnbs +pbs (n = ), dnbs+cat-msc (n = ), dnbs+tnf-α-cat-msc (n = ). the mice were sacrificed on day (for dss-induced colitis experiments) or day (for dnbs-induced colitis experiments), and colon tissues were collected for further processing. the disease activity index (dai) was determined by a scoring system described previously . briefly, the body weight loss (grades - ), stool consistency (grades - ), rectal bleeding (grades - ), and general activity (grades - ) were monitored every h. for histological analysis, colon tissue samples were fixed in % phosphatebuffered formaldehyde for - h, followed by embedding in paraffin, cutting into -μm sections, and staining with hematoxylin and eosin. ten fields per group were randomly selected and histological examinations were performed. colitis severity was calculated using the previously described scoring system. briefly, the extent of bowel wall thickening (grades - ), crypt damage (grades - ), and inflammatory cell infiltration (grades - ) were examined in a blind manner. tsg- and pge in the supernatants from tnf-α-pre-treated or nontreated cat-mscs were measured using a tsg- elisa kit (mybiosource, san diego, ca, usa) and pge elisa kit (cusabio biotech, md, usa), respectively. additionally, for in vivo experiments, total proteins were extracted from colon tissue samples using pro-prep protein extraction solution (intron biotechnology, seongnam, korea) and the concentrations of il- β, il- , and il- were measured using commercial elisa kits (all from ebiosciences) according to the manufacturer's instructions. to evaluate the mouse macrophage population, the following monoclonal antibody mixtures were used for the experiments: anti-f / -fitc and anti-inducible nitric oxide synthase (inos)-pe, or anti-f / -fitc and anti-cd -pe (santa cruz biotechnology, santa cruz, ca, usa) were incubated with cells isolated from digested colon tissues. flow cytometry was performed using a facs aria ii system (bd biosciences) and analyzed using flowjo software (tree star, ashland, or, usa). data are shown as the mean ± standard deviation. mean values among different groups were compared by one-way analysis of variance using graphpad prism software (v. . ; graphpad, inc., la jolla, ca, usa). p value < . was considered statistically significant. cells isolated from canine adipose tissue were assessed for msc characteristics. flow cytometry analysis showed that stem cell markers such as cd , cd , and cd were highly expressed in these cells. in contrast, there was no detectable expression of hematopoietic markers, including cd , cd , and cd (fig. a) . additionally, the cells could be differentiated into adipocytes, osteocytes, and chondrocytes (fig. b) . according to criteria established by international society for cellular therapy, the cells used in this study represent mscs. our previous studies demonstrated that secretory factors from canine mscs, such as tsg- and pge , play a key role in modulating inflammation. therefore, cat-mscs were stimulated with tnf-α, a proinflammatory cytokine, to produce more immunomodulatory factors. tsg- and pge concentrations determined from the cat-mscs pretreated with tnf-α were significantly higher than those measured from the naïve cat-mscs (fig. c) . next, we evaluated whether administering cat-mscs or tnf-α-stimulated cat-mscs could reduce colitis severity in mice. consistent with previous studies, administering cat-mscs resulted in a general reduction in body weight loss, dai, and colon length shortening in mice with dss-or dnbs-induced colitis ( fig. a -c, a-c). in addition, further improvement was confirmed in mice with colitis injected with tnf-α-stimulated cat-mscs ( fig. a-c, a-c) . additionally, histopathology in the inflamed colon tissue was evaluated. colons of mice treated with dss or dnbs showed severe submucosal thickening, crypt damage, and infiltration of inflammatory cells. in contrast, administering cat-mscs to mice with colitis resulted in a slight improvement, which was additionally enhanced by administering tnf-α-stimulated cat-mscs (fig. d, d) . we next explored whether cat-mscs or tnf-α-stimulated cat-mscs could improve the anti-inflammatory effects in dss-induced colitis. in the colons of mice treated with naïve cat-mscs, the production of il- β and il- was significantly decreased, and production of il- was increased considerably compared to that in the colons of mice treated with pbs, as expected from previous studies. similar to the above results, intestinal inflammation was further improved in colons from mice treated with tnf-α-stimulated cat-mscs (fig. a-c) . given that the cytokines (il- β, il- , and il- ) modulated in the above results are mainly secreted from macrophages, we further investigated whether cat-mscs or tnf-α-stimulated cat-mscs could alter macrophage phenotypes in inflamed colon tissue. our previous study demonstrated that tsg- secreted from cat-mscs could increase the number of m macrophages in the colons of mice treated with dss. consistent with these results, our study showed that the m macrophage population (f / + /cd + ) in colons of dss-induced colitis mice treated with cat-mscs was significantly increased compared with that in mice treated with pbs. in addition, the m macrophage population (f / + /inos + ) was decreased in the cat-mscs-treated group. the phenotypic population of macrophages was further altered in the tnf-α-stimulated cat-mscs-treated group (fig. ) . recently, a number of studies have shown that mscs can effectively ameliorate ibd in rodent models (gonzalez-rey et al., ; tanaka et al., ; wang et al., ) . furthermore, administering mscs as a therapeutic option for ibd in small clinical trials (both in humans and dogs) has also shown considerable promise (kim et al., ; perez-merino et al., ) . our previous studies have demonstrated that tsg- released from human and canine mscs plays a crucial role in immunomodulation by inducing an m macrophage switch song et al., b) . in addition, we have shown that canine mscs stimulated with tnf-α and ifn-γ released higher concentration of pge which exert anti-inflammatory effects (yang et al., ) . therefore, by up-regulating the secretion of theses soluble factors, mscs can enhance the immunomodulatory effects. overall, these findings highlight the efficacy of tnf-α-stimulated cat-mscs against dss-or dnbs-induced colitis in mice. in our study, we demonstrated that intraperitoneal injection of tnfα stimulated cat-mscs resulted in higher therapeutic efficacy than injecting naïve cat-mscs in two mouse models of ibd. for example, body weight loss and dai were further improved in the tnf-α-stimulated cat-mscs-treated mice by % and %, respectively, compared to the improvements in the naive cat-mscs-treated group. in addition, evaluation of colon length and histopathologic analysis highlighted the increased therapeutic effects of tnf-α-stimulated cat-mscs. in addition, concentrations of inflammatory cytokines in the inflamed colons were significantly altered in the tnf-α-cat-msc group compared with those in the cat-msc group. previous studies have revealed that human mscs stimulated with pro-inflammatory cytokines (such as tnf-α and il- β) can improve the secretory effects of immunomodulatory soluble factors (broekman et al., ; heo et al., ) . here, we also showed that tnf-α-stimulated cat-mscs released significantly higher concentrations of tsg- ( . -fold) and pge ( -fold), relative to naive cat-mscs. tsg- and pge are well-known immunomodulatory factors secreted from human and canine mscs, and recent studies have demonstrated that these factors play important roles in ameliorating atopic dermatitis, rheumatoid arthritis, acute pancreatitis, and ibd kim et al., ; mao et al., ; shin et al., ; song et al., a) . consistent with these studies, our results indicated that cat-mscs stimulated with tnf-α further reduced dss-or dnbs-induced colitis by releasing higher concentrations of tsg- and pge . macrophages are important innate immune cells that play a key role fig. . (a, b) cells isolated from canine adipose tissues were characterized before their use in this study by flow cytometry analysis (a), as well as adipogenic, osteogenic, and chondrogenic differentiation analysis (b). (c) canine adipose tissue-derived mesenchymal stem cells (cat-mscs) stimulated with tnf-α released higher concentrations of immunomodulatory factors such as tsg- and pge compared to levels released by naive cat-mscs. results are shown as the mean ± standard deviation of three independent experiments. ***p < . . in releasing inflammatory cytokines and transferring information to acquired immune cells such as t cells. it is well-established that two types of macrophages (m and m ) are observed in inflamed tissues (mosser and edwards, ; stout and suttles, ) and these cells play an important role in regulating inflammatory responses. melief et al. demonstrated that human mscs promote the transition of monocytes into cd + m macrophages, and consequently increase foxp + regulatory t cells (melief et al., ) . in addition, recent studies have revealed that soluble factors (such as tsg- and pge ) released from human mscs could promote the m macrophage fig. . canine adipose tissue-derived mesenchymal stem cells (cat-mscs) stimulated with tnf-α showed enhanced therapeutic effects on mice with dextran sodium sulfate (dss)-induced colitis. therapeutic abilities of cat-mscs were assessed by measuring body weight changes (a), disease activity index (b), colon length (c), and histopathologic analysis (d). four to six mice per group were used. results are shown as the mean ± standard deviation. *p < . , **p < . , ***p < . . fig. . canine adipose tissue-derived mesenchymal stem cells (cat-mscs) stimulated with tnf-α showed enhanced therapeutic effects on mice with dinitrobenzene sulfonic acid (dnbs)-induced colitis. therapeutic abilities of cat-mscs were assessed by body weight changes (a), disease activity index (b), colon length (c), and histopathologic analysis (d). four to six mice per group were used. results are shown as the mean ± standard deviation. *p < . , **p < . , ***p < . . phenotype and reduce inflammation in mouse models of rheumatoid arthritis, wound healing, and ibd (shin et al., ; song et al., b; zhang et al., ) . additionally, we previously demonstrated that tsg- released from canine mscs can induce m macrophage phenotypic changes in vitro and in vivo . in this study, tnf-α stimulated cat-mscs increased macrophage alteration to the m phenotype (f / + /cd + ) in the colons of mice with ibd, whereas numbers of m macrophages (f / + /inos + ) decreased in the inflamed colons. consistent with previous studies and our results, tnf-αstimulated cat-mscs reduced inflammation through altering macrophage phenotypic changes by secreting higher concentrations of tsg- and pge . recent studies have suggested other mechanisms by which mscs might help reduce colitis severity. for example, mscs may stimulate epithelial regeneration (sémont et al., ; sémont et al., ; valcz et al., ) . it is well-established that mscs upregulate ki + cells in inflamed tissues (nakagawa et al., ; wu et al., ) . in addition, chen et al. demonstrated that mscs increase lgr + intestinal stem cells in colonic tissues of ibd model mice (chen et al., ) . another potential mechanism of msc-dependent improvement in ibd involves microbiome changes. however, in this study, tnf-α-stimulated mscs, which release higher concentrations of soluble immunomodulatory factors, showed further improved colitis in mice (soontararak et al., ) . therefore, we demonstrated here that the anti-inflammatory effects of mscs play an important role in their overall therapeutic effects on colitis, although mscs might reduce ibd through various mechanisms. previous studies have revealed that a high frequency of results are shown as the mean ± standard deviation. *p < . , **p < . , ***p < . . intraperitoneally infused mscs aggregated with immune cells in the peritoneal cavity (sala et al., ; bazhanov et al., ) . in addition, our previous studies have shown that < . % of intraperitoneally injected mscs were detected in the heart, lung, liver, spleen, kidney, brain, and colon tissues (song et al., b; song et al., ) . based on these previous studies, it is tempting to speculate that most of intraperitoneally infused tnf-α-stimulated cat-mscs formed aggregates and ameliorated ibd at sites distant from the inflamed colon by releasing soluble factors such as tsg- and pge . it should be acknowledged that we were not able to perform microarray screening of tnf-α-stimulated cat-mscs, although we evaluated increased tsg- and pge from tnf-α-stimulated cat-mscs. however, it is well demonstrated that tsg- and pge secreted from mscs could induce macrophage phenotypic alterations. therefore, our findings suggest that increased tsg- and pge released from tnf-α stimulated cat-mscs play a key role in reducing inflammation in a mouse model of ibd. in summary, we demonstrated that tnf-α-stimulated cat-mscs further ameliorated ibd via their enhanced anti-inflammatory effects over naïve cat-mscs. additionally, we showed that cat-mscs pretreated with tnf-α could release higher levels of immunomodulatory factors such as tsg- and pge , which contributed to induce macrophage phenotypic alterations. these results may represent a novel cellbased therapeutic option for treating autoimmune diseases such as ibd. declarations of interest: none. this study was supported by the research institute for veterinary science and bk plus program for creative veterinary science research. pharmacokinetics and clinical efficacy of cyclosporine treatment of dogs with steroid-refractory inflammatory bowel disease exploring the role of soluble factors associated with immune regulatory properties of mesenchymal stem cells intraperitoneally infused human mesenchymal stem cells form aggregates with mouse immune cells and attach to peritoneal organs tnf-α and il- β-activated human mesenchymal stromal cells increase airway epithelial wound healing in vitro via activation of the epidermal growth factor receptor inflammatory bowel disease in the dog: differences and similarities with humans 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in colonic epithelial regeneration tgf-beta signaling-dependent alleviation of dextran sulfate sodium-induced colitis by mesenchymal stem cell transplantation mesenchymal stem cells enhance wound healing through differentiation and angiogenesis canine mesenchymal stem cells treated with tnf-α and ifn-γ enhance antiinflammatory effects through the cox- /pge pathway human gingiva-derived mesenchymal stem cells elicit polarization of m macrophages and enhance cutaneous wound healing we are very grateful to the research institute for veterinary science of seoul national university, and the bk plus program for creative veterinary science research. key: cord- -cxx pcln authors: papa, alfredo; papa, valerio; lopetuso, loris riccardo; gasbarrini, antonio; tursi, antonio title: covid- and the management of patients with inflammatory bowel disease: a practical decalogue for the post-pandemic phase date: - - journal: therap adv gastroenterol doi: . / sha: doc_id: cord_uid: cxx pcln the coronavirus disease (covid- ) pandemic caused by severe acute respiratory syndrome coronavirus (sars-cov- ) has raised several concerns for patients with chronic immune-mediated diseases, including inflammatory bowel disease (ibd). as the outbreak appears to be in the descending phase, at least in some part of the world, as in most european countries, guidance is urgently needed to provide optimal care for our ibd patients in order to gradually and safely reduce the gap in care that has been accumulated in the months of lockdown and to face all the backlogs. therefore, we have provided a decalogue of practical recommendations for gastroenterologists to manage patients with ibd in the post-peak phase of the covid- pandemic. they include all the aspects of ibd care, not only pharmacological ones but also endoscopy, surgery, psychological treatment, telemedicine, diagnostics and educational tasks provided by doctors and patient associations. dramatically. outpatient visits, colonoscopies and non-urgent surgery have been postponed, as recommended by the most qualified international societies and organizations with an interest in ibd, with the aim of avoiding patient contact with the hospital environment and allowing patients with ibd to maintain rigorous isolation and reduce the risk of contagion. [ ] [ ] [ ] [ ] as we gradually return to normal, at least in europe but also in other parts of the world where the numbers of the pandemic decrease significantly, we must face new challenges related to both uncertainty for the future, given the risk of possible new outbreaks, and the ability to reorganize clinical activities for our ibd patients to address the backlogs and reduce the possible "collateral damage" of covid- . however, for many ibd patients, the lack of face-to-face contact with the treating physicians, added to the uncertainty and fear about the future and the course of their disease, also exacerbated psychological symptoms. in this paper we provide a "decalogue" of practical advice for the management of patients with ibd in the post-pandemic phase based on the best available evidences and expert opinions. educate patients to continue to comply with the general rules for the prevention of the transmission routes of the sars-cov- include two main forms of transmission. the first is a direct person-to-person transmission route, including cough, sneeze, droplet inhalation; the second is a contact transmission, including contact with oral, nasal and eye mucous membranes and transmission by saliva or gastrointestinal secretions. since the beginning of the pandemic, the world health organization has provided general recommendations for the prevention of they include: to wash hands frequently and properly with soap or alcohol-based sanitizer, to maintain social distancing (at least m of distance), to avoid touching eyes, nose and mouth, to cover mouth and nose when coughing or sneezing, to seek medical care early when fever, cough or difficulty in breathing are recorded, to wear personal protective equipment (ppe), in particular the facial mask when social distancing is not possible to maintain or in closed places, to stay informed and follow any advice provided by own healthcare providers. all individuals, and among these particularly ibd patients due to their own frailty, must continue to adhere to these behaviors to prevent the spread of the infection. [ ] [ ] [ ] in fact, although the pandemic is in the waning phase, at least in some parts of the globe, the virus is still circulating, and the level of attention must not be lowered in order to avoid new epidemic waves. thus, gastroenterologists, in every circumstance and by all available means (outpatient visits, by telemedicine, social media, etc.) should always remind their patients to follow these recommendations to reduce the risk of spreading sars-cov- . this educational task is fundamental since prevention is still the best way to reduce the risk of contamination. the impact of the ibd treatments on covid- risk and outcome is still under debate. however, since the beginning of the pandemic, data have been accumulating and demonstrating that biological therapy, anti-tumor necrosis factor (tnf)α in particular, has not increased the risk of covid- as feared. a retrospective study from the united states including a large cohort of ibd patients reported that the incidence rate of covid- per ibd patients was of . in patients treated with anti-tnf-α and in those not in treatment with anti-tnf-α (p = . ). interestingly, a recent case report and larger studies , hypothesized that anti-tnf-α antibodies could guarantee a milder course of covid- . in fact, in the secure-ibd database (> patients enrolled), as of june , % of patients treated with anti-tnf-α agents required hospitalization, and only % experienced unfavorable outcomes, defined as intensive care unit admission, ventilator use, or death. one possible explanation for this beneficial effect is that anti-tnfα antibodies could achieve effective control of the so-called "cytokine storm" that occurs in severe covid- -related pneumonia, thereby mitigating the course of the disease. further support for the recommendation not to suspend ongoing anti-tnf-α treatment comes from a recent survey carried out on a cohort of chinese patients. ibd patients discontinued their medication during the pandemic and among these more than half ( . %) were in treatment with biological agents. the impact of medication discontinuation was devastating as . % and . % of patients with uc and cd, respectively, had an exacerbation of the disease with the need for hospitalization and surgical intervention in a not negligible percentage of cases. reassuring data also seem to be reported for tofacitinib, although on a limited number of patients. in conclusion, the data thus far suggest that anti-tnfα agents (and also the other biologics or tofacitinib) should not be discontinued since the available evidence shows that the benefits prevail in the risk/benefit ratio between the control of ibd disease activity and the potential risks of favoring sars-cov- infection. in addition, we also want to recommend not delaying the use of biologic therapies. indeed, the most important goal to obtain for ibd patients is to induce or maintain steroid-free clinical remission, particularly during the covid- pandemic. any delays in the access to biologic therapies could have a significant negative impact on disease outcomes, including a higher rate of hospitalizations, surgeries and emergency department visits related to ibd. [ ] [ ] [ ] prescribe steroids only if they are strictly needed and consider a rapid tapering of their dosage after the first description of the dramatic effect of steroids administration on the outcome of a patient with uc and covid- , also larger studies analyzed the impact of steroids on the prognosis of ibd patients affected by covid- . the secure-ibd database found that covid- cases in ibd patients treated with steroids had a worse prognosis. as of june , % of ibd patients taking oral or parenteral steroids needed hospitalization, with % experiencing unfavorable outcomes defined as intensive care unit admission, ventilator use, or death. these findings were further supported by the results of the ig-ibd study, which reported a significant association between use of steroids and covid- -related pneumonia (p = . ) and a trend towards increased mortality (p = . ). however, we cannot rule out that some confounding factors may have influenced these data. in fact, a co-existent severe exacerbation of ibd with need for steroid therapy could be the underlying factor associated with the worst prognosis of covid- . in conclusion, since there are no definitive data on whether the use of steroids increases the risk of severe covid- , it seems prudent to minimize their use and to think of therapeutic alternatives, such as biologics (e.g. anti-tnf-α) even as rescue therapy for acute severe uc or "first line" treatment for moderate-severe flare of cd or uc. however, this recommendation is based on clinical observations and expert advice and not on data from controlled studies. in addition, in patients already on steroid therapy it is advisable to progressively taper to the lowest possible dosage without any delay. keep your patients in follow-up by preferring remote methods: telemedicine, internet, social media ibds are generally managed by a traditional model, the classical outpatient face-to-face consultations. the majority of ibd centers offer the same type of routine follow-up visits for both cd and uc irrespective of demographics, disease history, geographic location and distance to the clinic. the only significant variation in the visits is the frequency of appointments, dependent on the characteristics of the disease (phenotype, activity and current treatment). as previously reported, the covid- pandemic has resulted in a dramatic shift in providing medical care. social distancing and lockdown led to a global suspension of traditional face-to-face outpatient visits, and ibd patients, as well as any patient suffering chronic disease, have suffered from this suspension. however, several digital technologies that can be applied to tackle major clinical problems and diseases are now available. these digital technologies include: the internet of things, with next-generation telecommunication networks; , big-data analytics; artificial intelligence that uses deep learning; , blockchain technology. of course, these technologies may work synergistically, enhancing the chance to manage health by using modified algorithms to ensure secured but traceable data. all these methodologies have been tested in ibd management during the covid- pandemic. in detail, during the pandemic phase, chinese clinicians communicated with ibd patients by extensively using social media (wechat) as an educational and instructional tool, with excellent results. a recent survey conducted on different countries across north and south america, europe, australia, asia and africa found that an almost complete suspension of face-to-face visits during covid- was reported by > % of the interviewed, and that telemedicine currently accounted for over half of all ibd consultations. most of them were performed by phone because . % of respondents did not have access to a video consultation setup, but many other non-integrated solutions such as gotomeeting, google, microsoft teams, wechat and whatsapp were widely used. finally, face-to-face provision remained the most popular format for ibd outpatient visits, even if it was significantly lower than at pre-pandemic times. the current question is: how has the pandemic changed the landscape of the management of ibd patients? the answer is that the covid- outbreak can be an opportunity to transform current systems of ibd care, increasing a not face-to-face management for selected indications. in the near future, until the desirable disappearance of the pandemic, a significant proportion of ibd outpatient visits should be performed through telemedicine, preferably by video consultation, in order to maintain social distancing and to avoid contact with healthcare professionals at the same time addressing backlogs. we are convinced that even in the immediate post-pandemic phase, the use of non-invasive monitoring of disease activity in patients with ibd, combined with the use of telemedicine, should be a priority pursued by gastroenterologists. therefore, we suggest for assessing clinical activity the use of patient-reported outcomes (pros) and non-invasive tests/exams such as blood tests [c-reactive protein (crp)], fecal biomarkers (calprotectin) and intestinal ultrasound. in fact, the use of non-invasive alternatives to endoscopy, at least for the monitoring of the effectiveness of therapy and to adhere to the "tight control" of disease activity, was a topic discussed even before covid- . , in detail, the symptoms to be evaluated in the pros are the number of bowel movements and abdominal pain for cd and the number of bowel movements and rectal bleeding for uc. it should also be emphasized that the symptoms of ibd exacerbation, especially if fever is present, may be difficult to distinguish from those of sars-cov- infection. furthermore, sars-cov- has been reported to cause colitis. therefore, especially if the patient needs an outpatient visit or hospitalization, it is advisable to perform a nasopharyngeal swab for the molecular research of sars-cov- as a part of the ibd flare workup. advise patients to seek psychological support if necessary mental health disorders are a current problem since a considerable number of subjects will develop severe psychological problems such as mood disorders, anxiety disorders, or posttraumatic stress disorders. in this context, ibd patients require greater attention to avoid adverse disease-related outcomes. indeed, a cross-sectional survey explored the emotional state, perception and concerns of saudi patients with ibd during the covid- crisis. many patients expressed symptoms of anxiety, although not depression. female patients, patients educated up to a high school diploma and those with indeterminate colitis were more likely to develop anxiety. another considerable issue is linked to pediatric disease and therapeutic compliance. a recent report detected preliminary consequences related to covid- lockdown in ibd children. it found a considerable percentage of patients underreporting ibd symptoms and an increased risk of reduced compliance to immunoregulatory therapies. it did not identify any new incident ibd cases, suggesting a potential diagnostic delay. of note, it did not register specific health-related quality of life red flags, with overall scores not differing from previous cohorts. these results may be partially explained by decreased stressful events, such as school, or by an increased parental closeness. in this scenario, a mindfulness-based approach could represent a powerful tool to manage ibd outcomes. mindfulness-based interventions, understood as journals.sagepub.com/home/tag an awareness of the experience of the present moment and emphasizing the attention paid to one's thoughts, bodily sensations and emotions, have shown to be effective among patients with ibd. they often combine meditation with contemporary cognitive-behavioral approaches, and several mechanisms behind their effectiveness have been identified. in fact, their positive effects on various mental health conditions have been reported in diverse clinical and non-clinical populations. overall, mindfulness-based therapy administered as part of standard clinical practice has been shown to effectively improve inflammatory biomarkers in patients diagnosed with ibd. during the lockdown until to the flattening of the pandemic curve, endoscopic examinations have been drastically reduced and limited only to the not deferrable cases. , currently, with the containment of the pandemic, the previously not performed endoscopies should be rescheduled. this scenario inevitably implies a very delicate and demanding task consisting in the shift from an open access endoscopy to a filtered access. , endoscopic exams will be rescheduled according to an order of priority decided on a case-by-case basis. in addition to emergencies, other indications are added at this stage for patients with ibd that can no longer be postponed, as they could lead to medium-and long-term negative consequences that can become irreversible. a list of the main indications, although not exhaustive, is as follows: (a) to confirm a suspected new diagnosis of ibd with moderate-to-severe symptoms; (b) to change therapy (also considering enrollment in clinical trials) or to pone indication to surgery in patients with known ibd refractory to ongoing medical treatment; (c) to assess a cd postoperative recurrence in symptomatic patients; (d) to screen for dysplasia in high-risk patients [with co-existent primary sclerosing cholangitis (pcs), persistently active disease, previous detection of dysplasia]; (e) patients with colonic stenosis documented by imaging techniques; (f) severe pouchitis. obviously, the resumption of endoscopic activity must be carried out by observing the most scrupulous recommendations for the reduction of the risk of contagion for patients and healthcare professionals. , , in particular, before access to the endoscopy room, the pretest probability of covid- should be established based on a standard questionnaire including epidemiologic and clinical risk factors, such as the appearance of suggestive symptoms in the last days (fever, cough, sore throat, loss of smell or taste, shortness of breath). as reported in point of this article, no infusion therapy should be discontinued. the same precautionary rules adopted in the pandemic peak phase must be adopted in the so-called descending phase of the epidemic curve. before access to the infusion clinic all patients should have body temperature measured and they should complete a history questionnaire (symptoms compatible with covid- ? contact with individuals with sars-cov- infection?). in addition, we retain that it is advisable, even in the absence of symptoms or previous contact with covid- patients, to propose to all patients at their first access to an infusion clinic after the pandemic to undergo serological testing for sars-cov- . for patients that will not interrupt infusion treatment it is advisable to collect serology for sars-cov- as soon as the test is available in the clinic. obviously, to ensure adequate social distancing, we recommend dividing the patients who should be given the infusion of biological therapy into -h blocks. the infusion seats should be spaced at least m apart and both the medical staff and the patients should wear face masks and wash their hands before the infusion. similarly to what happened during the lockdown period, accompanying persons should not be allowed access to the infusion clinic and the number of staff (doctor and nurse) should be limited to the minimum necessary. surgery is an irreplaceable resource in the therapeutic armamentarium for the management of patients with ibd. with the progressive reactivation of previously suspended surgical services one of the most important issues is to implement strategies to restore surgical activity safely. during the pandemic, patients with ibd on the waiting list for a planned intervention were forced, due to its cancellation, to manage with extreme difficulty the symptoms that would have been resolvable only with surgery. just to mention the most frequent clinical situations which need to prioritize surgery we remember: (a) perianal active disease with the presence of abscesses and/or perianal fistulas with local septic complications; (b) patients with cd with recurrent subocclusive episodes not responsive to medical therapy; (c) patients with uc refractory to medical therapy or with evidence of high-grade dysplasia (pending colectomy or subsequent interventions in the case of proctocolectomy with ileoanal pouch construction in two or three surgical times); (d) stenosing or penetrating cd not responsive to medical therapy. obviously, this is a non-exhaustive list of possible indications for surgery that had accumulated during the pandemic and that will be progressively disposed of based on the priority assessed case-by-case, preferably after a multidisciplinary evaluation. in the current phase, with sars-cov- still circulating, it is necessary to implement all prescribed precautions to avoid infection for both patients and surgical staff. the main recommendations, in addition to the correct indication to surgery, are: reduce the surgical staff to the bare minimum, use the prescribed ppe (surgeons should be equipped with masks with a microparticle filter as fpp or fpp ), test the patient for sars-cov- before the hospital admission with nose-pharyngeal swab (in addition to collecting the history of covid- symptoms and/or contact with infected subjects) and perform the surgery (if programmed) only after obtaining the result of the test. in order to optimize the protection of caregivers from contagious pathogens in the operating room (or), a negative-pressure or has been recommended for patients who are positive for covid- or suspected of having the infection. the use of an active smoke evacuator connected to a proper filter has been recommended for laparoscopic or robotic procedures during the covid- pandemic to reduce the spread of viral particles. while some authors have suggested that it is potentially safer to perform a closed procedure than to have an open abdomen with fluids and smoke able to spread widely, others have stated that it is safer to avoid laparoscopy because of the risk of viral spread in the pneumoperitoneum. we believe that if the surgeon cannot access the recommended precautionary tools, such as airtight ports and filters, it may be safer to offer laparotomy using full ppe. however, even during laparotomy, great care must be taken to ensure complete immediate evacuation of all diathermy/electrocautery smoke plumes. thus, the negative-pressure or again shows its value in helping achieve this safety goal. in this context, patient associations are a key link between physicians and patients and should be increasingly involved in patient education. close interaction could allow greater patient compliance with the recommendations of healthcare providers and could also help to establish longlasting, trusting relationships. patient associations generally use all available modalities such as internet, video chat, whatsapp, et cetera to obtain the maximum diffusion of information for educational purposes. we believe that this approach is most appreciated by patients in order to be kept up to date on any changes in the treatment modalities in their ibd treatment centers and about the pandemic more generally. therefore, we strongly recommend, if ever there was a need, that patient associations increase information and educational tasks with the support of their medical consultants at this particular time. journals.sagepub.com/home/tag pending the discovery of a vaccine or effective therapy against sars-cov- , we cannot rule out future re-ignitions of covid- outbreaks. however, in countries where the trend of the pandemic is waning, albeit with still circulating virus, the priority is to resume clinical activity, approaching as much as possible pre-covid- standards. therefore, as regards patients with ibd, it is necessary to provide practical guidance to gastroenterologists in order to obtain an optimal standard of care, while ensuring the safety of patients and healthcare professionals. this may require further changes in the planning of healthcare activities, both by using different prioritization criteria for outpatient visits, diagnostic tests and surgical interventions and by continuing to use treatment strategies that have worked well during the pandemic such as telemedicine, psychological support to patients and the educational function of patient associations (table ) . author contributions ap, vp, lrl, ag and at: made a substantial contribution to the concept or design of the work; or acquisition, analysis or interpretation of data; ap, vp, lrl, ag and at: drafted the article or revised it critically for important intellectual content, and approved the version to be published. table . the "decalogue": practical recommendations for the management of patients with ibd in the post-pandemic time. educate patients to continue to comply with the general rules for the prevention of covid- according to the indication of the who. remind patients at all times (outpatient visits, phone calls, chat, email, etc.) to comply with the recommendations for pandemic control provided by the who. do not discontinue undergoing biological therapy or delay new prescriptions. continue prescribing subcutaneous biological therapy and outpatient infusions. start biological therapy, if indicated, without any delay. prescribe steroids only if they are strictly needed and consider a rapid tapering of their dosage. use steroid therapy judiciously. keep your patients in follow-up by preferring remote methods: telemedicine, internet, social media. use telemedicine with its different applications for the follow-up of patients in stable remission and therapy. prefer non-invasive methods to monitor disease activity. use calprotectin, patients' reported outcomes and intestinal ultrasound as non-invasive tools for treat-to-target strategy. advise patients to seek psychological support if necessary. offer patients psychological support and the possibility of using mindfulness techniques even remotely. in more severe cases, ask for a consultation with the psychiatrist. schedule (or re-schedule) endoscopic examinations if you recognize a non-deferrable indication, giving priority to the most urgent cases. gradually resume normal endoscopic procedures for patients with ibd using a prioritization criterion assessed on a case-by-case basis. use the appropriate ppe to protect patients and healthcare professionals from the risk of contagion. infusion clinic: consider testing for sars-cov- all patients. resume (or continue) the activity of the infusion clinic, ensuring the safety of healthcare professionals and patients. recommended ppe, spacing and subjecting patients to serological tests for sars-cov- on first access or return to the clinic after lockdown. schedule (or re-schedule) surgical interventions, giving priority to the most urgent cases. promote urgent surgery postponed for the lockdown, if possible, test the patient with naso-pharyngeal swab for sars-cov- before hospitalization; gradually insert all the scheduled interventions in the list. continue educational initiatives (not necessarily related to covid- ) by involving patient associations. patient associations should offer webinars, videos, chats and other informative material to inform patients about the recommendations to be followed in "real time" to safely continue their treatments and diagnostic controls. ibd, inflammatory bowel disease; ppe, personal protective equipment; who, world health organization. coronavirus situation report clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china epidemiological, clinical and virological characteristics of cases of 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crosssectional survey on the psychological impact of the covid- pandemic on inflammatory bowel disease patients in saudi arabia epub ahead of print clinical and psychological issues in children with inflammatory bowel disease during covid- pandemic self-awareness, self-regulation, and self-transcendence (s-art): a framework for understanding the neurobiological mechanisms of mindfulness the association between anxiety, traumatic stress, and obsessive-compulsive disorders and chronic inflammation: a systematic review and metaanalysis the effect of a mindfulness-based therapy on different biomarkers among patients with inflammatory bowel disease: a randomised controlled trial endoscopy in inflammatory bowel diseases during the covid- pandemic and post-pandemic period the impact of covid- pandemic on ibd endoscopic procedures in a high planning phase two for endoscopic units in northern italy after the covid- lockdown: an exit strategy with a lot of critical issues and a few opportunities. dig liver dis. epub ahead of print covid- : long-term planning for procedure-based specialties during extended mitigation and suppression strategies. gastroenterology. epub ahead of print proposal for the return to routine endoscopy during the covid- pandemic elective surgery cancellations due to the covid- pandemic: global predictive modelling to inform surgical recovery plans covid- : elective case triage guidelines for surgical care coronavirus pandemic and colorectal surgery: practical advice based on the italian experience laparoscopy at all costs? not now during covid- outbreak and not for acute care surgery and emergency colorectal surgery: a practical algorithm from a hub tertiary teaching hospital in northern lombardy the authors declare that there is no conflict of interest. this research received no specific grant from any funding agency in the public, commercial, or notfor-profit sectors.orcid id antonio tursi https://orcid.org/ - - - key: cord- -keec fzo authors: barberio, brigida; buda, andrea; savarino, edoardo vincenzo title: should ibd patients be tested for active covid- prior starting a biological treatment? date: - - journal: gastroenterology doi: . /j.gastro. . . sha: doc_id: cord_uid: keec fzo nan we read with great interest the review article by rubin et al. aimed to provide recommendations regarding the management of patients with inflammatory bowel disease (ibd) in the era of covid- pandemic. given the rapid widespread of this infection from china to many countries in the world and our current knowledge about sarscov , these suggestions regarding patients with important comorbidity, such as immune-mediated diseases, and thus with poorer clinical outcome, are surely very welcome. in particular, although available data do not support an increased risk of contracting covid- infection in ibd patients, the international surveillance epidemiology of coronavirus under research exclusion (secure)-ibd registry reports a not reassuring % of case fatality rate and hospitalization in nearly one third of patients. moreover, a recent report from italy, showed a case fatality rate of % and an hospitalization rate of % among ibd patients with confirmed covid- followed-up at ibd referral units. we appreciate the recommendations by rubin et al, especially their clarity and practical suggestions. medications, in particular, is a priority area and the covid- pandemic has led to challenging decision-making about treatment in ibd patients. the aga suggests that ibd patients should not stop their current treatments in order to prevent infection or adverse outcome with covid- . considerations on specific drugs were also provided, although robust evidence on the impact of different immunosuppressive or immunomodulatory medications on the covid- related risk is still lacking. however, there was no specific international guidance with respect to covid- testing in patients starting immunosuppressive treatment, particularly if they are asymptomatic, and the article by rubin et al did not cover this topic either. should an asymptomatic patient be tested for sars-cov- before undergoing the induction regimen of a biologic drug? , higher concentrations of biologics are necessary for induction versus maintenance and induction dosing have been selected to minimize immunogenicity and provide serum levels higher than in maintenance. at this time the potential benefits of testing prior starting a biologic drug are not established, but there is plausible benefit to testing based on the following observations: . sars-cov- infection may be asymptomatic or minimally symptomatic; . the potential progression to severe disease of an asymptomatic infection in the setting of intensive immunosuppression; . according to disease severity, the opportunity to delay biologic treatment to allow resolution of sars-cov- infection. another important aspect to consider is the contribution of steroids, usually adopted concomitantly with infliximab during induction or as bridge therapy, to the risk of a negative outcome in patients with covid- active infection, as recently demonstrated by lukin and coworkers who showed that baseline disease activity and steroid therapy are the only two variables able to stratify the risk of covid- in patients with ibd. overall, we believe that screening for active covid- infection should be performed in order to avoid potential complications and to adjust therapy accordingly prior starting biological treatment. current recommendations for infection screening should be updated, at least temporarily, and testing for sars-cov- by oropharyngeal swab be included. j o u r n a l p r e -p r o o f aga clinical practice update on management of inflammatory bowel disease during the covid- pandemic: expert commentary secure-ibd registry: surveillance epidemiology of coronavirus (covid- ) under research exclusion outcomes of covid- in patients with ibd in italy: an ig-ibd study viral screening before initiation of biologics in patients with inflammatory bowel disease during the covid- outbreak are patients with inflammatory bowel disease at increased risk for covid- infection ? dig liver dis baseline disease activity and steroid therapy stratify risk of covid- in patients with inflammatory bowel disease key: cord- -pix hmpj authors: kennedy, nicholas a; hansen, richard; younge, lisa; mawdsley, joel; beattie, r mark; din, shahida; lamb, christopher a; smith, philip j; selinger, christian; limdi, jimmy; iqbal, tariq h; lobo, alan; cooney, rachel; brain, oliver; gaya, daniel r; murray, charles; pollok, richard; kent, alexandra; raine, tim; bhala, neeraj; lindsay, james o; irving, peter m; lees, charlie w; sebastian, shaji title: organisational changes and challenges for inflammatory bowel disease services in the uk during the covid- pandemic date: - - journal: frontline gastroenterol doi: . /flgastro- - sha: doc_id: cord_uid: pix hmpj objective: to determine the challenges in diagnosis, monitoring, support provision in the management of inflammatory bowel disease (ibd) patients and explore the adaptations of ibd services. methods: internet-based survey by invitation of ibd services across the uk from to april . results: respondents from ibd services completed the survey. the number of whole-time equivalent gastroenterologists and ibd nurses providing elective outpatient care decreased significantly between baseline (median , iqr – . and median , iqr – ) to the point of survey (median , iqr – . and median , iqr – ) in the -week period following the onset of the covid- pandemic (p< . for both comparisons). almost all ( %; / ) services reported an increase in ibd helpline activity. face-to-face clinics were substituted for telephone consultation by % and video consultation by % of services. a variation in the provision of laboratory faecal calprotectin testing was noted with % of services reporting no access to faecal calprotectin, and a further % reduced access. there was also significant curtailment of ibd-specific endoscopy and elective surgery. conclusions: ibd services in the uk have implemented several adaptive strategies in order to continue to provide safe and high-quality care for patients. national health service organisations will need to consider the impact of these changes in current service delivery models and staffing levels when planning exit strategies for post-pandemic ibd care. careful planning to manage the increased workload and to maintain ibd services is essential to ensure patient safety. what is already known on this topic ► covid- pandemic is expected to pose a myriad of challenges to national health services. ► essential services for inflammatory bowel disease (ibd) patients including outpatient care, advice lines, endoscopy and infusion units may be affected. ► this survey evaluates the challenges to ibd services during the pandemic and the adaptations to meet these challenges. ► there is significant reduction in staffing resources for the ibd team and significant increase in ibd advice line contact. ► face-to-face consultations in outpatients, non-emergency endoscopies and elective ibd have been significantly curtailed. ► there is increased uptake of telemedicine, virtual multidisciplinary team meetings and non-invasive monitoring of patients. objective to determine the challenges in diagnosis, monitoring, support provision in the management of inflammatory bowel disease (ibd) patients and explore the adaptations of ibd services. methods internet-based survey by invitation of ibd services across the uk from to april . results respondents from ibd services completed the survey. the number of whole-time equivalent gastroenterologists and ibd nurses providing elective outpatient care decreased significantly between baseline (median , iqr - . and median , iqr - ) to the point of survey (median , iqr - . and median , iqr - ) in the -week period following the onset of the covid- pandemic (p< . for both comparisons). almost all ( %; / ) services reported an increase in ibd helpline activity. face-to-face clinics were substituted for telephone consultation by % and video consultation by % of services. a variation in the provision of laboratory faecal calprotectin testing was noted with % of services reporting no access to faecal calprotectin, and a further % reduced access. there was also significant curtailment of ibdspecific endoscopy and elective surgery. conclusions ibd services in the uk have implemented several adaptive strategies in order to continue to provide safe and high-quality care for patients. national health service organisations will need to consider the impact of these changes in current service delivery models and staffing levels when planning exit strategies for post-pandemic ibd care. careful planning to manage the increased workload and to maintain ibd services is essential to ensure patient safety. the covid- pandemic has significant implications for the diagnosis and management of patients with gastrointestinal conditions including inflammatory bowel disease (ibd). healthcare systems have had to adapt rapidly to maintain provision of core services and reduce unintended consequences from the necessary diversion of resources to focus on the pandemic. how might it impact on clinical practice in the foreseeable future ► there is urgent need to review models of care and staffing levels of ibd service in planning exit strategies in the post -pandemic period. ► insights gained from the rapid adaptations by services during the peak of the pandemic may present opportunities for positive changes in ibd services. the continued accumulation of cases positive for sars-cov- and the intervention from national governments to enforce strict social isolation ('shielding') and distancing have necessitated ibd services to dramatically changing and restructuring the way they provide care for ibd patients. in addition, the rapid increase in covid- hospitalisations along with restrictions in endoscopic and surgical facilities has resulted in the redeployment of clinicians and nurses to front-line services to care for these patients with resultant impact on the delivery of ibd care. in ibd, delays in diagnosis and therapy can have serious consequences including the need for emergency surgery. patients are understandably concerned about the impact of their ibd and its treatment on their risk of severe covid- disease. however, it is important that ibd patients continue to attend for inpatient, day case and outpatient hospital care for the management of active disease and complications and for therapies such as intravenous biologics. furthermore, given that immunosuppressive and biologic agents form the cornerstone of ibd management, concerns have been raised that patients with ibd may be more susceptible to sars-cov- infection and whether they may have poorer outcomes if infected with the virus. although there are, as yet, no specific data quantifying additional risk, specialist societies and expert groups have recommended heightened vigilance. [ ] [ ] [ ] in the uk, patients categorised as high risk have been recommended for isolation ('shielding') by uk department of health and social care, requiring individual ibd services to rapidly identify individuals in this group using hospital databases and registries. the uk has a strong record of providing personalised multidisciplinary care for patients with ibd. successive ibd audits have shown improvements in resource provision (including ibd nurses) and overall quality of care. more recently, a multidisciplinary stakeholder group has proposed key quality standards for ibd care in the uk. maintaining high-quality care during the covid- pandemic will remain a constantly evolving challenge. ibd clinicians and specialist nurses across the uk have formed an ibd covid- working group to share expertise and promote a collaborative and co-ordinated nationwide approach to meet the challenges posed by the pandemic. this has enabled the development of a uk consensus on management of ibd during the covid- pandemic. the impact of the covid- pandemic on provision of ibd care has not been previously evaluated. most centres have rapidly and independently reconfigured their services guided by local management decisions based on varying service needs, redeployment of some of their staff and reconfiguration of available healthcare facilities. there has been limited opportunity or time to share experience of service reconfiguration to determine the impact across regions. we surveyed adult and paediatric gastroenterology services caring for ibd patients in the uk to assess the impact of covid- on service delivery. we developed an internet-based survey using google forms (google, california, usa) to assess changes to ibd service provision in the covid- period. this was circulated to ibd services throughout the uk through the membership of the uk ibd covid- working group and social media. the royal college of nursing (rcn) ibd specialist nurse network and the service leads of the services participating in the ibduk self-assessment were also invited to participate. furthermore, the survey was emailed to the membership of the british society of paediatric gastroenterology, hepatology and nutrition (bspghan). survey participation was voluntary, and the option was given to provide the national health service (nhs) trust identity and contact details with the option of being contacted for future surveys related to this subject. the survey was carried out between and april , which corresponded to month after the uk government decision for lockdown. the survey (included in online supplementary appendix) covered the characteristics and staffing resources of the services, the changes instituted in provision of ibd care in preparation for the covid- pandemic and the impact of the pandemic on the provision of ibd services. data were collected in google docs and then exported for analysis to microsoft excel (microsoft, washington, usa) and r v. . . (r foundation for statistical computing, vienna, austria). where more than one response was received from the same ibd service, the most recent response was used, though paediatric and adult services were counted as separate. response frequencies were tabulated and expressed as percentages of total responses; where there were incomplete responses to a question, this is reflected in the relevant denominator. wilcoxon signed-rank tests were used to compare paired continuous variables. fisher's exact test was used to compare categorical data. we received responses representing ibd services (england , scotland , wales and northern ireland ) representing approximately % of the ibd services in the uk (paediatric services % of the total). respondents included ( %) adult gastroenterologists, ( %) adult ibd nurses, ( %) paediatric gastroenterologists, ( %) paediatric ibd nurses and ibd surgeon. only the most recent response for each service was used. fifty-seven per cent ( / ) were dedicated ibd services and % ( / ) were general gastroenterology services providing ibd care. fifty-seven per cent ( / ) of the services were based in a university teaching hospital, while % ( / ) were based in district general hospitals. all services who responded were based in public hospitals in the uk nhs. the overall number of whole-time equivalent (wte) gastroenterologists and ibd nurses providing elective outpatient care decreased significantly between baseline (median , iqr - . and median , iqr - ) to the point of survey (median , iqr - . and median , iqr - ) weeks following the onset of the covid- pandemic (p< . for both comparisons). the proportion of services with more than three wte gastroenterologists providing ibd care was % ( / ) at baseline but fell to % ( / ) as a result of reconfiguration (p< . ), with % ( / ) services having no dedicated ibd clinician in the covid- era. similarly, the number of services with more than one wte ibd nurse fell from % ( / ) to % ( / ) (p< . ). eight per cent ( / ) of services stated that they had no provision for ibd nurse care. the main reasons given for the reduction in gastroenterologists and ibd nurses were redeployment ( % ( / ) and % ( / ), respectively), self-isolation due to covid- symptoms ( % ( / ) and % ( / )) and belonging to the shielding category ( % ( / ) and % ( / )). the number of wte nurses in adult ibd services dropped significantly from median (iqr - ) to (iqr - , p-value< . ). in paediatric services, the median number of nurses was (iqr - ) prior to covid- and (iqr - ) in the covid- era (p= . ). the median number of wte gastroenterologists and ibd nurses required to provide ibd care for adult patients as self-assessed by our respondents were . (iqr . - . ) and . ( . - . ), respectively. for paediatric services, the median number of wte gastroenterologists and ibd nurses required to provide ibd care was reported as . (iqr . - . ) and . ( . - . ), respectively. when asked about the possibility of reduction in staff numbers below this required number as a result of covid- , % ( / ) of services thought this was certain or highly likely for gastroenterologists and % ( / ) for ibd nurses (figure ). only % ( / ) of services reported that their ibd nurses could maintain their normal service. ibd specialist nursing support for inpatients was either not available for % ( / ) or curtailed by % ( / ) of the services. similarly, nurse-led outpatient clinics had been suspended by % ( / ) and reduced in a further % ( / ) of services. significant changes were reported in the provision of outpatient ibd clinics. no service reported continuing normal activity with routine face-to-face appointments. nine per cent of services reported running face-toface clinics with reduced capacity, and % ( / ) reported complete cancellation of routine clinics. face-to-face clinics were substituted with telephone consultation by % ( / ) and video consultation by % ( / ) of services; most services ( / ) using video were also using telephone consultations. the proportion of patients reviewed using telephone clinics was % in half of services ( / ) and above % in a further % ( / ). in contrast, only % of services ( / ) reported having access to video consultation, with the majority ( / ) having access to video facilities reporting that they used them for less than a fifth of their consultations. we observed the use of patient apps in some services with % ( / ) and % ( / ) of respondents, respectively, reporting current use or in set up. drug infusion services were relocated to a 'safer area' away from acute services by % ( / ). the majority, % ( / ), reported performing a prescreening check list for covid- before patients were invited to attend infusion services for treatment. most services ( %, / ) reported maintaining infusion intervals with 'enhanced provisions' to reduce transmission but % ( / ) reported delaying treatment. masks were reported as being used by staff in % ( / ) of services and by patients in % ( / ). seven per cent ( / ) of services reported proactively switching their patients from intravenous to subcutaneous biologics. sixty-two per cent ( / ) of services reported patient-initiated cancellation of at least some infusions; the most frequently reported proportion was approximately % of patients. patient-reported reasons for cancellation included selfisolation due to covid- symptoms and fears and concerns about therapies. iron infusion services have been completely stopped by % ( / ) or curtailed by % ( / ) of services, respectively. the majority of services ( %; / ) report an increase in ibd advice line activity, with % ( / ) reporting a more than % increase and % ( / ) reporting a more than doubling of activity (figure ). services are adapting to this increased demand using strategies such as an automated email ( %; / ), voice message response ( %; / ), more contact options ( %; / ) and additional staff overseeing/ providing ibd advice line services ( %; / ). conversely, % ( / ) have reported a reduction in the number of staff providing advice line services. twenty-seven per cent ( / ) of services reported an inability to set up new homecare services for subcutaneous biologics and immune-modulatory therapy. in addition, % ( / ) also experienced disruption to the homecare delivery provision of therapies due to provider issues ( %; / ), blood monitoring issues ( %; / ), pharmacy issues ( %; / ) and a reduced number of nurses ( %; / ). in keeping with national guidance, endoscopy activity has been significantly curtailed for ibd patients with current provision only being available for defined high priority indications (figure ). in % ( / ) of services, all ibd-related endoscopy activities have been cancelled. potential elective surgery for ibd has been put on hold/withheld in all services surveyed. indications for surgery that may be permitted include emergency small bowel resections in % of services ( / ), colectomy for acute severe colitis in % ( / ), perianal surgery in % ( / ) and colectomy for ibd dysplasia in % ( / ). in % ( / ) of services, all ibd surgery has been stopped. all ibd mdts have been cancelled in % ( / ) of the services, while % ( / ) have converted them to virtual mdts. twenty-five per cent ( / ) of services are still running face-to-face mdts, but with reduced capacity and/or social distancing. a small proportion either have put in place alternative arrangements ( %; / ) or never had mdts to start with ( %; / ). less frequent blood monitoring regimens for patients on immunomodulators have been adopted by % ( / ) of services, while % ( / ) have stopped all routine blood monitoring. the remainder of services ( %; / ) reported that they were continuing normal monitoring arrangements. there was significant variation in the provision of laboratory faecal calprotectin testing. a quarter of services ( / ) have no access to faecal calprotectin, while a further % ( / ) have reduced access. point-of-care calprotectin has been introduced in % ( / ) of services and scaled up in % ( / ); however, most services do not have access to point-ofcare calprotectin analysis. only half of the services ( %; / ) are providing access to face-to-face flare clinics. however, % ( / ) have access to blood tests in secondary care and % ( / ) to blood tests in primary care, while % ( / ) of services report no access at all to blood tests for flare. fifty-eight per cent ( / ) had access to faecal calprotectin testing (home or laboratory for flare managements). endoscopy was only being used to assess suspected flares in outpatients with known ibd in % ( / ) of services. the uk government introduced guidance on / march to protect patients at risk of contracting covid- based on emerging world data/medical advice. the concept of shielding was introduced, requesting patients in the highest risk category to withdraw from society in their own homes for a period of weeks. to support this endeavour, specialist societies including the british society of gastroenterology developed guidance to risk stratify patients. nhs trusts and health boards then had to identify the highest risk patients based on these criteria. at the time of the survey, % ( / ) of services reported having undertaken identification of high-risk patients who meet the criteria for shielding in their ibd cohorts and % ( / ) have already communicated with their highest risk patients. furthermore, % ( / ) of the services have communicated with their moderate risk patients. seventy eight per cent of services ( / ) reported an intention to participate in the secure-ibd registry (https:// covidibd. org), which is recording the number of covid cases in ibd patients; this includes % ( / ) who had already entered patients. exploration of the variation in provision of services around the uk, including faecal calprotectin, endoscopy and surgery, did not reveal any particular clustering of loss of service into one region of the country (figure ). the covid- pandemic is having a pronounced impact on the lives of patients and healthcare colorectal professionals (hcps). ibd services in the uk meanwhile have also needed to adapt their priorities rapidly and modify current models of service to ensure delivery of a minimum standard of safe and effective care. this involved an urgent redesign of clinical services with clear communication among hcps to develop an iterative model of care, responsive to the challenges posed by the unpredictable pandemic. the aim of this survey from the uk ibd covid- working group was to explore, consider and disseminate examples of dynamic models of service provision. dramatic and significant reductions in staffing levels have inevitably impacted negatively on service provision and delivery. this has affected routine care for people with ibd including disease and treatment monitoring, clinical and endoscopic assessment, endoscopic surveillance, access to elective and semiurgent surgery and multidisciplinary team working. despite this, most services have been able to adapt and have been innovative with service delivery and models of care with the aim of providing safe and effective care. the unprecedented scale of this pandemic and uncertainties driven by the absence of 'effective' treatment for covid- has important implications for contingency planning with existing, evolving and aspirational models of care delivery. important elements such as staffing levels from redeployment, provision of 'adequate' monitoring, clear routes of access to specialist advice and urgent review, and the ability to start, continue and monitor effective therapies and outcomes will need careful consideration. the high level of contact with ibd services by patients since the start of the pandemic demonstrates the ongoing requirement for suitably staffed advice lines and access to expert review, whether by telephone, video or face-to-face clinics. wherever possible, arrangements should be made to facilitate some method of ongoing regular, scheduled mdt working, for discussion of complex or concerning cases needing consensus opinion. in addition, there will arguably be benefit to all team members of accessing peer and colleague support. videoconferencing platforms provide a means to facilitate such discussions while ensuring staff can practise social distancing and, where possible and appropriate, work remotely. where scheduled mdts, either virtual or face to face, are no longer feasible due to changes in work schedules, other models of delivering care could be considered. informal arrangements such as discussion by email involving a suitable mix of specialist can provide short-term alternatives. there has been rapid uptake by services of telephone clinics, and some centres have instituted video consultations. previous studies [ ] [ ] [ ] have assessed the impact of telemedicine systems in ibd assessing feasibility, patient acceptance, effectiveness and impact on healthcare utilisation. however, there are several potential barriers and further adoption and upscaling of teleconsultation tools are urgently warranted. there is an ongoing international survey of telemedicine in ibd in the covid- era organised by the international organisation for the study of ibd. ibd patients appear to be receptive to the idea of non-face-to-face review where appropriate, with low levels of non-attendance reported to telephone and virtual appointments. reviewing patients also provides an opportunity to check their understanding of the ongoing pandemic, the impact it has on them as individuals and any effect it may have on their treatment. this should promote adherence to therapy, as well as facilitating early management of disease flares. future surveys should assess patients' preferences for telemedicine as we plan services following the covid- pandemic. ibd endoscopy practice during the ongoing covid- pandemic appears to be broadly in line with national and international consensus. - endoscopy services have been rationalised to provide the most urgent information for the safe care of patients such as in the management of acute severe colitis. similarly, elective surgery has been stopped with a focus on emergency surgery for acute severe colitis, emergency small bowel resection and drainage for perianal sepsis. there are concerns about the potential impact of delaying elective endoscopies and operations in patients with ibd. services will need to make plans for appropriate prioritisation of delayed procedures including those needing diagnosis of new ibd or/and those needing surveillance to ensure safety in the postpandemic era. currently, the uk consensus guidelines do not recommend cessation of therapies such as biologics and immunomodulators in ibd patients who currently do not have covid- . in those who stop therapies during illness with sars-cov or following a positive test, current guidelines recommend that biologics and immunomodulators are recommenced soon after cessation of symptoms. the ibd services surveyed here appear to have taken prompt action to ensure continuity of treatments in infusion units, but logistical challenges with location and delivery of treatments remain; a significant proportion of services reported difficulties in delivery of infusions. this may worsen as the pandemic progresses over time due to patient factors such as shielding, isolation due to contact and fears about safety as well as staffing-related challenges depending on the duration of the pandemic or indeed new peaks in the pandemic. adoption of subcutaneous therapies among patients starting biologic therapy may reduce the pressures on infusion units and reduce patient footfall in the hospital site. however, a concerning number are reporting difficulties in starting new home care treatments and also in the delivery of ongoing treatments, which needs addressing urgently. ibd advice lines are an immensely valuable resource for patients with ibd, and this is more so when elective activity has been curtailed as evidenced in our survey. the marked increase in the number of patients accessing ibd advice lines, coupled with a reduction in the number of staff in more than a quarter of services, is likely to impact provision of prompt support and advice to patients, who are understandably concerned about the potential impact of ibd and their medications during the covid- pandemic. services have attempted to provide more online support, but the unprecedented increase in number of contacts may prove overwhelming. it is likely that requests for advice regarding social distancing and shielding will reduce, but the number of patients contacting ibd advice lines for flare management may rise as routine outpatient clinics have been stopped or have limited access in many centres. non-invasive assessment and monitoring of ibd are critical during the covid- pandemic. hence, it is unfortunate that several services report cessation of faecal calprotectin services, mainly due to concerns regarding risks to laboratory staff, although faecooral transmission is not confirmed yet. sars-cov- has been detected in faeces even in asymptomatic patients with covid- ; however, it is not clear if this represents live virus. one option for such services is to initiate and upscale the use of point-of-care faecal calprotectin testing. a major concern highlighted by this survey is the significant reduction in both clinicians and specialist nurses available to care for ibd patients. more than % of the respondents felt that the services would remain understaffed and unable to meet the needs of ibd patients. redeployment to front-line covid- duties appears to be the main factor, and this may need addressing at unit level with measures such as creation of a designated core team of clinicians and specialist nurses to deliver ibd care, as adopted by centres in italy. we should also ensure that, where the local covid- situation permits, ibd specialist nurses and doctors are able to resume their responsibilities in ibd care. our survey has some limitations. we could not capture responses of all ibd services in the uk. we were, however, able to reach out to approximately % of uk ibd services who registered for the recent unit and patient assessment of services under ibduk (https:// ibduk. org/ services-map). the epidemic is at different stages across the uk, but as can be seen in figure , we have good geographic coverage of the country. it is possible that some of the most underresourced ibd services may also have been less likely to have someone available to complete the survey. finally, the rapidly evolving nature of data and guidelines relating to covid- in ibd make future assessment of service provision important to ensure equitable access to high-quality ibd care across the country. in this survey, we provide a comparative reference to support consistency of care across the uk during a difficult time and to offer a template to centres in other countries, which have yet to undergo such alterations. it is our hope that this will allow services to make suitable arrangements to maintain high-quality uninterrupted care for patients with ibd. the ongoing covid- pandemic has and may continue to pose myriad challenges to healthcare systems across the globe. ibd services in the uk and other countries will face unique challenges both during the peak and postpeak pandemic period with respect to responsive and responsible adaptation of service delivery. these are unprecedented and challenging times. yet, even grim challenges present opportunities not in the least with insights gleaned from rapid adaptation of models of service delivery some of which are likely to be also suitable in a post-covid- world. indeed, there may be opportunities for positive changes in ibd services resulting from this difficult time. collaborators on behalf of the uk ibd covid- working group (see appendix for details). contributors the original project was conceived by ss and the survey developed by ss, cwl, nak, rh and ly. analyses were performed by rh and nak. the initial draft of the manuscript was written by ss, rh, ly and nak. all of the remaining authors contributed to data collection and to further writing of the manuscript. the other listed contributors performed data collection. all authors and contributors were given the opportunity to review the manuscript. the authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors. competing interests none declared. provenance and peer review not commissioned; externally peer reviewed. data availability statement data are available upon reasonable request. deidentified participant data are available by request from the corresponding author. this article is made freely available for use in accordance with bmj's 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andrade, raúl; parras, fernando alberca de las; balaguer, francesc; acosta, manuel barreiro-de; bujanda, luís; gutiérrez, ana; jorquera, francisco; iglesias-garcía, julio; sánchez-yagüe, andrés; calleja, josé luis title: resumption of activity in gastroenterology departments. recommendations by sepd, aeeh, geteccu and aeg date: - - journal: nan doi: . /j.gastre. . . sha: doc_id: cord_uid: clf f t abstract the set of measures proposed by sepd, aeeh, geteccu and aeg are aimed to help departments in their resumption of usual activity. we have prepared a number of practical recommendations regarding patient management and the stepwise resumption of healthcare activity. these recommendations are based on the sparse, changing evidence available, and will be updated in the future according to daily needs and the availability of expendable materials to suit them; in each department they will be implemented depending upon the cumulative incidence of sars-cov- infection in each region, and the burden the pandemic has represented for each hospital. the general objectives of these recommendations include: • to protect our patients against the risks of infection with sars-cov- and to provide them with high-quality care. • to protect all healthcare professionals against the risks of infection with sars-cov- . • to resume normal functioning of our departments in a setting of ongoing risk for infection with sars-cov- . each region, and the burden the pandemic has represented for each hospital. the general objectives of these recommendations include: • to protect our patients against the risks of infection with sars-cov- and to provide them with high-quality care. • to protect all healthcare professionals against the risks of infection with sars-cov- . • to resume normal functioning of our departments in a setting of ongoing risk for infection with sars-cov- . keywords: sars-cov . gastroenterology departments. recommendations. resumption. restablecimiento de la actividad en los servicios de digestivo. recomendaciones de la sepd, aeeh, geteccu y aeg el artículo recoge el conjunto de medidas propuestas por la sepd, la aeeh, geteccu y la aeg que pretenden servir de ayuda a los servicios en su reincorporación a la actividad habitual. hemos confeccionado una serie de recomendaciones prácticas respecto al manejo y a la reintroducción progresiva de la actividad asistencial. estas recomendaciones están guiadas por la escasa y cambiante evidencia disponible y serán objeto de futuras actualizaciones, en base a las necesidades diarias y la disponibilidad del material fungible para adecuarse a las mismas; y se podrán implementar en cada servicio en función de la incidencia acumulada de sars-cov- en cada región y de la carga que la epidemia ha ocasionado en cada uno de los hospitales. los objetivos generales de estas recomendaciones son: • proteger a nuestros pacientes de los riesgos de la infección por sars-cov- y prestarles una atención de calidad. • recuperar el normal funcionamiento de nuestros servicios en un entorno de riesgo continuado de infección por sars-cov- . palabras clave: sars-cov- . servicios de digestivo. recomendaciones. restablecimiento. infection with the sars-cov- coronavirus and its potentially resulting disease, designated covid- , is causing significant concern among the general population, and -needless to say-healthcare professionals and patients ( , ) . in this regard, it has had a highly significant impact on our gastroenterology and hepatology departments, which have reduced both their hospitalization activity (by more than %) and the number of diagnostic/therapeutic endoscopic procedures (by more than %, unpublished data). besides affecting our activity, it also affected our work, with high numbers of gastroenterologists being moved to covid areas. finally, some -in fact many-of our colleagues have fallen ill as a consequence of caring for patients infected with sars-cov- . let us not forget that some of the procedures we carry out on a daily basis are associated with a high risk for covid- transmission ( ) ( ) ( ) . even if its incidence diminishes considerably, it will stay with us over the coming months, which should prompt us to take extreme precautions in a micro-environment with a high risk for coronavirus transmission as is the case with hospitals. times of crisis are usually accompanied by opportunities or else appropriate to reformulate activities and the way they are performed. in this crisis we had to respond to the exigencies of covid- , but must also carry on providing essential care as defined within our specialty. because of this, this document also reflects on the opportunity to incorporate telemedicine into our usual practice in order to enhance page of j o u r n a l p r e -p r o o f the care we provide to our chronic patients. since the present situation lacks consistency (different autonomous communities, hospitals, sars-cov- incidences, public/private centers, etc.), the right time to implement these recommendations may vary. be it as it may, we propose that the transition from the current state of alarm, which has brought activity in our departments to a virtually complete standstill, to a more normal situation be accomplished in three phases: activity resumption phase, stabilization phase, and normalization phase. the length of these phases is difficult to foretell in such dynamic, highly changing scenario, but will not foreseeably be shorter than - months. furthermore, when will the human and space resources redeployed to caring for covid- patients be recovered by our departments remains yet unknown. the set of measures proposed by sepd, aeeh, geteccu and aeg are aimed to help departments in their resumption of usual activity. we have prepared a number of practical recommendations regarding patient management and the stepwise resumption of healthcare activity. these recommendations are based on the sparse, changing evidence available, and will be updated in the future according to daily needs and the availability of expendable materials to suit them; in each department they will be implemented depending upon the cumulative incidence of sars-cov- infection in each region, and the burden the pandemic has represented for each hospital. the general objectives of these recommendations include:  to protect our patients against the risks of infection with sars-cov- and to provide them with high-quality care. footwear (if possible, specific for hospital use; otherwise, with shoe covers). in addition to providing protection, these measures will prevent care providers from serving as vectors for transmission in and out of hospitals. c. all healthcare personnel with respiratory symptoms and/or fever and/or suspicion of recent contact with someone infected with sars-cov- must report it at the earliest possible time to the head of their department. under no circumstances whatsoever must they go to their workplace in case of suspicion. i. wherever possible, pharmacy departments shall facilitate drug dispensation for longer periods, even home delivery, as is now the case with some hospitals. resuming activity should lead us to pender over the structure of our traditional consultation schedules. the use of telematic tools (consultations over the phone, video calls, other) should be promoted both for patient care and work meetings, as it significantly decreases exposure for both patients and care providers ( ) . objectives will be dependent on the current phase: in the first phase the primary goal is to reduce the risk for sars-cov- contagion among patients and professionals. in the second phase, and most particularly in the normalization phase, objectives will include: a) reducing non-value-added, on-site care (reporting normal results, further prescribing the same therapy, ordering supplementary examinations, etc.); b) facilitating care for patients unable to attend for work reasons; and c) reducing usual overcrowding in our clinics. some of the requirements telemedicine must meet are as follows ( , ) :  telemedicine should be considered for all intents and purposes a medical act. this type of visit must be included in electronic records and appear in the agenda as an "off-site" visit. the electronic medical record may include screen captures of the prescriptions. having contact information available is important so that instructions and prescriptions may be mailed in writing and a follow-up strategy may be established.  ideally, telematic visits should be interspersed among in-person appointments in order to prolong intervals between the latter, thus reducing the number of people in waiting rooms.  appropriate coordination should be sought with primary care centers. we suggest appointing a department coordinator for each primary care center, who should currently favor telematic visits rather than referrals. a. differentiated circuits must be maintained for patients with and without covid- . b. all admissions other than those strictly necessary should be avoided. c. in all patients admitted to hospital infection with sars-cov- must be ruled out regardless of symptoms. pcr is currently the most suitable technique but each hospital should follow their own previously approved protocol. d. since the risk of community transmission still lingers on, further rapid testing for sars-cov- at - days after admission is advisable to minimize the risk for in-hospital outbreaks. similarly, patients discharged after more than [ ] [ ] [ ] [ ] [ ] days in hospital must be tested to prevent community outbreaks. e. for patients who remain hospitalized: -only one adequately equipped physician shall enter the room. stethoscopes and any other non-expendable materials coming in contact with patients will be subsequently cleaned with hydroalcoholic solution (or a disinfectant). -attempts should be made to monitor patients using telematic or telephony devices. -limit to a minimum all testing involving patient transportation within hospitals. -invasive procedures such as placement of nasogastric or bladder tubes should be avoided whenever possible, as well as ordering excessive lab tests. -foster early discharge and home hospitalization. f. the number of visits to inpatients must be minimized; this is particularly relevant for immunosuppressed patients. in no case should patients be accompanied by more than one person at a time. g. it is advisable that patients visiting day hospitals to receive intravenous medications be tested for temperature before entering the facility, and that infusion chairs be at least meters away from each other. turns should be established if this is unfeasible. chairs and rooms should be adequately cleaned after infusion completion ( ) . it is advisable that patients attend alone whenever possible. h. in case of day-hospital overcrowding efforts will be made to prescribe drugs with subcutaneous formulation. once the confinement phase is over, massive screening will likely be a most effective measure to gain insight into the population's immune status regarding sars-cov- infection. knowledge of this immune status will be particularly relevant in areas with high infection rates and, above all, among those who provide care for the rest of citizens. because of this we recommend: a. regular, universal screening of professionals. such screening will reveal immunization level, and will likely help establish the risk run by care providers, a key aspect for the management of a potential recurrence of the pandemic. b. universal screening of all patients who must undergo examinations involving sars-cov- infection transmission risks. c. systematic screening for sars-cov- among particularly vulnerable patients. ideally, such screening should be primarily offered to: i. patients on biologics or immunosuppressants: -inflammatory bowel disease. -autoimmune liver disease. -other. ii. patients with immunosuppression secondary to their underlying disease: -compensated and, most particularly, decompensated liver cirrhosis. iii. patients with liver cancer. iv. transplant recipients. two important considerations apply regarding the above screening:  obviously, a systematic screening of all these populations cannot be carried out simultaneously, hence we recommend setting up local screening plans.  screening is for the asymptomatic population; should a patient present with symptoms suggestive of infection with sars-cov- , he or she should be diagnosed (pcr and/or serologic tests and/or chest x-rays and/or chest ct scan); in no case should the patient undergo an elective dianostic test. in our view, the best screening strategy should be established at any given time depending on the availability of rapid antibody tests and/or pcr and/or serological techniques such as elisa, as well as on endemic evolution.  interpretation of results obtained in asymptomatic patients without previous close contacts. -igg negative, igm negative. individual with no prior exposure to sars-cov- . -igm positive, igg negative or positive. recent, potentially active infection. a pcr shall be made to rule out active infection.  activity shall be immediately resumed (first phase) for patients with urgent or preferential indications, and/or for undelayable therapeutic procedures.  the rate of activity resumption shall depend on local characteristics, both regarding pandemic incidence and healthcare personnel/ancillary staff availability.  transient elastography is only exceptionally an urgent procedure.  both the patient and physician must wear surgical masks during the examination.  activity shall be gradually resumed, preferentially starting in the second phase.  high-risk examinations beause of aerosol formation.  only exceptionally urgent.  resumption shall be held off until the third phase.  should an urgent indication arise in the first phase (highly unlikely), the course of action shall be the same as for gastroscopy, including screening for sars-cov- and using appropriate protective equipment.  high-risk examinations beause of aerosol formation.  only exceptionally urgent.  they may be substituted for by other testing modalities (fecal antigens, commercial tests).  these tests shall not be resumed until the third phase.  portal hemodynamics is a moderate-risk study regarding sars-cov- transmission (position and duration).  during the first phase of activity resumption in our departments a conservative attitude is advisable, prescribing this examination only in two situations: -liver biopsy in cases of severe acute liver failure where the test may play a key role. -urgent placement of tips for intractable bleeding secondary to portal hypertension.  in the second phase it seems reasonable to perform any necessary procedures to assess portal hypertension in patients with hepatocellular carcinoma potentially amenable to surgical resection.  finally, during the third phase (normalization) all indicated procedures will be carried out the same as before the crisis. the covid- pandemic represents an unprecedented challenge to our health system, and as regards specifically patients with inflammatory bowel disease (ibd) multiple concerns arise in connection with their management, as many are on treatment with immunity-impairing therapies. furthermore, ibd is a condition that evolves in flares alternating with remission periods, and may have potential complications that often require urgent, or at least preferential, care. a variable proportion of patients have digestive complaints such as nausea, vomiting, bowel habit changes, or abdominal pain ( ) . these symptoms are common in patients with ibd, hence the importance of excluding covid in our patients. furthermore, the virus has been reported to be present in the stools of covid- patients, regardless of the presence of diarrhea, and to persist there even after respiratory symptoms are over or detection in the oropharynx is no longer feasible, its significance being uncertain concerning infectivity during endoscopic procedures or potential fecal-oral transmission ( ) . the first question we posed ourselves from the start of the pandemic was whether patients with ibd are at increased risk of infection. patients with ibd do not seem to have a greater risk for infection with sars-cov- or for development of covid- . according to data from bergamo in lombardy, a region especially affected of italy, no patient among their cases of ibd was diagnosed with, or admitted to hospital for covid- ( ) . a possible reason explaining this lower number of cases of covid- in ibd patients may be this population's adherence to protective measures. another common question is whether covid- may cause an ibd flare-up; current evidence does not seem to support that, albeit available data are scarce and caution is here advisable ( ) . finally, the next question we posed ourselves was whether suffering from ibd may condition the course of covid- . answering this is difficult since multiple factors may play a role: age, comorbidities, inflammatory activity, and trestments received, the available information being limited about these. there is an international registry called secure-ibd ( ) that aims to collect the data of patients diagnosed with ibd where covid- has been confirmed (positive testing) ( ). at the time of writing these recommendations a total of patients have been recorded, of them from spain. the overall rate of hospitalization has been %, and those of icu admissions, ventilation requirement, and mortality have been %, %, y %, respectively. therefore, it seems that the course of covid- in patients with ibd is not worse than in the general population, but we should bear in mind that our ibd patients are younger than the general population. in this respect the fact should be highlighted that patients with moderate-high activity required icu care/ventilation or died (pooled variable) in % of cases, versus % for patients with remission or low activity, with % of subjects with an untoward outcome being on steroids. below we include a table with a theoretical stratification of the risk for poor outcomes based on recommendations by the british society of gastroenterology (bsg) ( ), although, again, the dearth of data available about the therapies used for ibd should be borne in mind (table ) . drug half-life must be taken into account, and so patients who discontinued immunosuppressants or biologics within the last months remained exposed to their effects when it comes to risk categorization. recommendations concerning the treatment of ibd are based on those issued by the international organization for the study of ibd (ioibd), bsg, and american gastroenterology association (aga) ( ) , differentiating between uninfected, sars-cov- infected, and covid- patients. general recommendations regarding the treatment of patients with ibd, which must remain in force during the gradual resumption of activities: a. patients must not discontinue medication or visits to the infusion center, or start self-medication, without consulting with their doctor first. b. medication must be available at home in case an isolation period is required. c. smoking should be stopped as it increases the risk and severity of covid- . smoking augments gene expression of angiotensin converting enzyme (ace ), the receptor for viral entry ( ) . a. in case of suspect symptoms and a negative sars-cov- pcr, the potential for false negative results should be considered, as well as a repeat test. b. treatment must be maintained to prevent non-adherence-related relapse, which may represent a higher risk of infection because of steroid or hospitalization needs. e. when planning to initiate therapy with biologics or immunosuppressants, it is advisable that sars-cov- testing be included in the previous assessment routine ( ) . f. steroids use should be minimized. if required, rapid tapering by mg/week is advisable. a. if the patient is on steroids, reduce dose to below mg or switch to budesonide should the clinical scenario allow. clostridium difficile, which requires specific treatment. ibd patients already underwent specific follow-up prior to the covid- crisis. most ibd units have clinics or free-access mechanisms in case of flare-ups to avoid visits to the emergency room (er) ( ) . two further characteristics should be considered: nursing consultation ( ) and telemedicine, long implemented in our ibd units pioneered in our country by geteccu platforms such as teccu ( ) . in an initial phase it is recommended that all scheduled follow-up visits take place telematically. the duration of follow-up for patients in remission according to their medication should be met or at least deviated the least possible from the standards published and accepted by geteccu ( ) . in the initial phase it is recommended that patients with severe disease requiring specific physical examinations attend. in the stabilization phase patient numbers will be adjusted according to the above indications. non-scheduled consultations. during the initial and stabilization phases, aiming to avoid physical visits to hospital, patients with urgent consultations should be advised to contact the ibd unit via the nursing clinic, telephone, or email. should the issue be serious or unsolvable through telematic means a visit will be scheduled according to the above indications. infection with sars-cov- must be ruled out if fever and diarrhea are present. ( ) . a. it is recommended that patients with severe crohn's disease or ulcerative colitis flare-ups refractory to outpatient management be admitted to hospital. also patients with subocclusion and with septic complications. b. in case of high suspicion of covid- despite a negative test result on admission, it is advisable to place the patient in a pre-covid- area and then repeat testing given the potential for false negative results ( ) . e. consider reducing and facilitating bureaucratic aspects using sponsor amendments given the care burden and activity reassignments of participating physicians. probably, from a healthcare perspective, managing endoscopy units is the most challenging activity of any gastroenterology department. patient presence is mandatory, work there involves risk for both patients and care providers (and society at large), and no published or accessible protocols deal with activity resumption. on the way back to normalization, we must bear in mind that the latter does not result from overcoming the pandemic but rather a reduction in infection rate allowing to decrease hospital overload, which may in turn permit a recovery of regulated activity. therefore, one must at all times recall that the risk of infection may persist both for patients and care workers. in general, social distancing and the use of adequate personal protective equipment must be maintained. it is crucial that consideration be given to the amount of hospital resources devoted to caring for patients with covid- , and how their recovery for the care of non-covid patients is anticipated. when opening agenda windows the hospital's contingency plan to reclaim non-covid areas as covid areas should a new peak occur has to be taken into consideration. furthermore, the number of professionals available at the unit itself and their risk of infection must also be weighed up. a key point is the need for endoscopy units to have available all the materials necessary for a potential increase in activity (at least ppes, for physician, nurse, and assistant, per procedure ( if additional staff is required: anesthetist, nurse, etc.). without this minimum of materials (defined below in the present document) no endoscopic procedure should be performed. in preparation for activity resumption we should bear in mind a number of variables: a key point is the adjustment of the appointment schedule, which depends on the duration of endoscopic procedures (table ). at present the times necessary to change clothes, clean instruments and room, disinfect, avoid waiting room overcrowding, etc., should be estimated. our proposal, considering transmisison risks and the need for cleaning and/or protective measures in case of examining a high-risk patient, is as follows ( ): a. phase i: attempt to reach % of usual activity at the endoscopy unit. b. phase ii and iii: attempt to reach % of usual activity at the endoscopy unit. in order to define intervals between procedures when examining high-risk patients at least minutes should be added to the established duration according to the eficad study (table ) . a. appointments: patients will be called up on the day before endoscopy to fill out a risk checklist, which will be done again on the day of the procedure. the ideal scenario we must doubtless pursue is the running of a sars-cov- test in all patients before the treatment (see above) ( , ) . b. access to the endoscopy unit: a surgical mask and gloves (or hydroalcoholic solution for hand washing) will be placed on the patient, and temperature will be measured. c. the patient shall attend at most with one companion, who will not enter the unit unless the patient requires specific help. social distancing is key.  risk by patient type. the risk of transmission by patient type may be seen in table .  risk by procedure type. two kinds of procedure must be differentiated according to their potential to generate aerosols ( ): i. aerosol-generating procedures, namely those involving upper endoscopy (ercp, gastroscopy, upper echoendoscopy, upper enteroscopy); these are deemed to be high-risk. when possible, sedation must be used for all upper examinations in order to reduce the risk for aerosol formation. ii. non-aerosol generating procedures, namely those involving lower endoscopy (colonoscopy, lower enteroscopy, lower echoendoscopy) or ostomy; these are deemed to be low-risk. a. some level of protection must be used during access to the endoscopy unit, including common areas (administrative area, corridor, living area, washing area, recovery room, etc.). a mask, body protection with scrubs and overcoat, and hospital-specific footwear. continuous use of gloves would not be required but regular hand washing would. b. once in the room the protective level will vary according to the risk allotted to each patient and procedure ( , , ) : -perform the endoscopic study in a hospital-designated room (usually in the surgical area). -perform the endoscopic study within the endoscopy unit. in this case, it would be advisable to designate one specific room for these patients, with the procedures being undertaken at the end of the agenda in order to allow time and resources to clean the specific room. properties, which will minimize transmission risk for any type of virus. b. channel cleaning brushes must be single-use, and plastic connections to aspiration must be disposed of. c. endoscopes must travel to cleaning areas in a closed container (e.g., plastic bag); upon entering the disinfection room they must undergo immediate manual washing before entering the automated washing system. with telematic and phone-based care, during the present pandemic we have learned that patient acceptance is very high and their response to disease has changed. patients are now more reluctant to undergo procedures unless they are absolutely necessary. hence, the possibility and/or need emerges to clear the scheduled wait list. in this context patients could be contacted before their assigned appointment by a unit physician to assess their need for the scheduled procedure according to indication and clinical status. the possibility that the procedure could be delayed because of the pandemic should be laid out for their consideration. recommendations in case of patients with stable chronic liver disease (table ) there is no evidence that patients with stable chronic liver disease of any origin will be more susceptible to infection with sars-cov- , even though many of them have comorbidities such as hypertension and diabetes mellitus, which are associated with greater severity, particularly in patients with advanced fat deposition disease ( immunocompromised patients might be more susceptible to sars-cov- infection, even though solid evidence is lacking in this respect. however, some data suggest that immune response is a key factor in pulmonary involvement, and so immunosuppression may even be protective ( , , ) . in fact, post-transplant immunosuppression has not represented a risk factor for mortality during the sars or mers c coronavirus pandemic ( in case the lt program was interrupted and resumption is under consideration, its restart will be subjected to the availability of an adequate number of icu/ccru beds and covid- -free hospitalization areas. the course of the pandemic shall always be taken into account.  regarding donation, we shall preferentially use excellent donors with no risk factors whatsoever for covid- .  in case of an offer, a coordinator will interview the potential recipient over the phone to assess the presence of covid- .  performing an rt-pcr test on nasopharyngeal exudate samples from both donor and recipient is key to rule out covid- ; also a pulmonary assessment of the recipient should be undertaken (this will be according to each transplant group). in all cases a second recipient should be ready for the procedure.  when possible, acording to each center, the candidate recipient should undergo testing for measuring antibodies (igm and igg) in capillary blood, which will supplement the information obtained with the rt-pcr. this obviously extends and complicates logistics, but is indispensable.  confirmed covid- cases must be excluded as donors until at least days after symptom disappearance and therapy completion. cases deemed cured are described in the ont document of april , ( ) , as well as other technical issues whose review we deem advisable. the recommendations expressed in this document are aimed at helping departments in the resumption of their usual healthcare activity, which has been almost completely postponed in some of them. we are facing a changing reality that demands considerable plasticity of all of us; a relevant part of our way of working will change, and we must play a leading role in this process. for the above recommendations we have relied on pragmatism, although the scarce, changing evidence available will require future updates. the start of this journey towards a changing normality in every department will depend on the cumulative incidence of sars-cov- infection in each region, as well as on the burden the pandemic has inflicted on each hospital. afectación en aparato digestivo por covid clinical characteristics of coronavirus disease in china protecting health care workers during the covid- coronavirus outbreaklessons from taiwan's sars response substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov- ) science covid- : protecting health-care workers. the lancet covid- and italy: what next? lancet telehealth seen as a key tool to fight covid- global telemedicine implementation and integration within health systems to fight the covid- pandemic: a call to action. jmir public health surveill clinical practice update on management of inflammatory bowel disease during the covid- pandemic: expert commentary evidence for gastrointestinal infection of sars-cov- . gastroenterology 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the operation of gastrointestinal endoscopy and gastroenterology units, concerning the current sars-cov- pandemic (march, ). rev espanola enfermedades dig organo of soc espanola patol dig esge and esgena position statement on gastrointestinal endoscopy and the covid- pandemic -european society of gastrointestinal endoscopy (esge) covid- ) outbreak: what the department of endoscopy should know reprocessing of flexible endoscopes and endoscopic accessories used in gastrointestinal endoscopy: position statement of the european society of gastrointestinal endoscopy (esge) and european society of gastroenterology nurses and associates (esgena) -update liver injury in covid- : management and challenges cancer patients in sars-cov- infection: a nationwide analysis in china clinical features of patients infected with novel coronavirus in wuhan, china coronaviruses and immunosuppressed patients. the facts during the third epidemic aasld clinical insights for hepatology and liver transplant providers key: cord- - hwmajt authors: occhipinti, vincenzo; saibeni, simone; sampietro, gianluca m.; pastorelli, luca title: impact of covid- outbreak on the management of patients with severe ibd: a domino effect date: - - journal: gastroenterology doi: . /j.gastro. . . sha: doc_id: cord_uid: hwmajt nan the international organization for the study of inflammatory bowel diseases (ioibd) recently published on gastroenterology a consensus about the management of ibd patients during the coronavirus disease (covid- ) pandemic, addressing several topics of interest such as the risk of infection in ibd patients, how to manage therapies and how to safely provide continuity of biological therapy. we read it with great interest and we highly appreciated the effort to provide a guidance to ibd care in these difficult days, even in the absence of evidence-based data. indeed, as ibd physicians working in one of the most severely affected regions of the world (lombardia region, in northern italy ), we had to face additional and unexpected difficulties, while managing severe ibd during the sars-cov- outbreak. here, we report the emblematic case of a -year-old man. because of episodes of bloody diarrhea, the patient underwent colonoscopy in january with evidence of mild proctosigmoiditis, with histology compatible with ulcerative colitis (uc). a gastroenterological consult was scheduled for the end of february, but not performed due to the covid-related limitations to non-urgent consultations and procedures. a short course of oral mesalamine therapy given by the general practitioner provided clinical remission, but no maintenance therapy was initiated. after two months the patient developed severe bloody diarrhea (> episodes/day), malaise and diffuse abdominal pain. for these symptoms, he called twice the emergency service who telephonically suggested to avoid access to the hospitals because of the covid- outbreak. after two weeks at home clinical conditions further deteriorated, and he was finally transported to the emergency room of our hospital (policlinico san donato, a university hospital in the southeastern region of milan metropolitan area). he appeared severely ill, tachycardic, with mild fever ( . °c) and a diffuse abdominal pain. laboratory tests showed markedly elevated c reactive protein ( mg/dl, normal values < . ), neutrophilic leukocytosis and hypoalbuminemia ( . g/dl). chest x-ray was normal, nasopharyngeal swab for novel coronavirus was negative. an urgent ct scan excluded significative colonic dilatation but showed markedly thickened and enhanced colonic walls. a rectosigmoidoscopy showed severely inflamed mucosa with multiple, deep ulcers; histology confirmed severely active ulcerative colitis. broad spectrum antibiotics, intra-venous corticosteroids and anti-thrombotic prophylaxis were promptly started. despite the absence of urgent surgical indications, we thoroughly pondered the potential risk of performing urgent colectomy with post-surgical icus converted into critical covid- units. thus, we decided to transfer the patient to a covid-free hospital with ibd-specialized gastroenterologists and surgeons (rho hospital, in the northern area of milan) for further management. in the end, the patient only partially responded to i.v. steroids, with dramatic fall in crp levels ( . mg/dl) but persistent bloody diarrhea with up to bowel movements. then, salvage therapy with infliximab mg/kg was started with satisfying clinical efficacy, thus avoiding urgent colectomy. this case clearly highlights some unanticipated difficulties in providing adequate care to patients with severe ibd in a high-prevalence area of covid- . the limitation to all non-urgent consultations and the extreme pressure on the emergency system may lead to wide diagnostic and therapeutic delays. moreover, many patients themselves may try to avoid access to hospitals even in presence of severe symptoms, because of the fear of getting infected. severe ibd flares require admission, tight monitoring and may require urgent surgery. all of these measures may become problematic during the pandemic. in our region, after the identification of the first covid- clusters at the end of february, in few days, several hospitals (included ours) were almost completely converted in covid- clinics with consequent deranging of physicians' organization chart and limitations of specialistic activities. as a third level ibd center we struggled to guarantee essential care to our patients, such as infusional therapies and urgent consultations, and to protect them from the risk of infection by instituting telephonic screening and / availability . however, with our gastroenterological ward closed and all gastroenterologists but one reassigned in newborn covid units, we turned unable to adequately manage patients with ibd flares. management of patients with crohn's disease and ulcerative colitis during the covid- pandemic: results of an international meeting coronavirus disease (covid- ) in italy challenges in the care of ibd patients during the covid- pandemic: report from a "red zone" area in northern italy key: cord- -xg swuzs authors: pugliese, daniela; papi, claudio; privitera, giuseppe; aratari, annalisa; festa, stefano; armuzzi, alessandro title: the management of inflammatory bowel diseases in the era of covid- pandemic: when “non-urgent” does not mean “deferrable” date: - - journal: dig liver dis doi: . /j.dld. . . sha: doc_id: cord_uid: xg swuzs nan all the authors contributed to the conception and the design of this letter. daniela pugliese and claudio papi wrote the manuscript. giuseppe privitera, annalisa aratari, stefano festa and alessandro armuzzi revised the manuscript critically. all the authors approved the final version of the manuscript. alessandro armuzzi oversaw the project and guarantees for the integrity of the work. none dear editor, the widespread diffusion of the sever acute respiratory syndrome-coronavirus- (sars-cov- ) infection has significantly reshaped all healthcare systems. the high contagiousness and the unpredictable request of admission for symptomatic patients has led hospitals to reconvert several units into coronavirus disease- (covid- ) facilities, requiring the recruitment of medical and nursing staff, taken from both medical and surgical departments for the treatment of pulmonary or infectious diseases. moreover, to reduce the risk of transmission, outpatients' visits are currently limited to those considered urgent according to the priorities code of the italian health system (i.e. requiring medical assistance within maximum hours or days). accordingly, inflammatory bowel disease (ibd) units throughout the country had to re-organize their own agenda, limiting the accesses to categories of outpatients: ) patients undergoing biological therapies, for whom the risk of disease relapse is considered more worrying than the risk of covid- infection; ) patients enrolled in clinical trials in order to guarantee the prosecution of therapies and ) patients complaining of acute symptoms, not manageable with telemedicine. - examples of "non deferrable" visits are for patients complaining of moderate-to-severe ibd symptoms, new onset/relapse of extra-intestinal manifestations, active perianal disease or sub-acute obstruction symptoms requiring surgery. all other schedules, including endoscopic, radiological and elective surgery procedures had to be indefinitely deferred. however, whether these defined "urgency criteria" would perfectly fit all ibd patients could be a matter of debate. treatment outcomes of crohn's disease (cd) and ulcerative colitis (uc) have progressively evolved beyond symptomatic remission to more challenging "deep remission" and eventually "complete disease control", that is prevention of bowel-damage and long-term disability. medical strategies have been enhanced by the introduction of early intervention, treat-to target and tight monitoring approaches. in this context, deferring all scheduled procedures could reduce the level of care below acceptable standards for many ibd patients. we consider different scenarios of "non urgent" patients, who, anyway, should not be indefinitely postponed even in a pandemic situation: diagnostic delay is still a challenging issue for cd patients, especially in case of nonspecific abdominal complaints (usually ascribed to irritable bowel syndrome) and is associated with higher risk of complications at diagnosis and of early need for surgery. moreover, in patients already diagnosed with cd, it is common experience to observe a poor correlation between clinical disease activity and mucosal lesions. accordingly, highly suspect cd patients should be promptly investigated in order to start early and effective treatment strategies, aimed to impact the long-term clinical course and to reduce the risk of complications. ) patients in clinical remission requiring prompt evaluation, tight follow-up and longterm strategies: a) uc patient discharged after a severe acute flare on oral steroids: up to % of patients, admitted for an acute severe flare of colitis, respond to intravenous steroids and, at discharge, according to the physicians' judgement, are placed on maintenance therapy with -amynosalicilates, thiopurines or biologics. however, up to % of these patients may experience a short-term clinical relapse during steroids tapering, requiring prompt escalation of therapy or change in the maintenance treatment. a short term outpatient evaluation after discharge should not be deferred in these patients. bowel resection in cd is strictly recommended for patients with at least one risk factor for recurrence, including tobacco use, history of previous surgery and/or penetrating behaviour of the disease. thiopurines or anti tnf-α agents seem to be the best options for preventing post-operative recurrence in high risk patients, and the treatment should be started early (usually within to months) after surgery, provided that there are no specific contraindications. moreover, endoscopic assessment should be performed within to months after surgery in order to promptly recognise endoscopic recurrence and consequently adapt the medical strategy. therefore, early introduction of prophylactic treatment and endoscopic evaluation within - months should not be deferred, even in asymptomatic patients. c) patients with recent introduction of thiopurines: thiopurines are still used as maintenance therapy in steroid-dependent ibd patients, as prophylactic therapy after bowel resection and in combination with anti tnf-α drugs. despite several limitations of these drugs (i.e. effectiveness and safety), responders are offered a low cost, simple, oral maintenance therapy. patients started on thiopurines require strict safety follow-up in order to promptly recognise intolerance or allergic toxicities (e.g. nausea, myalgia, pancreatitis) and to manage dose-dependent side-effects (e.g. leukopenia). checking metabolite levels, when available, can be helpful to assess therapeutic effect and compliance. transition from paediatric to adult health care setting is a crucial phase for ibd patients, requiring structured programs, joint paediatric-adult clinic and an approach tailored to the patients. the acquisition of decision-making and self-efficacy skills seems to be the key of a successful transition. for adults in transition to a tertiary centre, it is important to face potential barriers to efficacious transition, such as fear to receive lower attention because of high-volume or reluctance for the loss of the relationship with previous doctors. in both situations, assuring continuity of care with an established transition program can improve the engagement of patients and reduce the risk of non-adherence and loss to follow-up. a) ibd pregnant women: women with ibd often have insufficient knowledge about pregnancy-related issues. fears about potential side-effects can lead to inopportune medications withdrawal causing relapse. ibd flares-up during pregnancy are a great concern, because they are associated with an increased risk of pre-term birth. therefore, a period of at least months of steroid-free clinical remission prior to conception is strongly recommended, as well as a close monitoring (up to every weeks on flare) during pregnancy by gastroenterologists in order to promptly adapt the medical therapy. a counselling on mode of delivery is advocated for patients with cd and perianal involvement. b) ibd patients with cancer: the management of ibd patients with cancer requires a strong multidisciplinary collaboration between gastroenterologists and oncologists. risks and benefits of introducing/maintaining ibd medical therapies should be balanced for each patient, according to ibd clinical activity and cancer diagnosis. in case of withdrawal of ibd drugs, patients should not feel abandoned, but closely followed-up and engaged in an oncological treatment plan. a prompt intervention should be assured in case of ibd flare during anti-neoplastic therapies, to guarantee the prosecution of standard treatments. c) cd patients who require nutritional support: enteral or parenteral nutritional support is mandatory for cd patients unable to obtain adequate calories intake, such as those ones with history of multiple bowel resections or with jejunostomy or proximal ileostomy or short bowel syndrome. patients should be given dietary counselling and strictly followed-up in order to prevent de-hydration and nutrition-related disorders. participating in a clinical trial can be a challenging step for refractory ibd patients, who are torn between safety concerns, placebo risk and hopes for efficacy. physicians should adequately discuss the informed consent form, in order to avoid therapeutic misconception, to ascertain that patients understand the purpose and procedures of the study and to reassure the patients that refusal to participate will not compromise the doctor-patient relationship. in conclusion, sars-cov- pandemic has dramatically changed the level of assistance for patients affected by chronic conditions, with an almost complete preclusion to routine follow-up visits and procedures. ragrding ibd, several concerns are emerging about how to maintain "optimal" standard of care also for patients considered "deferrable". diagnostic delay, nonadherence and loss to follow-up, lack of optimal and timely treatment can represent concrete risks for such patients. in the era of ambitious therapeutic goals, treatment strategies and decisions cannot rely on symptoms only, but they require tight monitoring of disease including also objective inflammation markers. therefore, close follow-up is mandatory for many ibd patients, even if in clinical remission and not considered "urgent" according to symptoms alone. the level of care for ibd and other chronic conditions should not be reduced below optimal quality standard even during a pandemic with reduced dedicated staff and facilities. impact of covid- pandemic on the daily management of biotechnological therapy in inflammatory bowel disease patients: the reorganizational response in a high-volume italian inflammatory bowel disease center impact of covid- pandemic on gastroenterology divisions in italy: a national survey. dig liver dis. epub ahead of print crohn's disease systematic analysis of the impact of diagnostic delay on bowel damage in paediatric versus adult onset crohn's disease long-term outcome of patients with acute severe ulcerative colitis responding to intravenous steroids systematic review and network meta-analysis of medical therapies to prevent recurrence of post-operative crohn's disease european crohn's and colitis organisation topical review on transitional care in inflammatory bowel disease inflammatory bowel disease in pregnancy clinical care pathway: a report from the american gastroenterological association ibd parenthood project working group espen practical guideline: clinical nutrition in inflammatory bowel disease informed consent in ibd trials: where we are and where we need to go daniela pugliese received speaker fees from abbvie, msd, takeda and janssen, pfeizer. claudio papi received consultancy and educational projects from takeda, msd, abbvie, sofar, chiesi, and alfa-wasserman. giuseppe privitera received consultancies fee from alphasigma. stefano festa received lecture fees and consulting from takeda, ferring, sofar, and alfa-wasserman. alessandro armuzzi: consulting and/or advisory board fees from abbvie, allergan, amgen, biogen, bristol-myers squibb, celgene, celltrion, ferring, janssen, lilly, msd, mylan, pfizer, samsung bioepis, sandoz, takeda; lecture and/or speaker bureau fees from abbvie, amgen, biogen, ferring, janssen, msd, mitsubishi-tanabe, nikkiso, pfizer, sandoz, samsung bioepis, takeda; and research grants from msd, pfizer, takeda. all the remaining authors have no conflict of interest to delclare. key: cord- -mct erg authors: kornbluth, asher; kissous-hunt, michele; george, james; legnani, peter title: management of inflammatory bowel disease and covid- in new york city : the epicenter of ibd in the first epicenter of the global pandemic date: - - journal: inflamm bowel dis doi: . /ibd/izaa sha: doc_id: cord_uid: mct erg nan o ur clinical practice is located in new york city (nyc), new york, the global epicenter of coronavirus . at the time of this writing in early july , there have been over , cases and nearly , deaths in nyc in the months ending in june . as of early june we have managed patients with ibd and confirmed sars-cov- diagnosed by pcr-rna for the virus ( patients), by positive igg antibodies ( patients) , and by classic covid- presentations. as of june , the number of cases from this practice exceeds the number of cases reported from of any state outside of new york in the united states and exceeds the number of cases in of the countries reporting to the international secure-ibd registry of patients with ibd and sars-cov- infection. in this article, we share our real-world experience in the care of this large cohort of patients, describe our practice's approach, and offer suggestions to the practical care of the patient with ibd and covid- . we also hope to describe the sense of life in nyc at this uniquely tragic time. through the month of june, nyc had successfully passed through "phase of reopening," allowing most businesses to open with appropriate "social distance" and wearing of masks both indoors and outdoors. this followed a -month period of onerous and suffocating but effective shutdown of nyc. but by early july, some plans to further reopen nyc were abruptly halted by the recognition of major new outbreaks in states such as arizona, texas, and florida, where statewide closings came too late and reopening occurred too early. this has led, almost predictably, to a foreboding spike in cases, hospitalizations, icu admissions and deaths into august , in a pattern resembling the onset of the most ominous weeks in nyc in march, april and may. as the number of cases is now growing alarmingly throughout other areas of this country and around the world, we hope that sharing the lessons that we have learned to date will be of value to others. our extensive experience in caring for ibd patients with covid- stems from our practice in which we have a very large ibd patient population of ashkenazi orthodox and hasidic jews that comprises nearly % of our cohort. our observations and those of others have been that these groups most likely have the world's largest incidence of multiplex ibd families (dubinsky, personal communication, may , ), and outside of israel, they are located in greatest prevalence by far in new york city. many of these families have large numbers of children (more than is not uncommon), and many live in crowded apartments or homes. socially, they tend to be insular and very tight-knit communities, and there are several areas in new york city (williamsburg, borough park, new square, monsey, and kiryas joel) and in new jersey (lakewood) where several thousand families can live within walking distance of each other. families attend weekly sabbath and holiday services in often crowded synagogues and share attendance at many large communal holiday gatherings, weddings, and funerals. large clusters of cases arose at some of these events. the close contact source of infection in these communities is suggested by the finding that over % of these patients in our cohort can identify household members with sars-cov- (with anywhere between to family members affected), whereas patients in our cohort from outside these communities have known household contacts of only approximately %. the stunningly abrupt, chronologic tipping point in these communities was clearly on march , , on the jewish holiday of purim. the holiday is marked by a great deal of celebratory dancing, communal meals, and near universal delivering and sharing of gifts and delivery of food between families. within week of this holiday, the onslaught of cases, severe illness, and death began in these communities. because we advised our patients to stay at home isolation if they had typical symptoms, the great majority of our patients who tested positive for pcr of sars-cov- either had another physician order the test or tey had obtained it on their own. we had only selectively ordered pcr testing in the very first weeks of the pandemic when the disease course and presentation was less well described. more recently, most patients who were tested for sars-cov- antibodies were being tested if they were otherwise going to have labs drawn. in some of these communities, both the mayo clinic and the mount sinai medical center put out calls for volunteers, and over subjects presented to be tested for the sars-cov- antibody in the hope of being able to donate plasma for potential use for convalescent serum as treatment for patients infected with sars-cov- . in this report, we share our experiences and suggestions in the management of patients with ibd, in addition to sars-cov- and covid- patients in a clinical outpatient practice. on march , , a woman recently returned from iran, where the disease was already widespread and was diagnosed with the first case of sars-cov- in nyc. the following day, a -year-old attorney living in new rochelle (a suburb miles north of nyc) who traveled daily by train to his office in manhattan was diagnosed without any known exposure. within a single week that index case led to a cluster of over in his home community of new rochelle, which then became the first community in the united states to become an enforced "containment area"; the national guard briefly patrolled the streets, not to quell civil unrest but to assist in food deliveries and to provide the personnel to disinfect public areas. the first death in ny was reported on march , and then began an unfathomable, exponential explosion of cases, hospitalizations, and death that shook and devastated nyc. in the months since the first case, nyc has become the global epicenter of covid- , with over , reported cases leaving nearly , dead. to put this into context, this number represents greater than times the number killed on september , in the world trade center attacks. the unrelenting, massive torrent of patients overwhelmed nyc hospitals, intensive care units, and staff. overall, in individuals tested in nyc were positive, and in residents of nyc died. east new york, an urban area with an almost entirely black and latino population, with a median annual household income of $ , , had the highest death rate in the city: in residents were infected and in residents of that area died of covid- . citywide, the death rate of blacks and latinos was twice that of white residents. major medical centers in nyc such as mount sinai and new york university would be managing over covid- patients at once, and lobbies were converted into patient's rooms, and operating rooms and postop recovery areas into intensive care units. massive refrigerated trucks serving as overflow morgues were parked outside some of nyc's municipal hospitals that serve the city's most impoverished patients. samaritan's purse, a charitable rescue-mission organization, arrived on a saturday night, march , and by that tuesday had set up cavernous medical tents equipped to handle overflow inpatients on the pastoral lawn of central park directly across the street from mount sinai (see figs. and ). inside mount sinai, "platoons" of medical teams were deployed in sequential waves of attendings, fellows, and residents. early on, there were variable appalling shortages of personal protective equipment (ppe), further heightening risk to the staff. in some of the most deprived municipal hospitals, staff with direct patient care were told to wear rain ponchos in lieu of proper gowns. a number of hospitals imposed gag orders on their staff from speaking to the press about ongoing ppe shortages. each of our gastrointestinal (gi) fellows served on the covid- floors for multiple rotations at mount sinai and thus denying them of months of training. early on, we each volunteered to be on call for inpatients with gi bleeding to relieve the fellows physically and emotionally, reduce additional covid- exposure risk for them, and save on ppe in that only the attending would be present during a procedure. frankly, we were relieved when our nights and weekends of being on call passed without any patients requiring emergency hospital procedures. in nyc, a number of physicians, nurses, and residents contracted the virus and were hospitalized, and a few among them died. outside the hospitals, the boroughs of nyc, home to . million residents, were put on "lockdown" on march . the boisterous, rambunctious, often ornery, and cantankerous city overwhelmingly observed all designated social prohibitions. the city became eerily silent overnight, with all nonessential businesses shut down, with many likely never to reopen. unemployment soared and , nyc residents became unemployed within a space of . months. at its worst, at midday one could drive down entire ghostly avenues and streets devoid of any people outdoors. nonetheless, the prevailing mood was of mutual camaraderie, support, and empathy. many local restaurants delivered daily free meals to the hospital's entire medical staff. the sacrifices of the health care workers and first responders did not go unnoticed. they were loudly lauded and appreciated throughout the city on the (nearly empty) streets and in the media. at : pm nightly, many new yorkers stepped out onto their stoops, their sidewalks, and their balconies and applauded all the first responders of nyc. though the physicians in our practice all hold academic faculty positions at the icahn school of medicine at mount sinai, our clinical practice is run independently of the medical center, and thus we are faced with all of the management, financial, and personnel issues of any "private practice." with the loss of income from a decline in office visits, greatly reduced procedural volume, and loss of income from a partial ownership in an ambulatory surgery center (asc), we benefitted greatly from receipt of a paycheck protection plan (ppp) loan, and all provisions were made to fulfill all stipulations to ensure loan forgiveness in the future. frankly, absent this grant, the financial strain on the practice would have been prohibitive, given our commitment to the staff and the typically very high costs of maintaining a practice in manhattan (monthly rents can approach $ per square foot). nevertheless, the loan is designed to cover only weeks of expenses, and we are already well beyond that period. office infusions of biologics (infliximab, vedolizumab, and ustekinumab) became the main source of revenue for the practice during that time. upon declaration of the nyc shutdown, we immediately made a commitment to our entire staff that they would remain fully employed for the duration of the crisis, in recognition of their devotion to the practice for anywhere from months to years. no more than half of the staff were present in the office on any given day, while the remainder worked from home, and the staff area is situated in a fashion that all staff, wearing masks, are at least feet apart. we made (variably successful) efforts to avoid having all staff present in the office on any given day in case there were an office-wide exposure to sars-cov- . as with all other gastroenterology practices, "telemedicine" has replaced the vast numbers of office ibd consultations and follow-up visits. we have termed these as video office visits or telephone office visits for our patients. we wanted to avoid the term "virtual," which could give the connotation that these visits were virtual rather than real, and we avoided the generic term of "telemedicine" because this term has been used to describe a variety of forms of remote care. the volume of consultation and follow-up visits was approximately % of our usual number, as would be expected in a practice caring for a large number of chronic patients with ibd. we have found that nearly all of our patients found these visits worthwhile and would opt for remote visits in the future for some or many of their routine follow-up visits. on the other hand, the welldescribed "zoom fatigue" set in rapidly and largely negated the hoped for "leisure" of working from home several days a week. as with most other gi practices, we performed a very small number (ie, about %) of our usual number of procedures in an ambulatory endoscopy setting. these were done for disease assessment in patients with severe disease for whom a change in therapy was being planned or for patients who required stricture dilation. meticulous care and great efforts were taken to secure adequate ppe, and all cases were done fully gowned, with n masks (one per day), eye protection, and full face shields. rooms were equipped with air filtration devices, and minutes were spent between cases disinfecting the endoscopy rooms. the video visits, as in real life, consist of a dialogue with our patients, and we have largely avoided sending patients out for labs of any type. however, we have increasingly relied on commercial labs to send an overnight home stool collection kit for calprotectin that can then be picked up by one of the express mail services eliminating the patients' need to leave home. imaging studies have likewise been largely deferred except for the patient with a bowel obstruction or concern for abscess. the patient with a perianal abscess may be a tough call to make. an on-screen demonstration of a perineum is often less than optimal, and we have some of these patients go straight to a colorectal surgeon for a video visit and examination under anesthesia if an intervention is thought to be necessary. we deferred all routine screening and surveillance colonoscopy, any planned follow-up procedures to assess for routine response to therapy, and the -month postoperative ileocolonoscopy to assess for crohn's disease (cd) recurrence. refreshingly, we have relearned what our beloved mentor, dan present, would say in his inimitable brooklyn accent, "if you wanna know how a patient is doin' all you need to do is say, 'how you doin'?" depending on old-fashioned clinical judgment has never seemed as reliable as it is now. a significant portion of each visit at this time is spent describing what we know on that given day about covid- and the interface of covid- and ibd. we preface these remarks by informing patients that this information is accurate as of the day and hour of our discussion because frequently our knowledge has changed by that afternoon or evening. we have communicated with our patients in a variety ways. our electronic medical record allowed us to generate email blasts to our entire practice. the first of these, in late march, described the general symptoms of covid- and the importance of adhering to all the risk mitigation programs at that time, which have since then been further expanded. we described the possible gi manifestations, chiefly nonbloody diarrhea, and asked patients to contact us with these or any other gi symptoms. we discussed that in general, our ibd patients should not discontinue their medications but should contact us for individual recommendations. also, our email informed them that they should contact us to discuss whether any routine follow-up labs should be deferred, which we most always advised. a second email in late may reviewed the emerging nature of our knowledge of the disease course, and the role of both repeated pcr and antibody testing-topics which are now evolving on a weekly or even daily basis. a technique that we have found helpful is to place relevant educational information in an "away vacation" message that is generated with every routine patient email to us, which they would see along with our email reply. the chief source of information to which we directed the patients were the websites of the crohn's & colitis foundation and the center for disease control (cdc). a number of the major gi societies, the crohn's & colitis foundation, british society of gastroenterology, european crohn's and colitis organization, the american gastroenterology association, and the international organization for the study of inflammatory bowel disease (ioibd) have published guidelines regarding treating the ibd patient with sars-cov- and covid- . they are largely very similar, and they are concisely compared in a recent correspondence. the key features are that the patient without proven or suspected sars-cov- should continue on their current medications with aggressive attempts to reduce steroid usage because this is the only single agent that has been associated with increased poor outcomes with covid- , defined in the secure registry as a composite score of hospitalization, intubation, or death. patients are informed that the underlying diagnosis of ibd does not increase the risk of acquiring sars-cov- (although this, as with most published recommendations at this time, has not yet been borne out by rigorous data). in patients with proven or suspected sars, it is recommended that all of the biologic drugs, thiopurines, and methotrexate be held and aggressive efforts made to wean steroids. however, accumulating data in the secure database suggest that patients on antitumor necrosis factor (tnf) drugs, vedolizumab, and ustekinumab do not have a higher incidence of a poor outcome, and in fact, patients on theses agents may have a numerically lower incidence of poor outcomes. on the other hand, the use of combination therapy with a thiopurine and an anti-tnf drug may slightly increase the likelihood of a poor outcome. in a multivariate analysis, steroids were associated with a significant increase in poor outcomes, as were older age and the presence of multiple comorbidities. what has been a consistently surprising finding in the registry is that the use of mesalamine drugs, even when controlled for disease activity, may be associated with a worse outcome. thus far, this has not led us to discontinue these drugs, unless the patient is already in remission on an anti-tnf drug and the discontinuation of mesalamine has not been associated with a disease exacerbation. for patients receiving in-office infusions of biologics, we follow the precautions well outlined in the ioibd guidance. patients are contacted in advance of their infusion to ascertain that they have no symptoms suggestive of covid- . they are screened again at arrival to the office, including a temperature check. the patient must be wearing a mask before entering the office and must enter the office unaccompanied. each patient is given their own wrapped, unopened hand sanitizer to keep and is brought directly into an infusion room. unless there is a specific indication for an examination, we remain masked and conduct our office visit at least feet away from the patient. all furniture and equipment are washed copiously with bleach between patients. we aim to use so much bleach that the following patient is struck by its odor and confident that we have spared no cleansing agent. we participate in a number of pharmaceutical industrysponsored, double-blinded, randomized controlled drug trials. variable allowances were made by the sponsors to defer in-person visits, allow remote visits, and lengthen intervals between procedures. in no case was an elective study procedure mandated during the height of the pandemic between late march and early june. in any case, the patients and we, on their behalf, would have refused to do so. in our cohort, with ibd patients with a sars-cov- confirmed infection, our experience has been of a slightly lower incidence of poor outcomes than in the reported secure database as a whole ( . % vs % respectively), despite a similar median age of (see table ). this may be due to having only ( %) patient on more than mg of daily prednisone compared with % of patients in the secure registry. the percentage of our patients on anti-tnf drugs is similar to the most recent report of the secure database ( % vs %, respectively). we had patients, with pcr-documented sars-cov- who were hospitalized for covid- -related complications: a -yearold man with ulcerative colitis (uc) on vedolizumab who developed bilateral pneumonias was intubated and recovered; a -year-old man with uc on sulfasalazine and budesonide who developed adult respiratory distress syndrome was intubated for weeks, and recovered; and a -year-old man with uc and pneumonia was treated with high flow oxygen and recovered. no patients died. notably, patients with active crohn's ileitis, who initially refused hospital admission because of concerns of being in a hospital with a very high prevalence of covid- , developed acute ileal perforation, and both had surgery (one with a temporary diverting ileostomy) and had uncomplicated postoperative courses and no covid- -related complications. another patient on no ibd medications developed severe uc while infected with sars-cov- , was admitted to the hospital, treated with infliximab and intravenous hydrocortisone, improved, was discharged, and sustained no covid-related complications. two pregnant patients with crohn's disease in remission contracted sar-cov- . the first developed symptoms from weeks to and had an uncomplicated vaginal delivery at week ; she recovered uneventfully. another patient developed mild covid- symptoms from week into her third trimester currently and had no adverse outcome during her ongoing pregnancy now at weeks ( table ) . our concern has been in holding biologic therapy in the patient with active ibd and sars-cov- infection. in these patients with resolving symptoms of covid- or with minor symptoms without fever or dyspnea, we generally continue to treat or restart early their anti-tnf drug, vedolizumab, or ustekinumab, as long as they are afebrile without antipyretics for at least hours. the now well-recognized symptoms of loss of sense of smell or taste can persist for weeks after all other symptoms have resolved, and we therefore do not consider those patients as persistently infected if they are otherwise asymptomatic. as a rule, we did not attempt to alter combination therapy if we were not otherwise intending to do so. in those patients with prolonged interruptions in their biologics, we consider using a full reinduction regimen, as our greater concern has been loss of response and the subsequent possible need for prednisone (as occurred in a single patient); and our greatest fear, now abating, was that patients would need hospitalization during this crisis when mount sinai hospital had over covid- inpatients. we have not typically relied on documenting viral clearance with testing for a negative pcr or positive sars-cov- antibody. the ability to document clearance of active viral infection has been severely limited by problems with availability of pcr testing (now easing) and the finding of some patients with persistently positive viral pcr, which in many patients may represent nonreplicating, noninfectious rna debris. the advent of more widely available sars-cov- antibody testing is likewise not without interpretive flaws. one can easily be misled by accepting results from unknown labs, and we have been very cautious in accepting results of antibody testing without knowledge of the lab used. over antibody tests have been brought to the market, many with absolutely no fda oversight of accuracy, and as of may , , the fda had only issued emergency use authorization for of these assays; of had sensitivity and specificities of > %, respectively. many other assays have been found to be entirely unreliable and have been withdrawn from the market by fda edict. even with reliable antibody assays with high sensitivity and specificity, there is insufficient data at present to know whether the presence of antibodies are in fact neutralizing, and at what titer-if and for how long-they may confer immunity. in the absence of either negative pcrs or positive antibodies, we have relied on clinical resolution of symptoms of covid- for at least for hours as a guide as to when to restart therapies that we may have held. a recent ioibd guidance statement highlights the importance of considering the relative severity of both the underlying ibd and the covid- course in informing the decision regarding the timing of restarting biologics. we are now participating in the development of a database that will follow patients after clearance of the sars-cov- virus to determine the courses and outcomes of the ibd and of any sequelae or recurrence of covid- after any drug therapy has been suspended. perhaps never in the history of medicine has the advancement of knowledge about a given disease proceeded with such disconcerting and often terrifying speed. we spend countless hours devouring the news and ceaseless new published data. we have found the around-the-clock new york times online coronavirus coverage, offered free of charge, to be a superb resource-not just for ongoing news coverage and multiple updates throughout the day on demographics of new cases and fatalities but more importantly for its very deep reporting of any breaking scientific news in this era when reading a manuscript from a month ago feels like reading a journal article from the s. the times and the washington post to a lesser extent have many useful links to articles in press, in the journals, and sites we are the most familiar with (eg, new england journal of medicine, lancet, science, nature, johns hopkins coronavirus resource, cdc, fda, who etc., all also offering covid- information free of charge), in addition to online symposia and journals with very relevant, up-to-the-minute breaking news in clinical trials, microbiology, vaccine development, epidemiology, public health, and yes, even aerosol particle physics. medrxiv is a source of pre-peer reviewed manuscripts jointly established by cold spring harbor laboratory, bmj, yale. many important subsequently published papers have appeared there first, but many manuscripts posted there are never published or subjected to peer review and so caveat emptor. with the lockdown of nyc in march, the severely limited ability for all to travel, our greatly reduced in-office staff, and the near closure of our outpatient endoscopy center, we initially anticipated that our workdays would be shorter. we were mistaken. our patients continued to call, many with heightened anxiety regarding coronavirus and concerns regarding potential interruption in their familiar medical routines of follow-up care, monitoring, and continued medication treatment. the patients with ongoing active disease were terrified at the prospect of an emergency room visit or hospitalization. patients were particularly grateful for the unsolicited emails we sent and educational materials we shared with them via the crohn's & colitis foundation website, which is always accessible and constantly updated. we are certain that video office visits will become a staple of ongoing care for routine follow-up visits and for some patients with mild-moderate flares. most patients came to appreciate the avoidance of time and money spent on travel. how and when the payers rescind their reasonable and enlightened approach to appropriately compensate us for these visits remains to be seen. on a personal level, the severely reduced income, the confinement of our kids and families at home for nearly months, and the constant heightened vigilance we had to maintain for our staff and ourselves to prevent infection while at work in nyc amounted to a degree of stress to which we thought we would be immune. we look back to the outset of all of this, when some of us naively hoped we could contract the infection, quickly recover and just be done with it. watching some colleagues fall quite ill and hearing of deaths of others disabused us of this notion. here in nyc, we are hopefully, and perhaps wistfully thinking, that we are permanently emerging from the era of months that have felt like so many long and very dark years. it has been a time of unimaginable and unmitigated devastation to our city, with the poorest blacks and latino individuals in our city, as always, being vastly disproportionately affected and dying. we have been humbled by our overwhelming limitations in overcoming what we had anticipated would be just another viral epidemic passing us by and affecting others less fortunate than us. we hope that while we await the arrival of the "new abnormal" that the race for effective therapies and the dreamed-of vaccines will arrive soon enough, that we can learn enough, and be wise enough, to somehow do better for our patients and for ourselves for now, and for the next tie around. authors would like to acknowledge the commitment and bravery of all of our mount sinai gi fellows who took months away from their training to rotate on all of the various covid- ward and intensive care units. they want to thank all of the mount sinai hospital staff that have gone largely unrecognized: the housekeeping porters washing every bed, the transporters who take the patients to and from the icus and too frequently to the morgues, and the respiratory therapists who are perhaps the ones who put themselves at the greatest risk of all. our patients, their families, and we all owe them a huge debt of gratitude for their courageous and tireless service. secure-ibd database public data update new york times corona virus map and cases count covid- (coronavirus): what ibd patients should know bsg) advice for management of inflammatory bowel diseases during the covid- pandemic aga clinical practice update on management of inflammatory bowel disease during the covid- pandemic: expert commentary ioibd covid- webinars and guidelines. accessed management of inflammatory bowel disease during covid- . summary of recommendations from gastrointestinal societies but not tnf antagonists, are associated with adverse covid- outcomes in patients with inflammatory bowel diseases: results from an international registry stopping mesalamine therapy in patients with crohn's disease starting biologic therapy does not increase risk of adverse outcomes covid- and post infectious immunity: limited evidence, many questions serology assay to manage covid guidance for when to restart inflammatory bowel disease therapy in patients who held immunosuppressant medications during covid- the new york times corona virus coverage the washington post the corona virus coverage covid- sars-cov- preprints from medrxiv and biorxiv key: cord- -z onmkrn authors: bezzio, cristina; saibeni, simone title: severe ibd flares and covid- : expand the gastroenterology-surgery team to include an infectious disease specialist date: - - journal: gastroenterology doi: . /j.gastro. . . sha: doc_id: cord_uid: z onmkrn nan severe acute respiratory syndrome coronavirus (sars-cov- ) is a new virus that was first identified in wuhan, china, and has now spread worldwide. it causes a potentially fatal infectious respiratory syndrome called coronavirus disease (covid- ), which has rapidly evolved into a pandemic ( ) . covid- is a major health emergency that has had a substantially impact on everyday clinical practice. it has raised several new questions and concerns about its potential impact on patients with chronic illnesses, especially those treated with immunomodulating drugs. in the field of inflammatory bowel disease (ibd), the british society of gastroenterology ( ) and american gastroenterological association (aga) ( ) have recently provided guidance on the management of patients with crohn's disease and ulcerative colitis during this complicated period. for example, aga recommends following standard therapeutic algorithms for patients hospitalized for ibd who also have mild or incidentally identified covid- ( ). it also recommends scheduling the usual surgical consultation, but acknowledges that it is reasonable to medically attempt to postpone surgery during the pandemic. perhaps because of the lack of empirical evidence, these guidelines did not specifically address the challenging case of ibd patients hospitalized due to a severe flare of disease with subsequent or concomitant pneumonia due to sars-cov- . especially in ulcerative colitis, a severe flare of disease is a life-threatening emergency that requires prompt recognition, hospitalization, early initiation of treatment and close monitoring ( ). the outcome of severe flares is measured in terms of surgery and mortality rates. therefore, a multidisciplinary approach involving a gastroenterologist and a colorectal surgeon is essential. this professional pair shares the responsibility in correctly identifying the most effective type and timing of rescue therapy or surgery. however, in the covid- pandemic, this professional pair may not be enough to effectively manage the infection in an ibd patient with a severe disease flare. we recently published a prospective, observational study of ibd patients with covid- ( ) . the study found that active disease, age > years and comorbidities associated with worse assessing the risk-benefit ratio we decided to start with infliximab at mg/kg. we do not yet know what will be the final effect of this treatment and the outcome for the patient. nonetheless, we think that the issues raised by this case are of great interest to physicians managing ibd patients during the covid- pandemic. in particular, we suggest that an infectious disease specialist join the gastroenterologist and surgeon on the team managing ibd patients hospitalized for a severe flare of disease. in this way, they will become "three of a perfect pair". gut , accepted key: cord- - v lfb authors: neurath, markus f title: covid- and immunomodulation in ibd date: - - journal: gut doi: . /gutjnl- - sha: doc_id: cord_uid: v lfb the current coronavirus pandemic is an ongoing global health crisis due to covid- , caused by severe acute respiratory syndrome coronavirus . although covid- leads to little or mild flu-like symptoms in the majority of affected patients, the disease may cause severe, frequently lethal complications such as progressive pneumonia, acute respiratory distress syndrome and organ failure driven by hyperinflammation and a cytokine storm syndrome. this situation causes various major challenges for gastroenterology. in the context of ibd, several key questions arise. for instance, it is an important question to understand whether patients with ibd (eg, due to intestinal ace expression) might be particularly susceptible to covid- and the cytokine release syndrome associated with lung injury and fatal outcomes. another highly relevant question is how to deal with immunosuppression and immunomodulation during the current pandemic in patients with ibd and whether immunosuppression affects the progress of covid- . here, the current understanding of the pathophysiology of covid- is reviewed with special reference to immune cell activation. moreover, the potential implications of these new insights for immunomodulation and biological therapy in ibd are discussed. ► the current covid- pandemic is an ongoing global health crisis due to severe acute respiratory syndrome coronavirus and provides major challenges for patient care in ibd. ► severe patients with covid- may suffer from progressive pneumonia, acute respiratory distress syndrome and multiorgan failure due to hyperinflammation and a cytokine storm syndrome; the interleukin- r antibody tocilizumab is currently tested for clinical therapy of severe covid- . ► the receptor ace is highly expressed in ileal and colonic tissue and ace levels are induced in patients with ibd. ► there is currently no evidence for an increased frequency of covid- cases in ibd; a recent study suggesting decreased disease activity in ibd requires further investigations. ► based on the currently available limited data, immunomodulatory and biological therapies can be continued in patients with ibd in remission; however, close attention to new results should be paid in the dynamic pandemic situation. ► future cases of patients with ibd suffering from covid- should be documented in the new secure registry (https://covidibd.org/). the current coronavirus pandemic is an ongoing global health crisis due to covid- , caused by severe acute respiratory syndrome coronavirus . although covid- leads to little or mild flu-like symptoms in the majority of affected patients, the disease may cause severe, frequently lethal complications such as progressive pneumonia, acute respiratory distress syndrome and organ failure driven by hyperinflammation and a cytokine storm syndrome. this situation causes various major challenges for gastroenterology. in the context of ibd, several key questions arise. for instance, it is an important question to understand whether patients with ibd (eg, due to intestinal ace expression) might be particularly susceptible to covid- and the cytokine release syndrome associated with lung injury and fatal outcomes. another highly relevant question is how to deal with immunosuppression and immunomodulation during the current pandemic in patients with ibd and whether immunosuppression affects the progress of covid- . here, the current understanding of the pathophysiology of covid- is reviewed with special reference to immune cell activation. moreover, the potential implications of these new insights for immunomodulation and biological therapy in ibd are discussed. the current coronavirus pandemic is an ongoing global health crisis due to covid- . this infectious disease represents a key challenge for gastroenterologists. one of the major questions relates to the clinical handling of covid- in the presence of inflammatory disorders of the gi tract such as in patients with ibd, particularly in the presence of immunosuppressive or immunomodulatory therapy. therefore, this review will address the following questions: ► what is the evidence for intestinal covid- disease and how does covid- induce mucosal immune cell activation and a cytokine release syndrome? ► is there an increased or a decreased risk for patients with ibd to suffer from covid- ? ► what is the effect of corticosteroids and classical immunosuppressive agents such as methotrexate (mtx) and azathioprine on covid- ? ► what is the effect of biological agents and cytokine blockers on covid- ? a newly identified beta-coronavirus, denoted severe acute respiratory syndrome coronavirus (sars-cov- ), caused a cluster of pneumonia cases in wuhan, china, in december . [ ] [ ] [ ] [ ] [ ] the disease caused by sars-cov- was subsequently named by who as covid- . genome sequencing of sars-cov- showed that the virus is closely related (with % identity) to two bat-derived sars-like coronaviruses, but displays more distant similarities to sars-cov (about %) and middle east respiratory syndrome coronavirus (mers-cov) (about %). structural analyses of sars-cov and sars-cov- identified amino acid substitutions between these coronaviruses, which may cause functional and pathogenic divergence between these viruses. another recent study suggested that sars-cov- uses mutations and recombination in different genomic regions including the membrane, envelope, nucleocapsid and spike glycoprotein regions to become a novel infectious agent. in fact, nine putative recombination patterns were identified in the spike glycoprotein, rdrp, helicase and orf a regions as well as six recombination regions in the s gene that may control evolutionary survival and antigenicity thereby allowing sars-cov- to cross borders from its original host (bats) to humans and to adapt to a human host. figure hypothetical pathogenesis of covid- . severe acute respiratory syndrome coronavirus (sars-cov ) infects ace expressing epithelial cells in the lung and/or the intestine. this is followed by production of mediators causing immune cell activation. overwhelming immune cell activation may lead to severe complications including acute respiratory distress syndrome (ards), shock and kidney or multiorgan failure. b, b lymphocytes; iec, intestinal epithelial cell; ilc, innate lymphoid cell; m, monocyte/macrophage; n, neutrophils; teff, effector t cells; treg, regulatory t cell; type i p, type i pneumocytes; type ii p, type ii pneumocytes. the ace/ace receptor system (modified according to zhang et al ) . the classical renin-angiotensin-aldosterone system ace/angiotensin ii/angiotensin type / receptor (at -r) and ace / ang - /mas- receptor (mas -r) systems are shown. [ ] [ ] [ ] [ ] the ace/ at -r system has been mainly implicated in pro-inflammatory immune responses and tissue injury. in contrast, the ace /masr system appears to play a key role in many anti-inflammatory pathways controlling tissue protection. several studies have analysed the mechanisms by which sars-cov- may enter the human host. these studies showed that the surface spike glycoprotein (s protein) on the surface of the virus binds to the receptor ace , a monocarboxypeptidase controlling cleaving of several peptides within the reninangiotensin system that is highly expressed on pneumocytes (type ) in the lung, particularly in smokers (figures - ). in this context, the cleavage of the s protein regulates viral uptake and is regulated by the cell surface-associated transmembrane protease serine protease (tmprss ), a key enzyme for s protein cleavage and priming. subsequently, the receptor binding domain of the s protein is recognised by the extracellular peptidase domain of ace mainly through polar residues thus providing a molecular basis for coronavirus recognition and infection. bioinformatical studies indicated that sequence mutation of sars-cov- with methylation of the '-cap structure may affect ace binding and facilitate viral rna escape recognition by the host innate immune system. after membrane fusion between the virus and the host cell, the viral genome rna reaches the cytoplasm followed by the translation of accessory and structural proteins. newly generated envelope and nucleocapsid proteins as well as genomic rna then form viral particle buds followed by the formation of virion-containing vesicles that may fuse with the cell membrane resulting in the release of viruses in the local microenvironment. clinical symptoms in covid- vary between patients but most individuals have a mild form of the disease with no or flu-like symptoms including dry cough, fever, a runny nose and fatigue. additional symptoms may comprise shivering, throat pain, anosmia, headache, joint pain, nausea and diarrhoea. in more severe forms of the disease, marked inflammation and progressive pneumonia occur leading to difficulties in breathing. finally, respiratory failure due to pneumonia, acute respiratory distress syndrome (ards) and multiorgan failure may occur in severe cases and are associated with high mortality in patients with covid- - (figure ). the severity of the disease is modulated by co-factors such as the age and comorbidities. covid- is more severe in older patients as well as in patients suffering from diabetes, hypertension or obstructive lung disorders. finally, smokers were suggested to be . times more likely to have severe symptoms of covid- and approximately . times more likely to be admitted to an intensive care unit, need mechanical ventilation or die compared with non-smokers. thus, smoking is most likely associated with progression of covid- . the infection with sars-cov is currently diagnosed by sars-cov nucleic acid amplification tests using oropharyngeal swabs. [ ] [ ] [ ] [ ] however, the combination of the amplification test with ct scanning (figure ) has been shown to improve the diagnosis of covid- . ct scans can detect disease even in the presence of a negative nucleic acid amplification test and a subgroup of patients with covid- has pneumonia in spite of absent clinical symptoms. ace is expressed on pneumocytes of the lower airways possibly explaining the high frequency of pneumonia in affected patients. [ ] [ ] [ ] although initial studies suggested that gi symptoms, such as diarrhoea ( %- . %), nausea and vomiting ( %- . %), are not very common in covid- , - a recent study showed that gi symptoms are more frequently present than initially thought. in fact, analysis of patients revealed that % of patients present with nausea, vomiting or diarrhoea. severe types of covid- were more frequently seen in patients with gi symptoms ( %) as compared with patients without gi symptoms ( %) consistent with the possibility that intestinal manifestation of covid- may augment mucosal cytokine production and clinical outcome of the disease. a subgroup of patients with covid- with gi symptoms also had significantly higher rates of fever, shortness of breath and headache, respectively. it was suggested that gi symptoms may cause patients with covid- to be more prone to electrolyte disturbances including decreased serum sodium levels that may aggravate the disease. a recent study in > outpatients with mild courses of covid- demonstrated the presence of diarrhoea in approximately % of patients suggesting that diarrhoea may be a frequent hallmark of mild disease. the presence of diarrhoea could be due to direct infection of gi cells. in this regard, gastric, duodenal and rectal epithelial cells rather than cells in the oesophagus were shown to express the sars-cov receptor ace . covid- led to infection of these cells followed by expression of the viral nucleocapsid protein indicating that sars-cov- may spread from infected to uninfected cells in the gi tract. infection was not associated with marked macroscopic inflammation on endoscopy. however, in the lamina propria of stomach, duodenum and rectum, numerous infiltrating plasma cells and lymphocytes with interstitial oedema were seen in a covid- patient indicating mucosal immune cell activation. in addition to local enteric infection, viraemia following lung infection may occur in few patients (approximately %) and may lead to a secondary attack of sars-cov on ace target organs such as the kidney and the intestine. rna from sars-cov- was detected in samples from faeces of patients with covid- , suggesting a possible faecal-oral transmission route. moreover, infectious sars-cov- was isolated from stool of these patients indicating the presence of infectious virions in the gi tract that may contribute to viral spread. thus, these observations suggest that sars-cov- may actively infect and replicate in the gi tract. interestingly, in a subgroup of patients with sars-cov- (> %), viral rna in faeces could be detected even after negative conversion of the viral rna in the respiratory tract. these findings are consistent with a model in which faecal-oral transmission may even occur after viral clearance in the airways. future studies will have to confirm this pilot study and to determine whether testing of stool samples for sars-cov- is essential in covid- . the potential presence of sars-cov- in stool is critical for our understanding of covid- , as a recent study did not find any evidence for the presence of infectious virus in stool samples from affected patients in spite of the presence of sars-cov- rna. there is overwhelming evidence that covid- results in innate and adaptive immune cell activation in the infected host. the release of mediators and chemokines by infected cells rapidly leads to a local accumulation of neutrophils at the site of the infection. while such neutrophils may exert important antiviral effector functions, they also secrete cytokines and chemokines that attract further immune cells such as monocytes and t lymphocytes (figure ). thus, they may contribute to exaggerated immune responses and covid- non-survivors had higher levels of neutrophils than survivors. also t-cell activation is present in patients with covid- . while cd + t cells are important in providing b cells help and mediating protective humoral immunity by stimulating b cells to produce virus-specific antibodies, cd + t cells have been suggested to provide key cytotoxic function and to limit local infection. insufficient clearance of the infection, however, leads to progressive hyperinflammation. in fact, many patients with covid- with progressive pneumonia, ards and multiorgan failure suffer from hyperinflammation and a cytokine storm syndrome. [ ] [ ] [ ] it has been suggested that this cytokine storm in covid- may lead to secondary haemophagocytic lymphohistiocytosis, a hyperinflammatory disorder triggered by viral infections that is associated with multiorgan failure and high lethality. this hyperinflammation may be particularly deleterious in older patients with t-cell immunosenescence; a chronic low-grade proinflammatory state of the immune system that affects many older individuals, even when they are apparently healthy and free of risk factors. various recent studies described the immune cell dysregulation in covid- by using peripheral blood mononuclear cells. a study in patients with severe covid- demonstrated that affected individuals exhibit lower lymphocyte counts, higher neutrophil counts, an elevated neutrophil-lymphocyte ratio as well as decreased percentages of monocytes, eosinophils and basophils. neutrophilia has been suggested as risk factor for the development of ards and progression from ards to death in covid- . additionally, a reduction of nk cell percentages was noted in covid- , while b cells were within the lower level of normal range. lymphocytopenia was a hallmark finding in patients with severe covid- cases and present in . % of the patients on admission. both cd + and to a lesser extent cd + t-cell numbers were reduced in severe covid- cases, although the cd +/cd + t-cell ratio remained normal. moreover, within the cd + t-cell subset, decreased numbers of effector memory t cells (cd ro+) and regulatory t cells (cd +cd low) were noted, while the percentage of naive t cells (cd ra+) increased. the higher naïve/memory t-cell ratio in severe as compared with mild covid- cases suggested marked immune cell alteration in these patients. moreover, the suppression of regulatory t cells in severe cases indicated insufficient counterregulation of proinflammatory immune responses that may further aggravate hyperinflammation and tissue injury. with regard to b-cell activation, total levels of iga, igg and igm were similar between severe and mild covid- cases suggesting no major general impairment in b-cell activity. however, no tests for protective antibodies in serum were made in this study. finally, no differences in complement levels were noted between both groups. analysis of inflammation markers in serum showed elevated levels of procalcitonin, c reactive protein, d-dimer and ferritin in covid- . moreover, in severe cases, significantly higher serum levels of tumour necrosis factor (tnf), interleukin (il)- r, il- , il- and il- were detected as compared with milder cases, while il- beta levels and the number of ifn-gamma producing cd + and cd + t cells remained unchanged. a comparison of cytokine and chemokine levels among covid- intensive care unit (icu) patients (n= ), non-icu patients (n= ) and healthy controls further defined the plasma changes associated with the cytokine storm syndrome. icu patients had significantly higher levels of il- beta, il- ra, il- , il- , il- , il- , il- , il- , il- , il- , il- , fibroblast growth factor, granulocyte colony-stimulating factor (g-csf), granulocyte macrophage-colony stimulating factor, interferon (ifn)-gamma, tnf, vascular endothelial growth factor, ip- , monocyte chemoattractant protein (mcp)- , macrophage inflammatory protein (mip)- alpha, platelet-derived growth factor (pdgf) and mip- beta than healthy controls, while levels of eotaxin, regulates upon activation normal t cell expressed and secreted (rantes), il- , il- and il- were unchanged. this finding is probably due to production of cytokines and chemokines by numerous cells including epithelial cells, neutrophils, monocytes and t cells and mirrors the broad overwhelming hyperresponsiveness of the immune system in covid- ( figure ) . moreover, icu patients had significantly higher levels of il- , il- , il- , tnf, g-csf, mcp- and mip- a than non-icu patients indicating that these cytokines with known immunoregulatory and/or chemotactic functions may regulate the progression of the disease. although il- levels were not significantly higher in the icu group versus the non-icu group, another study in patients suggested that the combination of d-dimer and il- differentiates between mild and severe covid- cases. based on these findings, il- was suggested as potential biomarker of disease activity. the covid- receptor ace is particularly highly expressed in intestinal epithelial cells from the terminal ileum and to a lesser extent in the colon, where mucosal inflammation in patients with ibd (crohn's disease (cd); uc) is frequently detected. in this context, ace may act as a co-receptor for nutrient uptake, in particular for amino acid resorption from food. moreover, immunohistochemical analyses revealed higher ace protein expression in inflamed ibd samples from the terminal ileum and colon as compared with controls (no differences between inflamed and non-inflamed regions). furthermore, ace activity in the colon was elevated in non-inflamed colon in ibd as compared with controls and active ibd. finally, the average expression of soluble ace was shown to be increased in patients with ibd (mainly in cd) and a higher ace :ace ratio in plasma was noted in patients with ibd as compared with controls without ibd. cytokines expressed in ibd, such as ifn-gamma, can potentially induce ace expression by cytokine signalling events driving ace promoter activity consistent with the idea that mucosal inflammation may increase expression of ace . a study using tandem mass tag-based shotgun proteomics additionally showed higher levels of ace in inflamed intestinal areas in patients (cd) as compared with patients with uc. finally, the fusion of sars-cov with the host cell membrane is critical for uptake in cells and is modulated by the s protein. activation of the s protein via proeolytic cleavage is controlled by host trypsin-like proteases, whose activity is upregulated in ibd, and this effect might facilitate infection in patients with ibd. collectively, these findings suggested the possibility that patients with ibd might be particularly susceptible to covid- . however, there is no evidence so far that patients with ibd are highly susceptible to covid- . in contrast, a recent study from wuhan studied patients with ibd ( uc and cd) during the local outbreak of the disease and did not report any covid- cases. the reasons for this observation are not entirely clear but might relate to the local adjustment of protective measures to prevent infection, the particular awareness of the ibd patient cohort to hygiene and infection prevention and the modulation of immunosuppressive therapy (eg, stop of treatment with immunomodulators and biologicals). alternatively, patients with ibd might be less susceptible to covid- and further studies in this regard are highly warranted. is there a theoretical rationale to assume that patients with ibd might be less susceptible to inflammation? there is currently no direct evidence that altered expression of ileal and colonic ace and tmprss directly regulates viral entry in ibd and expression levels of these proteins may depend on the local inflammatory activity and the degree of epithelial damage. further studies are essential to address these points. however, in addition to the full-length, transmembrane ace protein on the surface of epithelial cells, a soluble form of ace can be generated by proteolytic cleavage that lacks the membrane anchor and circulates in small amounts in the blood. it has been suggested that the soluble form of ace acts as a competitive binding partner for sars-cov and thus sequestrates viruses and prevents their binding to the cellular full-length ace protein. interestingly, the cleavage of membrane ace into the soluble form is regulated by the tnf-α convertase adam , a protease upregulated in patients with active ibd. consistently, elevated levels of ace were seen in plasma from patients with ibd, suggesting the possibility that increased levels of soluble ace may limit infection progression and modulate susceptibility to infection. additionally, expression of ace in ibd may control anti-inflammatory immune responses, as discussed in the next paragraph. the ace receptor has been shown to be part of the reninangiotensin-aldosterone system (raas) that comprises the classical raas ace/angiotensin ii/angiotensin type / receptor (at -r) and ace /ang - /mas- receptor (mas -r). [ ] [ ] [ ] [ ] while the ace/at -r system has been mainly implicated in proinflammatory immune responses and tissue injury, the ace / masr system plays a crucial role in many anti-inflammatory pathways controlling tissue protection (figure ). functional studies in murine experimental colitis models indicated that modulation of ace expression may affect the severity of colitis activity. in fact, deficiency in the ace cleavage controlling protein adam aggravated dextran sulphate sodium (dss) colitis activity. moreover, ace expression was shown to be elevated in the colon during experimental dss colitis as compared with controls. a chemical inhibitor of ace (gl ) reduced dss colitis severity suggesting that ace plays a pathogenic role in colitis. in contrast, ace deficiency caused enhanced susceptibility to dss colitis probably due to altered immune cell trafficking, changes in innate immunity and cytokine production as well as alterations of the gut microbiota. in this study, dietary tryptophan primarily absorbed via an ace -dependent epithelial transport pathway regulated the expression of antimicrobial peptides via mammalian target of rapamycin (mtor), either directly through nutrient sensing and/or through the tryptophannicotinamide pathway. the findings suggested a direct link between dietary amino acid metabolism and innate immunity, the composition of the intestinal microbiota, the susceptibility to colitis and malnutrition. collectively, these results were consistent with a model in which ace is protective in experimental colitis. consistently, angiotensin (ang - ) ameliorated colitis activity in the dss colitis model, while blockade of the mas- r aggravated disease, indicating a protective role of the ace / ang - /mas- r pathway. several immunotherapies in patients with ibd have been associated with an increased risk of infections as they may inhibit intracellular signalling cascades needed for the host to fight pathogens. [ ] [ ] [ ] corticosteroids, for instance, are potentially associated with an increased risk for infections and have been widely used in studies from china for treatment of ards and hyperinflammation in covid- . although these studies were not designed to evaluate the effects of steroids, one study even suggested that treatment with methylprednisolone decreases the risk of death in covid- . however, as corticosteroids suppress lung inflammation but may also inhibit antiviral immune responses and pathogen clearance, the use of steroids during covid- is controversial. studies during previous coronavirus outbreaks in mers and sars suggested delayed viral clearance and potential complications such as diabetes and psychosis in patients receiving high doses of corticosteroids. in the absence of controlled clinical trials on the use of corticosteroids, interim guidelines from who advised against the use of steroids unless indicated for another reason. in agreement with this concept, a recent uncontrolled study in covid- suggested that patients with high steroid use (n= ) have significantly worse clinical outcomes (ards, shock, kidney injury, continuous renal replacement, secondary infection) than patients without steroid use (n= ). however, no data on low-dose and short-term steroid use are currently available. thus, the potential advantages and disadvantages of steroid use should be carefully considered in individual patients until more data become available. thiopurine (azathioprine, -mercaptopurine) and jak / inhibitor (tofacitinib) treatment can potentially reduce the number of activated t cells and affect t-cell activation and effector function. although no data are currently available in this context in ibd, this might affect the course of covid- , as lymphopenia was associated with worse prognosis in this disease. moreover, tofacitinib has an increased risk for certain viral infections (eg, herpes zoster infection). [ ] [ ] [ ] thus, treatment indication needs to be discussed in individual cases. at this point, no evidence for stopping these treatments in patients with remission exists. similarly, there are no specific data available on mtx. anti-tnf antibodies are frequently used for ibd therapy. [ ] [ ] [ ] as tnf inhibition may potentially affect antiviral immunity and has been shown to affect hepatitis b virus reactivation, tnf blockade could regulate the susceptibility to covid- . however, analyses of tnf levels in covid- led to different results. one study showed no effect on tnf levels in severe covid- cases in spite of the regulation of other proinflammatory cytokines. in contrast, another study reported that covid- icu patients had significantly higher serum levels of tnf than non-icu patients. tnf may also exert pathogenic effects in covid- by augmenting the expression of ace or by augmenting lymphopenia through induction of direct leucocyte death via tnf/ tnfr signalling in t cells. these findings argue for a potentially protective effect of tnf inhibition in covid- and further studies are needed to address this point. the alpha /beta -specific antibody vedolizumab blocks immune cells homing by preventing the interaction between these integrins and madcam- , thereby preventing immune cell trafficking and suppressing mucosal inflammation in patients with ibd. [ ] [ ] [ ] direct effects have been shown on homing of t helper (th) , th , th , th and treg cells to the inflamed intestine. no marked increase of viral infections was noted in patients with ibd receiving vedolizumab therapy. moreover, no viral reactivation was seen in patients with hepatitis. furthermore, vedolizumab treatment in addition to antiretroviral therapy led to sustained virological control in siv-infected macaques. these findings encouraged a phase i clinical trial of vedolizumab in patients with hiv and this study did not show effects on the viral load in this disease on antibody therapy. ustekinumab is a cytokine antibody blocking the p subunit of the two cytokines il- and il- . this antibody has been approved for clinical therapy of patients with ibd. [ ] [ ] [ ] [ ] mechanistically, this antibody prevents th t-cell priming via il- and simultaneously inhibits perpetuated activation of il- responsive th cells. [ ] [ ] [ ] [ ] [ ] no increase in viral infections in patients with ibd has been noted on ustekinumab therapy. it is tempting to speculate that il- blockade in covid- may suppress the pathogenic th response in the cytokine storm syndrome, although no studies have addressed this concept so far. thus, there are currently no major concerns on the use of this antibody for clinical therapy in patients with ibd due to the current covid- risk situation. although cytokine blockers and jak inhibitors have raised theoretical concerns with regard to ibd therapy, it should be noted that these agents are currently considered for clinical therapy of covid- cases with hyperinflammation and ards. jak inhibitors blocking jak have been suggested to block viral entry and the th part of the cytokine storm syndrome without altering interferon signalling (this has not been demonstrated for the jak / inhibitor tofacitinib so far). additional studies suggested potential benefits of cytokine blockade in sepsis. re-analysis of a phase iii trial in sepsis suggested that blockade of il- signalling via anakinra may result in significant survival benefits in patients with hyperinflammation and elevated cytokine production. moreover, tnf inhibition has been suggested to be helpful in selected patients with hyperinflammation and high il- levels in sepsis. finally, the il- r antibody tocilizumab is currently considered for therapy of hyperinflammation and ards in covid- and a prospective phase iii trial has been initiated ( www. clinicaltrialsarena. com/ new/ roche-actemracovid- -trial) to clarify its effects on cytokine storm syndromes in the disease. this antibody had been previously tested in a small phase ii trial in cd and the results suggested potential efficacy and safety. however, blockade of il- signalling has recently raised concerns in cd, as il- antibodies (pf- ) led to reduction of inflammation but the formation of abscesses and perforation in a subgroup of patients with ibd. in summary, there is currently no evidence for an increased risk or aggravated outcomes in patients with ibd in the context of covid- consider to restructure your ibd patient care under consideration of the local situation ► strictly separate areas for care of patients with suspected/ proven covid- and patients with ibd in the hospital. ► assign-specific physicians to inpatient care, endoscopy and remote monitoring. ► convert multidisciplinary team meetings into virtual meetings. ► consider to avoid elective surgery. ► avoid crowding in the waiting area of the outpatient unit. ► consider to wear personal protective equipment and follow the who recommendations to prevent any contamination. ► consider to limit the number of patients in the outpatient clinic by focusing on infusion treatment. ► secure home delivery and adequate drug supply to all patients given subcutaneous and oral drugs. ► provide help in individual cases by using emails and telephone calls. ► send newsletters or information material by email. ► secure communication between local gastroenterologists and primary care doctors with your centre. ► consider alternative and safer ways of administration and remote monitoring of patients with ibd. modified according to danese et al and fiorino et al. have been published so far. in general, few studies on the effect of covid- on pregnancy are available and it has been suggested that it is necessary to carefully monitor suspected pregnant women before and after delivery as well as newborn children in this context. other covid- risks situation comprise older patients with ibd with comorbidities as well as patients suffering from malnutrition who may be at risk for infections and severe courses of the disease, respectively. - furthermore, dependent on the local situation, there could be an increased risk for covid- in the context of ibd surgery in the hospital. finally, experimental covid- treatment with hydroxychloroquine or remdesivir may increase the risks for drug-drug interactions with established ibd medications ( www. drugs. com: potentially increased risk of combination therapy with hydroxychloroquine and adalimumab/infliximab for nerve damage). with regard to the effect of ibd on covid- , it should be pointed out that further studies are required in this highly dynamic situation. there is no evidence to suggest that patients with ibd should discontinue ibd-specific medications. however, older patients with ibd with comorbidities such as diabetes mellitus, obstructive lung disease, coronary heart disease and hypertension might have an increased risk for covid- and further studies are urgently needed to address this point. in this context, there is an ongoing international programme initiated by the international organization for the study of ibd to register covid- cases in patients with ibd in the secure registry (https:// covidibd. org/) in order to obtain further insights into the disease susceptibility and effect of 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(covid- ) in pregnancy; a narrative review clinical analysis of pregnant women with novel coronavirus pneumonia nutritional aspects in inflammatory bowel diseases espen guideline: clinical nutrition in inflammatory bowel disease nutritional approach as therapeutic manipulation in inflammatory bowel disease systematic review with meta-analysis: biologics and risk of infection or cancer in elderly patients with inflammatory bowel disease implications of covid- for patients with pre-existing digestive diseases management of ibd during the covid- outbreak: resetting clinical priorities inflammatory bowel disease care in the covid- pandemic era: the humanitas, milan, experience key: cord- - nbi ws authors: segal, jonathan p; quraishi, mohammed nabil; bhala, neeraj; brookes, matthew james title: prevention of covid- in patients with ibd date: - - journal: lancet gastroenterol hepatol doi: . /s - ( ) - sha: doc_id: cord_uid: nbi ws nan we read with interest the correspondence from ping an and colleagues describing their efforts to prevent coronavirus disease in patients with inflammatory bowel disease (ibd) in wuhan, china. the team reported that of the patients who were registered with ibd during the covid- pandemic in the wuhan region, only five patients were admitted to hospital because of ibd, and none were reported to have covid- . this information was obtained by use of social media and online educational materials, as well as by contacting % of their ibd population. in the uk, this approach would prove difficult, especially as our understanding of all at-risk groups in this pandemic evolves. indeed, the national health service, in conjunction with the british society of gastroenterology (bsg), relied on individual health-care trusts to highlight patients at high risk with ibd so advice could be delivered by post regarding shielding and stringent physical distancing. a further challenge for the uk is the large difference in ibd incidence and prevalence between china and the western world. the age-standardised prevalence of ibd in china is · ( % uncertainty interval · - · ) per population compared with · ( · - · ) per in the uk; the prospect of protecting this population from covid- is likely to be a much greater challenge. with concerns regarding a second wave of covid- cases, it is imperative that we protect the most sick and susceptible in our society. an and colleagues show the importance of ibd registries and the ability to contact at-risk groups via innovative means such as social media and the internet. these methods can result in rapid development of virtual telephone clinics, but they still ultimately require people to run them. the uk and other countries should therefore urgently seek to improve their ibd digital resources and staff resources to potentially reduce the burden of further waves of covid- . of further interest, an and colleagues also reported measures to avoid immunosuppression, including ceasing infliximab infusions in exchange for aminosalicylates or thalidomide. bsg guidance suggests that patients should continue on their current medications, including infliximab, as active disease remains the biggest risk to a patient with ibd. furthermore, the use of thalidomide for patients with ibd in the uk is uncommon, with a systematic review highlighting that there is insufficient evidence for its use in ibd and that it is potentially associated with adverse effects. it would therefore be of interest to see the long-term implications of this practice to guide future health-care systems in their approach to their patients with ibd at risk from covid- . as further evidence accumulates, our understanding of covid- -related risks in ibd populations globally will improve. we could potentially be overprotecting patients with ibd, but overprotection is better than undue risks given the current uncertainties. mjb reports grants, travel support, conference fees, and honoraria from vifor international, grants, travel support, and conference fees from tillotts pharma, grants from national institute for health research (nihr) uk research, nihr hta funding stream, and nihr health foundation, outside the submitted work. all other authors declare no competing interests. department of gastroenterology prevention of covid- in patients with inflammatory bowel disease in wuhan, china british society of gastroenterology. bsg covid- guidance on ibd patient risk groups a systematic analysis for the global burden of disease study british society of gastroenterology guidance for management of inflammatory bowel disease during the covid- pandemic systematic review: thalidomide and thalidomide analogues for treatment of inflammatory bowel disease key: cord- -ydbyonbg authors: spagnuolo, rocco; larussa, tiziana; iannelli, chiara; cosco, cristina; nisticò, eleonora; manduci, elena; bruno, amalia; boccuto, luigi; abenavoli, ludovico; luzza, francesco; doldo, patrizia title: covid- and inflammatory bowel disease: patient knowledge and perceptions in a single center survey date: - - journal: medicina (kaunas) doi: . /medicina sha: doc_id: cord_uid: ydbyonbg background and objectives: spreading of sars-cov- infection from china to countries with a higher prevalence of inflammatory bowel disease (ibd) has generated concern among gastroenterologists and patients. the aim of this survey is to evaluate knowledge about clinical importance of covid- , disease management, prevention measures, and anxiety level during pandemic among patients with ibd. material and methods: from th march to th april , a questionnaire survey was administered to patients with ibd by email or phone application. the questionnaire consisted of five sections: ( ) anthropometric, demographic and clinical characteristics, ( ) knowledge about clinical importance of covid- , ( ) ibd management, ( ) prevention measures, ( ) anxiety level during pandemic. results: one hundred forty two questionnaires were completed. ninety-seven patients ( . %) were males with a mean age of years (sd ; range – ). fifty-four individuals ( %) were affected by crohn disease and ( %) by ulcerative colitis. most patients reported high knowledge about clinical importance of covid- ( %), ibd management ( %), and prevention measures ( %). sixty-two percent of them showed moderate-high level of anxiety. high education level was independently associated with high knowledge about clinical importance of covid- (odds ratio [or] , % confidence interval [ci] . – . , p = . ) and older age (or , %, ci . – . , p = . ), while the receipt of e-format educational material with low knowledge about clinical importance of covid- (or , %, ci . – . , p = . ). displaying an active disease appeared to be independently associated with low knowledge of ibd management (or . , % ci . – . , p = . ) and no variables other than an older age was independently associated with higher level of anxiety (or . , % ci . – . , p = . ). conclusions: high educational level and aging promote knowledge about clinical importance of covid- , while e-format educational material does not. taken together with findings that an active disease status compromises knowledge of ibd management and the high level of anxiety related to increasing age, these data suggest the need of further supporting patient-oriented strategies in ibd during covid- pandemic. following the first reports of cases of acute respiratory syndrome in the chinese wuhan municipality at the end of december , chinese authorities have identified a novel coronavirus as the main causative agent. on february , the novel coronavirus was named severe acute respiratory syndrome coronavirus (sars-cov- ) while the disease associated with it is now referred to as covid- [ ] . on march , world health organization stated that the outbreak might be uncontrolled and made the assessment that covid- can be characterized as a pandemic. as of june , , , cases of covid- were reported worldwide in more than countries and regions, with , total confirmed deaths. considering the ratio of individuals tested positive to the covid- over population, italy is the fourth worst-affected european country in the pandemic, with , cases and , deaths [ ] . however, these data are constantly updated on international data resources. from the available evidence, covid- infection causes a mild disease (i.e., no pneumonia or mild pneumonia) in about % of cases and in most cases it resolves. about % have a more serious illness and % have a critical illness [ ] . the vast majority of the most serious diseases and deaths have occurred among the elderly individuals and those with pre-existing chronic conditions. patients with inflammatory bowel disease (ibd) are at increased infection risk, especially when being treated with steroids, immune-suppressants or biologics. the nature and magnitude of this risk vary with the type of immunosuppressive drug and with the patient's sex and age [ ] . the overall available evidence suggests that patients with ibd do not have an increased risk of developing covid- and should stay on ibd medications. patients receiving immune-suppressors should be carefully monitored for the occurrence of symptoms and/or signs suggesting covid- [ ] . spreading of the outbreak from china to countries with a higher prevalence of ibd has generated concern among gastroenterologists and patients, however, a recent systematic review has shown that patients with ibd do not appear to be more susceptible to sars-cov- infection and there is no evidence of an association between ibd therapies and increased risk of covid- . ibd medication adherence should be encouraged to prevent disease flare but, wherever possible, high dose systemic corticosteroids should be avoided [ ] . at the time of writing, there are patients with covid- and ibd worldwide. the countries with the most reported cases are us, uk, and spain. in italy about cases have been reported showing active ibd, old age and comorbidities were associated with a negative covid- outcome, whereas ibd treatments were not [ ] . only a recent survey online on european federation of crohn's & ulcerative colitis associations (efcca) [ ] yet not published and conducted on patients, reported concern and fear of patients about management of ibd and normal daily activities, during pandemic. anxiety level in patients affected by chronic immune-mediated inflammatory diseases (imids) has been widely quantified through several patients reported outcomes showing higher level in comparison with general population [ ] . actually, various studies evaluated the anxiety level in general population during pandemic. a cross-sectional survey on the psychological impact of the covid- on patients with ibd has shown that about half ( %) expressed symptoms of anxiety [ ] . the aim of this study is to perform a survey about knowledge about clinical importance of covid- , knowledge of disease management, prevention measures, and anxiety level during the pandemic among patients with ibd. from march to april , a questionnaire survey was administered to patients with ibd by email or phone application. patients were affected by crohn's disease (cd) and ulcerative colitis (uc) and were followed by gastroenterology and digestive physiopathology unit of "mater domini" university hospital. diagnosis of cd or uc was established according to standard clinical, endoscopic, histological, and radiological criteria [ , ] the questionnaire was defined and organized into five sections, dealing with: ( ) demographic and clinical characteristic, ( ) knowledge about clinical importance of covid- , ( ) knowledge about management of ibd, ( ) knowledge of prevention measures, ( ) anxiety level during pandemic, respectively. first section was composed by anthropometric, demographic, and clinical data of patients, including age, gender, educational level defined as having a high school diploma, marital status, and parental status, number of cohabitants, employment, type of occupation and workplace, use of smartphone or personal computer (pc) as source of covid- information, reception of educational material by gastroenterologist to inform patients about management of ibd during covid- emergency and, finally, any presence of a family member with covid- . clinical data included disease type, duration and prescribed treatment. disease activity was measured by harvey bradshaw index for cd [ ] and partial mayo score for uc [ ] . furthermore, disease activity was dichotomized in "low" with a total score below for partial mayo score and below for harvey bradshaw index, "high" with a score above for partial mayo score and above for harvey bradshaw index. finally, eventual diagnoses of psychiatric diseases and other chronic diseases were recorded. in the second section, as shown in previous studies [ ] , questions were used to collect information regarding knowledge about clinical importance of covid- . each answer was graded as (incorrect answers) and (correct answers). maximum score was and minimum . the score was dichotomized in "low degree" when ≤ and "high degree" when > . in the third section, questions were used to collect information regarding patients' knowledge about management of ibd during pandemic. each answer was graded as (incorrect answers) and (correct answers). maximum score was and minimum . the score was dichotomized in "low degree" of knowledge when ≤ and "high degree" of knowledge when > . in the fourth section, as shown in previous studies [ ] , questions were used to collect information regarding patients' knowledge of prevention measures during pandemic. each answer was graded as (incorrect answers) and (correct answers). maximum score was and minimum . the score was dichotomized in "low degree" of knowledge when ≤ and "high degree" of knowledge when > . fifth section evaluated anxiety level during covid- emergency by state-trait anxiety inventory (stai) [ ] : a self-completed questionnaire of items which aims to assess separately the state of anxiety (defined as temporary and influenced by the contingent situation in which the respondent notes how he feels right now) and the anxiety trait (propensity to be anxious where the respondent see how it feels "generally") with items each. the total score ranges from - ; score less than was classified as no anxiety, - mild anxiety, - moderate anxiety and - severe anxiety. furthermore, anxiety levels were dichotomized in "low anxiety" with score less than and "moderate-high" with score higher than . data were described using means and standard deviation, numbers, and proportions as appropriate. univariate analysis was performed by chi-square test and independent sample t test to analyze factors influencing outcomes of interest. a multivariate analysis was performed by logistic regression models adjusting for type of ibd, gender, age, educational level, marital status, parental status, job status, use of smartphone/pc, educational material received by gastroenterologist, disease duration biological therapy active disease, anxiety. the odds ratio (or) was used as a measure of association between the results of the questionnaires and the presence of a certain variable. all statistical analyses were performed using the statistical package for social sciences (version . ; spss, inc., chicago, il, usa). p values < . were considered as statistically significant. this study was approved by the local ethics committee of magna graecia university on march (protocol number / ). this study was conducted in compliance with the principles outlined in the declaration of helsinki. informed written consent was sent together with the questionnaire and obtained from each participating patient. a total of questionnaire were sent. out of these, were completed, with a response rate of %. detailed anthropometric, demographic and clinical characteristics of the patients are shown in table . one hundred fourteen patients ( %) have shown high degree knowledge about clinical importance of covid- , while an older age has been found independently associated with it (or . , % ci: confidence interval . - . , p = . ) ( table ) . most patients ( . %) reported that covid- is a viral infection, originated from asia ( %), with human-to-human transmission mainly via droplets ( %) and an incubation period between and days ( %). moreover, almost all ( %) the patients with ibd correctly answered about clinical manifestations of covid- , indicating fever, cough, and dyspnea as the most common symptoms while reporting ( %) that oro-nasopharyngeal swab was used for diagnosis of covid- . participants were also informed about the lack of effective therapy ( %) or available vaccine ( %), and they correctly reported also that elderly people were at increased risk of infection ( %). patients with high knowledge about clinical importance of covid- reported high educational level more often than patients with low knowledge about clinical importance of covid- ( % vs. %, p = . , respectively). this was consistent with the highlight that high education level was independently associated with high knowledge about clinical importance of covid- (or , % ci . - . , p = . ) ( table ). the receipt of informative material by gastroenterologist was more frequent in patients with low knowledge about clinical importance of covid- compared to those with high knowledge about clinical importance of covid- ( % vs. %, p = . , respectively) (table ) . accordingly, receiving educational material on covid- by gastroenterologist was independently associated with low knowledge about clinical importance of covid- (or , % ci . - . , p = . ) ( table ). furthermore moderate-high level of anxiety was independently associated with low level of knowledge (or , % ci . - . , p = . ) ( table ). most participants ( %) reported a high knowledge concerning the management of ibd. just over half ( %) declared that their intestinal disease could increase the risk of infection with sars-cov- and much more ( %) that it could worsen the outcome of covid- infection. although almost all patients ( %) considered appropriate to continue their current medical therapy, fewer ( %) believed that the treatment does not increase the risk of infection with sars-cov- . most of the patients ( %) agreed on the remodulation of hospital activity during covid- pandemic, with suspension of routinely visits and almost all ( %) considered appropriate going to the hospital only in case of clinical relapse. an active disease status was independently associated with a low knowledge of ibd management during pandemic (or . , % ci . - . , p = . ) ( table ) . nevertheless, the remaining variables were not associated with the level of knowledge of ibd management during pandemic. almost all patients with ibd ( %) presented a good knowledge of prevention measures such as utility of frequent hand washing ( %), use of disinfectant ( %), use of masks ( %), and importance of social distancing ( %). eighty-eight ( %) participants reported a moderate-high level of anxiety. older age was independently associated with moderate-high level of anxiety (or . , % ci . - . , p = . ). more patients with a low level of anxiety were employed ( % vs. %, p = . ) and used smartphone or pc as a source of covid- information ( % vs. %, p = . ). however, these associations disappeared when adjusted for the other variables. at the same time, no other variable was associated with the level of anxiety (table ). in the current context of covid- pandemic, ibd units have been forced to dramatically change and restructure management of patients with ibd [ ] . this study aimed to investigate the level of knowledge about clinical importance of covid- and perceptions among patients suffering from ibd. as expected, a high knowledge about clinical importance of covid- was found to be associated with high educational level. consistently, a previous study [ ] among chinese residents demonstrated a strong association between degree of information about covid- and higher educational level. nevertheless, this is the first study dealing with the knowledge about clinical importance of covid- in a cohort of patients with ibd. from the beginning of the pandemic in italy, several ibd units attempted to improve remote contact with patients providing them with educational material containing relevant information about covid- and ibd [ ] . surprisingly, in this study, patients who had received this type of information displayed a lower knowledge about clinical importance of covid- compared to those who had not, and this was confirmed to be an independent association. a possible explanation could be that the choice of a digital format was not the preferred way in our cohort of patients. likewise, this suggest that doctor-patient interpersonal interaction is an unquestionably important feature in the health system in order to improve knowledge, health-related behavior change, and self-management of patients with ibd. the finding that a higher level of anxiety was independently associated with a lower level of knowledge about clinical importance of covid- was not surprising. that is, anxiety significantly impairs patient performance in getting the right available information in the field. as a consequence, gastroenterologists should take into account the high level of anxiety in the context of covid- pandemic to further support the related needs of the patient with ibd. furthermore, it was not surprising that increasing age was independently associated with high level of knowledge about clinical importance of covid- . indeed, older patients are more prone than younger to pay attention on health-related issues which normally increase with aging. at date, several ongoing studies evaluate management of ibd patients during pandemic reporting as cd and uc does not increase risk of infection [ , ] and of severe forms of covid- [ , ] . moreover, the actual risk of infection or of development of covid- due to medical therapy is unknown [ ] . long-term data are needed to define the best strategy, considering that stopping therapy exposes patients to a greater risk of disease recurrence and therefore this decision should be individualized [ , ] . all european hospitals intensively reduced routine activities to prepare for high numbers of admissions of patients with covid- and there was a readjustment of care and resetting of clinical priorities [ ] . however, patients with ibd should be reminded that this interruption is temporary and alternative solutions have been found, including remote monitoring, drug home delivery, and limitations for infusion units [ ] . as far as we know, no validated instruments have been developed in order to measure patients' knowledge of ibd management during covid- pandemic. therefore, we built a questionnaire according to the available published data [ , ] . the high rate ( %) of patient who declared to have high level of knowledge of ibd management during pandemic is probably due to the widespread information by the medical system and ibd units as well as the several media channels and forum they certainly attended in that period. nevertheless, the sole variable that showed to be independently associated with a low knowledge of ibd management resulted to be the activity status of the disease. this is probably due to the fact that the deeper patients are involved in their disease (i.e., active disease), the lower is their knowledge about how properly manage it. once again, consistently with previous study [ ] , this finding suggests that gastroenterologists have to pay more attention at patients with ibd in active disease status, further addressing the knowledge of how to manage ibd during pandemic in this setting of patients. several studies have been published about psychological impact of the outbreak in the general population and demonstrated an increase of anxiety level during pandemic. furthermore, many patients seeking medical consultation for gastrointestinal problems show an associated affective disorder [ ] . patients with ibd are - times more likely to develop anxiety disorders [ ] [ ] [ ] . according with recent study [ ] we confirmed that ibd patients have a high level of anxiety during covid- pandemic. however, no variable other than an increasing age has been shown to be independently associated with this findings. it is not surprising that mood disorders are more frequent in older than younger patients. even thought to be employed and the use of smartphone/pc have been shown to be associated with lower level of anxiety, they both still not maintained statistical significance when adjusted for the other variables. limitation of the study was constituted mainly by the relatively small sample size, because of the fact that data collection was performed exclusively through email as routinely visits were suspended. the non-prospective evaluation of patients did not allow us to highlight the impact of the pandemic on disease management, but this was beyond the scope of this survey. in conclusion, this study shows that in patients with ibd a high educational level promotes knowledge about clinical importance of covid- while an active disease status lowers the knowledge of ibd management. taken together with the high level of anxiety and its relationship with increasing age, this suggests that gastroenterologists have to further support the needs of patients with ibd with targeted strategies during covid- pandemic. epidemiology of coronavirus disease outbreak: the italian trends inflammatory bowel disease management during the covid- outbreak: a survey from the european crohn's and colitis organization (ecco) second european evidence-based consensus on the prevention, diagnosis and management of opportunistic infections in inflammatory bowel disease are patients with inflammatory bowel disease at 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during the rapid rise period of the covid- outbreak: a quick online cross-sectional survey views of patients with inflammatory bowel disease on the covid- pandemic: a global survey support for the reliability and validity of a six-item state anxiety scale derived from the state-trait anxiety inventory affiliations inflammatory bowel disease care in the covid- pandemic era: the humanitas, milan experience. j. crohn's colitis maintaining the quality standards of care for inflammatory bowel disease patients during the covid- pandemic protection of inflammatory bowel disease patients from the outbreak and rapid spread of covid- infection in wuhan uneventful course in ibd patients during sars-cov- outbreak in northern italy ioibd update on covid for patients with crohn's disease and ulcerative colitis withdrawal of immunosuppressant or biologic therapy for patients with quiescent crohn's disease dop ecco expert consensus and topical review on treatment exit strategies in inflammatory bowel disease. j. crohn's colitis management of ibd during the covid- outbreak: resetting clinical priorities inflammatory bowel disease in the covid- pandemic-the patients' perspective. j. crohn's colitis state and trait anxiety and depression in patients affected by gastrointestinal diseases: psychometric evaluation of patients referred to an internal medicine outpatient setting gastrointestinal symptoms of and psychosocial changes in inflammatory bowel disease: a nursing-led cross-sectional study of patients in clinical remission depression and anxiety in people with inflammatory bowel disease the prevalence and risk factors of undiagnosed depression and anxiety disorders among patients with inflammatory bowel disease this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - o nx z authors: bernstein, charles n; ng, siew c; banerjee, rupa; steinwurz, flavio; shen, bo; carbonnel, franck; hamid, saeed; sood, ajit; yamamoto-furusho, jesus k; griffiths, anne; benchimol, eric i; travis, simon; lopes, susana; rubin, david t; kaplan, gilaad g; armstrong, david; gearry, richard title: worldwide management of inflammatory bowel disease during the covid- pandemic: an international survey date: - - journal: inflamm bowel dis doi: . /ibd/izaa sha: doc_id: cord_uid: o nx z background and aims: persons with inflammatory bowel disease (ibd) may be particularly vulnerable to covid- either because of their underlying disease or its management. guidance has been presented on the management of persons with ibd in the time of this pandemic by different groups. we aimed to determine how gastroenterologists around the world were approaching the management of ibd. methods: members of the world gastroenterology organization (wgo) ibd task force contacted colleagues in countries largely beyond north america and europe, inviting them to review the wgo website for ibd and covid- introduction, with links to guideline documents, and then to respond to ancillary open-ended management questions. results: fifty-two gastroenterologists from countries across continents completed the survey (april to may , ). they were all adhering for the most part to published guidelines on ibd management in the covid- era. some differences and reductions in services related to access, and some related to approach within their communities in terms of limiting virus spread. in particular, most gastroenterologists reduced in-person clinics ( of ), limited steroid use ( of ), limited elective endoscopy ( of ), and limited elective surgeries ( of ). if a patient was diagnosed with covid- , immunomodulatory therapy was mostly held. conclusions: in most countries, the covid- pandemic significantly altered the approach to persons with ibd. the few exceptions were mostly based on low burden of covid- in individual communities. regardless of resources or health care systems, gastroenterologists around the world took a similar approach to the management of ibd. inflammatory bowel disease (ibd) is a global disease with high prevalence in the western world and rapidly rising incidence in newly industrialized countries in asia, africa, and latin america. [ ] [ ] [ ] the management of ibd may differ by geography with economically more advanced countries having greater access to health care resources such as endoscopy and expensive therapies like biologics. geographic differences and inequities in access to ibd management may be heightened during the coronavirus disease (covid- ) pandemic, as lockdown measures have placed significant strain on government and personal finances. there has been uncertainty to what extent persons with ibd may be at increased risk of being infected with the severe acute respiratory syndrome coronavirus (sars-cov- ), whether their outcomes from covid- would be different from the general population, and whether immunomodulatory therapies (including both immunosuppressive and biological therapies) used in the treatment of ibd pose a significant increased risk for adverse outcomes from covid- . despite the uncertainty, there has been some agreement reached among international ibd specialists, including the international organization for the study of ibd (ioibd), the american gastroenterological association (aga), the british society for gastroenterology (bsg), and the european crohn's and colitis organisation (ecco). recommendations include continuing all immunomodulatory medications in patients with ibd, reducing steroid doses as much as possible, limiting elective endoscopies, and encouraging telemedicine in place of in-person clinic visits. although the most severe aspect of covid- infections are respiratory symptoms, gastrointestinal (gi) symptoms are common. a meta-analysis suggested that gastrointestinal symptoms may be seen in %- % of persons presenting with covid- . moreover, a report from hubei province, china (wuhan is the capital city), the initial covid- epicenter, suggested that nearly % of affected persons had gastrointestinal symptoms. in large american series, gastrointestinal symptoms were seen in % of persons with covid- in california and in % of affected persons with covid- from new york city. in this new york city study, having ibd or being on immunosuppressive therapy were not associated with presenting with gastrointestinal symptoms. however, the possibility that gastrointestinal symptoms are part of a covid- infection poses a particular challenge in managing persons with ibd. for persons with ibd presenting with increasing gastrointestinal symptoms, the challenge for the clinician is whether these symptoms reflect a flare of ibd or covid- . the world gastroenterology organization (wgo) has a mandate to deliver education and information to clinicians worldwide. an introduction to management challenges for persons with ibd was presented on the wgo website with links to some of the reports described previously. we aimed to develop an understanding of international management approaches to persons with ibd during the pandemic, with an emphasis on countries that were less likely to be represented in guidelines put forth by the ioibd, aga, bsg, or ecco. in particular, we wanted to determine how management of ibd was being handled in countries with fewer resources and to what extent management differed from north america and europe. members of the wgo who have worked on global ibdrelated practice guidelines (wgo ibd task force) reached out to colleagues across the world to review current recommendations regarding management of persons with ibd and to answer ancillary questions about their practices in their communities during the covid- pandemic. most of these colleagues had leadership roles in the management of ibd in their respective communities. it was not the intention to get as many gastroenterologists as possible to respond but rather to get as many countries represented as possible, especially from countries outside of canada, the united states, and europe. gastroenterologists mostly from north america and europe participated in the position papers from the aga, bsg, ecco, and ioibd. [ ] [ ] [ ] [ ] hence, it was the goal to reach out to gastroenterologists who would be practicing in countries less likely to be represented in these guidelines. some gastroenterologists from north america and europe were included. nine questions were sent electronically and allowed for open-ended responses. the questions posed to participants were responses were received between april , , and may , . all responses were collated by one author (cnb), and responses were inputted into tables. where there were unique responses underscoring an important management issue, this was highlighted in the report. fifty-two gastroenterologists from countries across continents completed the survey (due to potential resource allocation differences, hong kong was considered separate from china, and puerto rico was considered separate from the united states; fig. ). three respondents were pediatric gastroenterologists. the results are summarized after each question and also in the tables (tables - ). table also presents whether the country is a high income country or not, as per the world bank. )what is the effect of resource availability on recommendations (table ) ? countries across the world were mostly able to abide by the recommendations, but respondents identified several challenges. first, the availability of personal protective equipment (ppe) was limited. limited supply of ppe was reported from bangladesh, china, mexico, puerto rico, and thailand. further, a discrepancy between private and public hospitals for availability of ppe was reported in brazil. second, travel restrictions and home isolation affected availability of resources. for example in china, travel restrictions affected access to medications that was rectified by increased use of e-commerce no effect yes a)no india (n = ) center had no effect center had limit on testing to facilitate drug delivery. in south africa, there was concern that patients' fears of infection reduced their willingness to pick up prescribed medications, leading to patients to use their own personal supplies of prednisone for treatment of flares. from puerto rico, there was a comment on limited ability to work from home and limited numbers of employers allowing for proper distancing. finally, a respondent from india highlighted the problem of limited access to testing. ) are you still having in-person clinics ( table ) ? the vast majority ( of ) reported a marked reduction in in-person clinics, restricting clinics to persons who were at least moderately ill. practices were effectively closed to in-person outpatient visits in nepal, new zealand, philippines, puerto rico, the uk, in practices from canada (a pediatric practice), in of clinics in china, in one of in brazil, in of in portugal, and of in the us surveys. practices from indonesia, korea, myanmar, taiwan, and vietnam and of from china and of from brazil were open to seeing outpatients without reduction. ) are you conducting any elective endoscopy in ibd patients, and if so, for which indications have you been undertaking endoscopy ( bangladesh (n = ) no cases yet (n = ) limited surgery availability (n = ) urgent surgeries only (n = ) use more thiopurines and if using steroids patient is in isolation (n = ) taper rapidly, reduce dose (n = ) korea (n = ) d/c immunomodulatory therapy d/c steroids there was consensus to stop or rapidly taper steroids. the slim majority of respondents ( of ) would stop or reduce immunomodulatory, therapy but responses varied, including some who reported they had yet to experience such cases and did not present a plan in case that occurred. one american suggested continuing ustekinumab and vedolizumab, a respondent from mexico would continue vedolizumab and budesonide, and a respondent from taiwan discontinued steroids but maintained other immunomodulatory drugs. respondents from saudi arabia, singapore, and india suggested the approach to the immunomodulatory drugs would depend on the severity of the covid- infection and the severity of the ibd. one respondent from malaysia reported continuing usual maintenance therapy, and respondent from malaysia reported stopping immunomodulatory therapy. when specified, -asa was consistently continued. a respondent from kuwait reported switching intravenous biological therapy to subcutaneous. a respondent from pakistan reported that the fear and stigma of testing positive for sars-cov- infection inhibited patients in his jurisdiction from accessing medical care. several respondents stated that they had yet to encounter ibd cases with covid- , so they did not state a specific plan of management. ) what is the role of surgery in the face of covid- ? does covid- affect the indications for surgery or the scope of surgical interventions (table ) ? broadly, only urgent surgeries were undertaken. however, in indonesia, kenya, taiwan, and israeli center (a pediatric center), approaches to surgery did not change, suggesting that elective surgeries in ibd continued. in center each from china, indian, pakistan, and vietnam, surgery was undertaken after a negative covid- test. it was not clear whether emergency surgery would have been undertaken in these centers in a patient with a positive test, especially if asymptomatic. ) if in a low resource area and there is no access to biologicals, how does one address steroid use in the current environment of covid- concern (table )? some respondents declared avoidance of steroids, but the majority declared selective steroid use and a more rapid taper than usual. the use of steroids in ulcerative colitis (uc) was singled out as more likely. vietnam, portugal, of the pakistani sites, of chinese sites, and both israeli sites would country how should one investigate and manage someone presenting with potentially new onset ibd? how is this affected by resource availability? considering that at least % of persons with covid- present with isolated gi symptoms when a patient with ibd presents with worsening gi symptoms are you routinely testing for covid- ? what is the role of noninvasive markers in assessing disease if endoscopy is not available? for example, esr, crp, fecal calprotectin, wbc / platelet count, radiology? how does resource availability affect this testing?* romania (n = ) no change no all testing done saudi arabia (n = ) no change except for covid test first yes all testing done singapore (n = ) no change no, but yes before biological started all testing done south africa (n = ) avoid endoscopy, steroids and thiopurines endoscopy being done (n = ). covid- testing first (n = ) empiric asa(n = ) no (n = ) yes (n = ) all testing done except limited imaging vietnam (n = ) no change except for covid test first no all testing done abbreviations: n refers to number of respondents from that country. "?" refers to unclear response on that question. colonoscopy was still being performed for diagnosis of ibd in of us centers, of indian centers, of israeli centers, of malaysian centers, of pakistani centers, and centers in canada, indonesia, kenya, korea, portugal, romania, singapore, and taiwan. in one of the portuguese centers, there was a preference for imaging and noninvasive testing such as fecal calprotectin. in the philippines, endoscopy was undertaken in more active cases. in center each in china, hong kong, korea, saudi arabia, taiwan, and vietnam, endoscopy was only undertaken after negative covid- viral testing. some centers were avoiding endoscopy even in this scenario of new onset disease, including centers in brazil, south africa, thailand, the uk, and of centers from china, of centers from india, and of centers from malaysia. in cases of likely colitis, responses from of us centers, of pakistani centers, and the uk reported use of empiric treatment, particularly with -asa. canadian respondents reported avoiding intravenous biologicals in favor of subcutaneous dosing when biologicals were being initiated to avoid attendance at in-person infusion clinics. respondents from taiwan, uruguay, vietnam, and a pediatric center in israel reported no change in their approach to ibd management. ) considering that at least % of persons with covid- present with isolated gi symptoms when a patient with ibd presents with worsening gi symptoms are you routinely testing for covid- (table ) ? clinicians from countries did not test for covid- on flare of gastrointestinal symptoms. this included both high resource countries like canada, korea, and taiwan and lower resource countries like vietnam. in korea and taiwan, this was a response to the relatively low community prevalence of covid- in their countries. in canada, though, clinicians did not have open access to testing, so it was limited to persons with respiratory symptoms. respondents from countries, both low and high resource regions, reported that they routinely tested persons with ibd with new gastrointestinal symptoms. in brazil, india, malaysia, pakistan, portugal, and the united states, there were those who reported testing and those who did not. hence, it was not uniform in all countries. indications for testing have been rapidly changing, so these approaches may have even been different for those who responded at the beginning of the study period compared with the end of the period of survey collection. ) what is the role of noninvasive markers in assessing disease if endoscopy is not available? for example, esr, crp, fecal calprotectin, wbc, platelet count, radiology? how does resource availability affect this (table )? blood testing, fecal calprotectin, and imaging were widely performed. however, imaging access was limited in of brazilian centers, of canadian centers, both american centers, puerto rico, and thailand. in some canadian regions, access and funding for fecal calprotectin was limited. in qatar, patients tended to avoid clinics, and therefore, it was difficult to investigate them. this was also reported in response to question by a south african respondent and in question by an israeli respondent. table shows the number of covid- cases, the number of covid- cases per million people, and the number of covid- tests undertaken as of may , . the management of persons with ibd across the world in the setting of the covid- pandemic shares many similarities. gastroenterologists were heeding the recommendations of established gastroenterology bodies with adaptations based on local resources. of note, gastroenterologists from some high resource countries, such as canada, and from lower resource countries both reported impediments in access to ppe. some clinicians reported unique issues limiting patient access to clinics or completing diagnostic testing, such as traffic restrictions in china or patient fear of attending clinics in israel, qatar, and south africa. some gastroenterologists reported no change in their approaches to ibd patients including no reduction in endoscopy services. this was mostly reported in countries with low covid- patient burdens and was true of both higher and lower resource countries. most countries had reduced outpatient clinics, reduced outpatient endoscopy and reduced surgery. several countries retained outpatient endoscopy for persons with ibd as part of management decision-making. nearly all who responded as to which endoscopies would be allowed reported undertaking endoscopy for urgent ibd scenarios such as active bleeding, severe colitis to assess for infection, certain bowel obstructions, and cancers. many centers were undertaking endoscopy for new onset ibd to assist with diagnosis. there was near uniform agreement on reducing or avoiding steroid use. most clinicians reduced or stopped oral immunomodulatory therapy or steroids. however, multiple clinicians from romania, singapore, taiwan, and uruguay and from malaysia did not change their management approaches. in the surveillance epidemiology of coronavirus under research exclusion (secure)-ibd database, steroid use was associated with worse outcomes in ibd patients who were affected by covid- . others reported not having dealt with a case of covid- in an ibd patient and hence did not respond as to how they would manage that dilemma. the use covid- testing as part of management approach for persons with ibd was variable. in taiwan and american center, covid- testing was undertaken before further ibd-related diagnostics; a respondent from vietnam would test for covid- if changing drug treatment; in bangladesh, they would only reduce steroids if the patient was covid- positive; and in center each from china, pakistan, and vietnam, they would undertake covid- testing before surgery. of note, when ibd patients had new or increased gastrointestinal symptoms, a minority reported testing for covid- . the survey was not structured to determine if this was because the jurisdictions within which they practiced had limited access to covid- testing or if the clinicians simply thought this was unnecessary. all centers had access to standard blood testing, most had access to fecal calprotectin (including some centers accessing a home version, which facilitated patient isolation practices), and most had usual access to imaging. some centers ( in canada, in the united states, in bangladesh, and in thailand) had limited access to cross-sectional imaging. in the case of canada and the united states, this was deliberate on the part of the health system to reduce patient contact with hospital staff for nonurgent testing. table shows the distribution of cases of covid- and of tests for covid- per million inhabitants. one can see that there were several countries with fewer than per million cases; however, there was a marked discrepancy in testing frequency. testing ranged from out of million inhabitants (myanmar) to , out of million inhabitants (new zealand). undoubtedly, the experience of respondents was impacted by the burden of disease in their communities. several had yet to experience cases of ibd in which patients were infected with covid- . it will be important to repeat this survey again to determine if views changed as the disease burden changes. this study had some notable limitations. clinicians within every country may have different practice approaches so that a survey response from or clinicians does not necessarily reflect what may be done by most clinicians in that country. it is unknown to what extent the responses reflect the majority of gastroenterologists managing ibd patients in each jurisdiction, especially because the respondents were leaders in ibd management in their respective countries. however, even with reports from or clinicians from each country, it allows insight into the uniformity or key differences in the practice of ibd around the world during the covid- pandemic. though some respondents reported limitations to ppe access or covid- testing, not reporting such limitations does not mean that they did not exist in those jurisdictions. in several countries, there are dual private and publicly funded health care systems. respondents were not asked to distinguish their answers for each of the systems, although some respondents did report those differences. in summary, regardless of resources and health care system, gastroenterologists around the world were generally taking a similar approach to the management of ibd. for the most part, they were adhering to published guidelines on ibd management in the covid- era. there were some notable differences in care approach due to reduced service availability, resource availability, and community prevalence of covid- . countries that had or more respondents showed how responses could vary within a country, and so these results should not be taken to reflect a consensus of an entire country's approach to managing ibd. increasing incidence of inflammatory bowel disease with time and among regions, based on systematic review the worldwide incidence and prevalence of inflammatory bowel disease in the st century: a systematic review of populationbased studies progression of inflammatory bowel diseases throughout latin america and the caribbean: a systematic review changing global epidemiology of inflammatory bowel diseases: sustaining health care delivery into the st century ioibd update on covid for patients with crohn's disease and ulcerative colitis | ioibd aga clinical practice update on management of inflammatory bowel disease during the covid- pandemic: expert commentary bsg expanded consensus advice for the management of ibd during the covid- pandemic inflammatory bowel disease management during the covid- outbreak: a survey from the european crohn's and colitis organization (ecco) aga institute rapid review of the gi and liver manifestations of covid- , meta analysis of international data, and recommendations for the consultative management of patients with covid- don't overlook digestive symptoms in patients with novel coronavirus disease (covid- ) high prevalence of concurrent gastrointestinal manifestations in patients with sars-cov- : early experience from california gastrointestinal and hepatic manifestations of novel coronavirus disease in a large cohort of infected patients from new york: clinical implications management of patients with inflammatory bowel disease worldometer covid- coronavirus pandemic world bank country and lending groups but not tnf antagonists, are associated with adverse covid- outcomes in patients with inflammatory bowel diseases: results from an international registry key: cord- -dnsdg n authors: nan title: poster sessions date: - - journal: eur j immunol doi: . /eji. sha: doc_id: cord_uid: dnsdg n no abtract the humoral pattern recognition receptors of innate immunity include collectins, ficolins and pentraxins. ptx , the prototype of long pentraxins, plays a nonredundant role in resistance against a. fumigatus lung infection. the model proposed suggests that upon binding, ptx facilitates recognition, phagocitosis and killing of a. fumigatus conidia by alveolar macrophages, dendritic cells and neutrophils and the subsequent development of a properly th -oriented adaptive response. actually, ptx -deficient mice are highly susceptible to aspergillosis and develop th skewed responses; moreover, ptx -deficient resident macrophages and neutrophils show defective conidia phagocytosis. both in vitro and in vivo defects can be rescued by the administration of recombinant ptx , which does not show direct activity on fungal cells. finally, ptx alone or in combination with antifungal agents, induces a curative response in mice with aspergillosis, even when given prophylactically. in the present study, we investigated the mechanisms underlying the ptx -mediated opsonic activity and the involvement of complement, complement receptors and fcg receptors, by in vitro studies and genetic approaches in vivo. in vitro ptx amplified the complement-dependent effects on a. fumigatus conidia phagocytosis by human neutrophils, activated through the alternative pathway. accordingly, in the presence of ptx -opsonised conidia, cd b activation, internalization, recruitment to the phagocytic cup and cd b-dependent phagocytosis were increased. as pentraxins interact with fcgreceptors, which in turn can control cd b activation, the phagocytic assay was performed in the presence of fcgr blocking abs. data obtained strongly suggest that upon conidia opsonisation with ptx , fcgriia/cd mediates inside-out activation of cd b and consequently phagocytosis of c b-opsonised conidia. in vivo phagocytosis experiments performed with c q-and fc common gamma chain-deficient mice and complement inhibitors support in vitro data. these data confirm and extend the paradigm of cooperation among innate receptors, in particular among the humoral arm of innate immunity (complement, ptx ) and the cellular arm (fcgrs, cr ). moreover, they confirm previous studies on the interaction between pentraxins and fcgrs and support the idea that pentraxins behave as predecessors of antibodies. innate immunity is the first line of defence against pathogens and plays a key role in the initiation, activation and orientation of adaptive immunity. the humoral arm of the innate immunity includes soluble pattern-recognition receptors (prrs) such as collectins, ficolins, complement components and pentraxins. the prototypic long pentraxin ptx is rapidly produced and released by diverse cell types in response to proinflammatory signals. ptx binds selected microorganisms such as aspergillus fumigatus and restores protective immunity against this pathogen in ptx -/-mice. neonates have an immature innate immune system and are more susceptible to bacterial infection than older children or adult. a beneficial effect of breast feeding on newborn health is highly demonstrated. this protective effect is mediated by nutrients, immunomodulatory mediators (ifn-g, tnfa, or tgf-b), innate immunity factors (soluble cd , immunoglobulins, lactoferrin), and leukocytes contained in milk that can penetrate the newborn circulation. we thus hypothesized that milk may contain ptx . we found high concentration of ptx in human colostrum ( . ± . ng/ml at day post-delivery) compare to the one found in human serum ( x ng/ml). the presence of ptx in human colostrum seems to be due to the secretion of ptx by human mammary gland since we report the production of ptx by these cells. this prr is also found in human milk cells (hmc), mainly in leukocytes, and penetrate into newborn tissus after suckling. furthermore, human colostrum upregulated the ptx production by adult and neonate immunocompetent cells and we demonstrate that neonate mice present a deficit in their ptx production after lps injection. collectively, these data demonstrate that newborn have three distinct ways of ptx supplying by breast feeding: (i) soluble ptx in colostrum (ii) hmc that can secrete ptx upon stimulation in the specific tissue, (iii) an increase of ptx production by immune cells in the presence of colostrum. thus, soluble or cell-derived ptx may participate to the beneficial role of breast feeding on the newborn health. a. m. baru , j. stephani , h. wagner , t. sparwasser twincore, institute for infection immunology, hannover, germany, technical university of munich, institute for medical microbiology, immunology and hygiene, munich, germany toll-like receptors (tlrs) represent the best characterized pattern recognition receptor family in mammalian species. the family currently comprise of receptors in humans (tlr - ) and in mice (tlr - , - ). as transmembrane receptors, tlrs are expressed on the cell surface (tlr , , , , , ( ) ( ) ( ) ( ) and at endosomal membranes (tlr , , and ) . toll-like receptors recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity. bacterial dna has been shown to possess immunomodulatory activity about a decade prior to the identification of cpg motifs. about years later to this, toll-like receptor (tlr ) was identified and shown to be the receptor for unmethylated cpg dna which is present mainly in non-vertebrate genome. studies have defined potential role of tlr as adjuvant enhancing protective immune responses against tumours and infectious diseases in murine models. although promising results are obtained from a few human clinical trials, overall efficacy and safety could not yet be translated entirely from murine studies to human trials. one explanation for these discrepancies could be the fact that expression of human-tlr (hutlr ) is restricted to b-cells and plasmacytoid dendritic cells (pdcs) whereas murine-tlr (mutlr ) is also expressed on conventional dendritic cells (cdcs). consequently, tlr ligands induce distinct cytokine profiles in mice and human thereby probably regulating immune responses in a different manner. by employing bacterial artificial chromosome (bac) technology, we generated transgenic mice with hutlr (henceforth called as hut mouse) integration in their genome under human epigenetic control. to avoid effects seen due to overlapping ligand specificities, we crossed this mouse onto mutlr knock-out background. we expect that hut -mutlr -/mice mimic the human specific expression pattern of tlr , i. e. exclusively in b-cells and pdcs, allowing us to investigate detailed in vivo functions of hutlr . by studying infection and tumour models as well as models for autoimmunity, allergy and transplantation we could then define appropriate and safe implications for employment of tlr ligands in human immunotherapy. the fractal analysis provides unique physical insights into the interactions between c q and the prp protein. if one may take the liberty to extend this to cellular surfaces, where presumably these reactions are taking place, then one has access to a possible avenue by which one may control these reactions in desired directions. if this is true, then surely, this is worth exploring further. any effort, no matter how small that assists in help providing better insights into these debilitating and neurodegenrative disorders such as alzheimers is defintely worth the effort. interleukin- is a heterodimeric cytokine consisting of the two subunits p and p . the main inducers of il- p are microbial components activating toll-like receptors with the magnitude of il- p induction depending on the specific tlr engaged. differential induction of il- p upon tlr stimulation correlated with striking differences in the kinetics of nfkb activation. cpg-dna strongly induces il- p due to its outstanding capacity (i) to induce nucleosomal remodelling in proximal il- p promoter region and (ii) to stimulate prolonged rela activity. here we were interested in further changes in chromatin structure of the il- p promoter upon tlr triggering. we did not observe a change in dna methylation, but using chormatin immunoprecipitation (chip) we were able to detect a strong increase in histone and acetylation in specific regions of the proximal promoter region. acetylation of h showed a specific distribution pattern and occured mainly in regulatory elements within the il- p promoter, whereas acetylation of h took place over all regions analyzed. tlr tolerance has been reported to be associated with specific chromatin alterations. methylation status of lysine residue on h turned out to be important for the inhibition of gene expression upon repeated stimulation. modifying the chromatin structure of gene promoter regions therefore seems to be a sensitive mechanism to modify cytokine expression to exogeneous stimuli in innate immune cells thereby allowing adaption of innate immune responses. a. d. koepruelue , w. ellmeier medical university of vienna, institute of immunology/division of immunobiology, vienna, austria macrophages are important in innate and acquired immunity. failures are associated with inflammatory and autoimmune diseases. understanding their stimulation is the basis for therapeutic targeting. members of the tec kinase family (bmx, btk, itk, rlk and tec), expressed in the haematopoietic system, constitute the second largest family of non-receptor tyrosine kinases. mutations in btk represent the source of human x-linked agammaglobulinemia (xla). a mutation in the murine btk gene accounts for a similar syndrome, x-linked immunodeficiency (xid). although the tec family members tec, btk and bmx are expressed in monocytes/macrophages, little is known about their function there. tec kinases become activated upon signaling via divers receptors including antigen receptors, receptor tyrosine kinases or tlrs. several studies in xla or xid macrophages and in monocyte/macrophage cell lines implicated roles for tec kinases in tlr signaling and as well as other macrophage effector functions like phagocytosis. inspired by these findings, we aim to determine the role of tec kinases in bone marrowderived macrophages (bmm), during macrophage activation and in other macrophage functions such as recruitment or phagocytosis. in a comprehensive functional analysis of tlr-mediated bmm activation from mice deficient for one or more of the tec family members in vitro, we reveal which of the kinases play a role in which tlr pathway. based on the results of this analysis, we set the goal to further study how tec kinases regulate the respective signaling cascades. our study will contribute insights into the role of tec kinases in this important cell population of the innate immune system. g. lunazzi , m. buxadé , j. minguillón , r. berga , j. aramburu , c. lópez-rodríguez universitat pompeu fabra, department of experimental and health sciences (dcexs), barcelona, spain nfat is a transcription factor that regulates the expression of cytokines such as tnfa and lymphotoxin b in response to osmotic stress. in addition, nfat participates in multiple processes not linked to the response to hypertonicity. in this regards, it has been recently reported that nfat is required as a novel host factor that supports hiv replication in macrophages. given the established connections between nfat , the expression of certain inflammatory cytokines, and its role in the response to specific pathogens in macrophages, we aimed at studying whether nfat could be activated by receptors for pathogens expressed in macrophages. the activation of toll-like receptors (tlrs) is central to innate immunity. upon stimulation of tlrs, cells of the immune system induce signalling pathways that lead to the activation of different transcription factors. as a result of that, cells such as macrophages and dendritic cells induce the expression of genes that participate in the response to pathogens such as those encoding proinflammatory cytokines, antimicrobial products, survival factors or mediators of cellular migration. we have analyzed whether nfat is expressed in primary macrophages through the activation of different toll-like receptors. likewise, we have explored whether the activity of nfat is induced during the response to tlrs. in addition, we have studied whether the specific inhibition of different signalling pathways positioned downstream of tlrs could interfere with the expression of nfat . our work indicates that nfat is a novel transcriptional regulator acting in response to the activation of tlrs. our work extends the knowledge about mechanisms that participate during the innate immune response to pathogens and offers a new regulatory pathway as a possible target to modulate this response. objectives: compelling evidence support a link between inflammation, cell survival, and cancer, with a central role played by nf-xb, a master switch of inflammation. recent studies implicate some tlrs in tumor development or regression, and immune escape. however, mechanisms leading to tumor growth or apoptosis induced by tlr stimulation are not fully understood. several studies strongly suggest that chronic inflammation in lungs induced by chronic bronchitis, chronic obstructive diseases, emphysema, asbestos or tobacco smoke, increases the risk of carcinogenesis. we hypothesized that some tlrs can contribute to lung inflammation and tumor development in vitro and in vivo. methods: tlr expression in lung cancer was assayed by immunohistochemistry or flow cytometry. nfxb activation was determined by western blot and nuclear translocation assay after tlr stimulation. clonogenicity of stimulated cells was analyzed by colony assay. transcriptomic analysis were performed by taqman lda technology. tumor growth in vivo was analyzed in nod/scid mice after subcutaneously engraftment of human lung tumor cell lines. we have observed that primary human lung tumors express tlr , tlr , tlr and tlr and that stimulation of these receptors in lung tumor cell lines by poly i:c, lps, loxoribine or poly u induces nfxb activation through atypical signaling pathway, with phosphorylation of ixba without its degradation and nuclear translocation of p and p nfxb subunits. interestingly, we observed that tlr stimulation induces apoptosis depending of the histological type of the tumor. on the contrary tlr , tlr and tlr stimulation induces cell survival and increases clonogenicity. this is correlated with an up-regulation of bcl- expression. moreover, despite a common atypical activation of nfxb, our transcriptomic analysis revealed major differences in gene modulation after triggering of tlr , tlr , tlr and tlr . finally, in vivo tlr stimulation of human lung tumor cells dramatically increases tumor size and metastasis. conclusions: altogether, these data emphasize that tlr , tlr or tlr triggering can directly favor tumor development whereas tlr signaling can induce tumor cell death. these data suggest that anticancer immunotherapy using tlr adjuvants should take into account the expression of these tlrs in lung tumor cells. objective: dasatinib (bms- ) is a small molecule src/abl tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukaemia and philadelphia chromosome-positive acute lymphoblastic leukaemia. members of the src family of kinases are involved in normal physiological processes, and play a significant role in the induction and regulation of innate and adaptive immunity. the purpose of this study was to evaluate the inhibitory action of dasatinib on toll like receptor (tlr) signalling, natural killer (nk) cell cytotoxicity as well as antigen-specific cd + and cd + t cell function. methods: to analyse tlr signalling in vitro murine bone marrow derived (bmd) macrophages were stimulated with the tlr ligand lipopolysaccaride (lps) in the presence of dasatinib and tumour necrosis factor a (tnf-a) in the culture medium was measured. the response to tlr stimulation was also tested in vivo, dasatinib-treated mice were challenged with lps and tnf-a in the serum was quantified. in addition, the clearance of the rma-s cells, a mhc class i deficient thymoma sensitive to nk cell lysis, was analysed in mice undergoing dasatinib treatment. to investigate the inhibitory effects of dasatinib on adaptive immune responses, transgenic cd + and cd + t cells specific for ovalbumin were utilised to measure antigen specific t cell proliferation. endogenenous cd + and cd + t cell responses were determined following immunisation of dasatinib-treated mice with a nonreplicating recombinant virus. results: we show that dasatinib impairs: . innate immune response; dasatinib treatment reduced the (a) production of tnf-a following tlr stimulation of bmd macrophages in vitro, (b) production of tnf-a in vivo in response to lps and (c) ability of nk cells to eliminate mhc class i deficient cells in vivo . . adaptive immune response; dasatinib treatment inhibited (a) proliferation of antigen-specific murine transgenic t cells, (b) endogenous antigen-specific helper t cell recall-responses and (c) t cell-mediated cytotoxic effector function. conclusions: these findings suggest that dasatinib has the potential to modulate the host immune response and highlights scope for off target applications, for example therapeutic immunosuppression in the context of autoimmune pathogenesis, or in combination with other interventions for the treatment of endotoxic shock. i. zanoni , r. oatuni , m. collini , m. caccia , p. castagnoli , g. chirico , f. granucci university of milano-bicocca, biotechnology and bioscience, milan, italy, university of milano-bicocca, physics, milan, italy, singapore immunology network (sign), biomedical sciences institutes, immunos, singapore, singapore the recognition of mamps by tlrs expressed on dendritic cells (dcs) plays an essential role for the regulation of the immune responses. by recruiting different combinations of adapter proteins, individual tlrs turn on signal transduction pathways leading to the activation of different transcription factors. interleukin- (il- ) is one of the molecules produced by dcs shortly after stimulation with different tlr agonists. based on this observation and by analogy with the events following t-cell receptor (tcr) engagement leading to il- production, we hypothesized that the stimulation of tlrs on dcs might lead to activation of the ca +/ calcineurin and nfat pathway. we found that dc stimulation with lps induces extracellular ca + influxes, leading to calcineurin-dependent nfat activation. the activation of this pathway was independent of tlr engagement, depending instead exclusively on cd . we also found that lps-induced nfat activation in dcs was necessary for the efficient synthesis of cyclooxygenase- (cox- ) that, by generating prostaglandins (pgs), such as pge , regulates different dc functions including migration and polarization of t cell responses. our findings reveal novel aspects of the molecular signaling triggered by lps in dcs and define a new role for cd . given the essential involvement of cd in many diseases, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via cd represents a major step towards the development of potential treatments with modes of action involving interference with cd functions. we have examined the interaction of cd , a -kda glycosyl-phosphatidylinositol (gpi)-anchored membrane protein, with the monocyte signalling receptor, cd . human monocytes were isolated from healthy adult donor's peripheral blood. this involved labelling molecules at saturation with different coloured fluorophores and determining their positions separately by dual wavelength imaging. the cells were labelled at saturation with anti-cd antibody coupled to biotin visualised by qd- -streptavidin and anti-cd antibody coupled to allophycocyanin. the images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. single particle fluorescence imaging (spfi) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. the images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional gaussian function providing the basis for determining the dynamic and associated behaviour of receptors on living cells. changes in the numbers of receptors, and in the proportion of receptors showing colocalisation, indicated that lps promotes the interaction of cd and cd , suggesting a new functional role of cd as a member of a multimeric lps receptor complex. l. lundvall , r.r. schumann charité -universitätsmedizin berlin campus mitte, institute for microbiology and hygiene, berlin, germany meningitis is a life-threatening disease mainly caused by bacteria and viruses. bacterial components such as lipopolysaccharide (lps), lipoproteins or peptidoglycan breakdown products (i. e. mdp, mesodap) stimulate pattern recognition receptors (prrs), such as toll-like receptors (tlrs) and the intracellular nod-like receptors (nlrs) for an inflammatory response. we hypothesize that a synergistic effect of tlr-induced nf-xb activation and nlr-mediated caspase- induction leads to an increased release of mature il- b during bacterial meningitis in brain-derived cells. a mouse meningitis model with s. pneumoniae (d ) was established for assessing il- b induction during this disease. the murine raw . cell line, the human astroglial u- mg and the murine microglial cell-line bv- were stimulated with the tlr ligand lps, the tlr ligand pam cys, the nod ligand mdp, or the nod ligand c -ie-dap, and, as control, atp alone or in combination. we assessed il- b by elisa and caspase- and pro-il- b expression by western blot. furthermore, primary mouse astrocytes isolated from the cortices of mouse puppies were used for stimulation followed by sirna suppression of elements of the il- b induction pathway. s. pneumoniae (d ) infected mice showed a significant increase in il- b release after hours. in vitro, an increase in il- b levels after costimulation with lps or pam cys, and mdp or c -ie-dap was observed in a dose-dependent manner. a synergistic enhancement of il- b by tlr-and nlr-ligands was observed in raw cells, bv- cells, u- cells and primary astrocytes. active caspase- (p ) was induced by mdp or c -ie-dap, corresponding with high il- b responses when lps or pam cys was added. sirna experiments show that a knock-down of nod leads to a diminished il- b release after lps-and mdp-stimulation. the precursor forms of il- b and caspase- seem to be constitutively expressed in astrocytes and microglia. a synergistic enhancement between tlrs and nlrs in il- b release in brain-derived cells was observed. so a two-step stimulation seems necessary for the release of high levels of mature il- b by astrocytes and microglia. bacteria containing both, tlr-and nlr-ligands thus have the potential to induce high levels of il- b which may contribute to disease pathology and may point to novel intervention strategies. j. rosenberg toll-like receptors (tlrs), nod-like receptors (nlrs), and rig-i-like (rlrs ) are more well-characterized in their identity and expression as signaling markers which effect the ealry innate immune response and elicit adaptive immunity , . in the case of tlrs most sutides to date have delineated tlr expression and function on antigen presenting cells like dendritic cellof this research. extension of the profiling and presence of tlrs on cell characterized as adaptive immune cells such as t cells is the subject of this line of research. using a cd and cd activation model system -tlr presence on cd + cells is found in mouse t cells, human t cells and jurkat cell lines. following cd /cd activation for hours we have identified a small but distinct populationof tlr + cells. further characterization indicates these cells to be cd +cd + cells. further characterization of the expression and functional acitvity of the tlr + t cells indicates co-expression of tlr with md- indicating a functional tlr receptor. in addition lps activiation did not lead to upregulation of tlr expression in t-cells. the data indicate that tcr activation leads to tlr expression. the expression appears to be associated with cd +cd + cells and refelecting an activated t cell phenotype which will be further characterized as perhaps related to tregs or other tcell subsets. s. m. lehmann , d. kaul , c. krüger , f. zipp , r. nitsch , s. lehnardt charité-universitätsmedizin berlin, cecilie-vogt-clinic for neurology, berlin, germany, charité-universitätsmedizin berlin, institute for cell biology and neurobiology, berlin, germany the innate immune system is the first line of defense against various pathogens and requires the expression of toll-like receptors (tlrs). in macrophages, tlr plays a crucial role in immune responses elicited by gu-rich ssrna (i. e. ssrna ) as well as synthetic antiviral chemicals, including imidazoquinoline components (i. e. imiquimod) and some guanine nucleotide analogs (i. e. loxoribine). these compounds were initially described to activate mouse tlr (and human tlr ) and are potent immune response modifiers leading to important antiviral and antitumor activities. microglia serve as the major innate immune cells in the central nervous system (cns). employing various techniques including pcr, in situ hybridization, and immunocytochemistry, we demonstrate that tlr is expressed in these cells. incubation of microglia with all three of the above mentioned tlr ligands leads to activation of these cells displaying an ameboid shape and releasing inflammatory cytokines such as tnf-a and il -b in a dose-and time-dependent fashion. analysis of wild type (wt) and tlr knock out (ko) microglia by real- because neutrophil apoptosis plays a key role in resolving inflammation, identification of proteins regulating neutrophil survival should provide new strategies to modulate inflammation. using a proteomic approach, coronin- was identified as a cytosolic protein cleaved during neutrophil apoptosis. coronin- is an actinbinding protein that can associate with phagosomes and nadph oxidase but its involvement in apoptosis was currently unknown. in coronin- -transfected plb cells, coronin- overexpression did not modify the kinetics of granulocyte differentiation as assessed by cd b labeling. concerning apoptosis, increased coronin- expression in dmf-differentiated plb significantly decreased gliotoxin-induced mitochondrial depolarization as compared with controls. likewise, coronin- significantly decreased trail-induced apoptosis with less mitochondrial depolarization, caspase- and caspase- activities, but not caspase- or bid truncation suggesting that coronin- interfered with mitochondria-related events. to validate the prosurvival role of coronin- in a pathophysiological condition involving neutrophil-dominated inflammation, neutrophils from cystic fibrosis (cf) patients were studied. circulating neutrophils from cf patients had more coronin- expression assessed by immunoblotting or proteomic analysis of cytosolic proteins. this was associated with a lower apoptosis rate than those from controls evidenced by delayed phosphatidylserine externalization and mitochondria depolarization. in addition, inflammatory neutrophils from cf patients lungs showed an intense coronin- immunolabeling. we concluded that coronin- could constitute a potential target in resolving inflammation. p.-n. hsu national taiwan university, graduate institute of immunology, taipei, taiwan, republic of china human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (tnf) ligand superfamily members and their receptors. many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. recent evidence indicates that the tnf-related apoptosis-inducing ligand (trail) of the tnf ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated t cells. we show in this work that trail can induce osteoclast formation from human monocytes and murine raw . macrophages. we demonstrated that both cell models differentiate into osteoclast-like cells in the presence of trail in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (trap)-positive multinucleated cells and bone resorption activity. the trail-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. however, trail-induced osteoclastogenesis is dependent on activation of nf-xb, erk, and p map kinase. the trail-induced osteoclastogenesis was significantly inhibited by treatment with traf- sirna and traf- decoy peptide, indicating this pathway is traf- dependent. thus, our data demonstrate that trail induces osteoclast differentiation via direct engagement with the trail death receptor through a signaling pathway distinct from apoptosis. our results indicate that in addition to triggering apoptosis, trail induces osteoclast differentiation. it provides a novel role for trail in regulating osteoclast differentiation and in osteoimmunology. microglia are considered to be the local antigen presenting cells (apcs) of the central nervous system (cns) which are thought to play a crucial role in local reactivation of autoreactive t cells during cns autoimmunity e. g. in multiple sclerosis (ms) and its animal model experimental autoimmune encephalomyelitis (eae) . in this study we investigated if the anti-inflammatory nuclear transcription factor peroxisome proliferator-activated receptor gamma (pparg) that has been described to negatively regulate macrophage activation has an influence on microglia immunogenicity. sustained activation of pparg both reduced microglial signalling via mhc molecules and costimulatory molecules and concomitantly increased signalling via the coinhibitory molecules b -h and b -dc. moreover, also production of pro-inflammatory cytokines like tnf-a and il- was profoundly reduced if microglia were pre-treated with the pparg-agonist pioglitazone (pio). in contrast to this, the lack of pparg in microglia resulted in increased expression of pro-inflammatory cytokines not only following an inflammatory stimulus but also in the steady-state indicating that pparg might play a cell-intrinsic role in controlling microglia immunogenicity. importantly, if pparg was activated in microglia, the capacity to prime ovalbumin-specific t cells was impaired. t cells primed by pio-treated microglia produced reduced amounts of il- and ifn-g which could not be overcome by restimulation with acd . this indicates that t cells primed by pio-treated microglia did not undergo functional differentiation but were impaired in exhibiting effector functions. furthermore, microglia were able to induce antigen-specific differentiation of naive cd t cells into t helper (th ) cells, which have been associated with autoimmune pathogenicity during eae. however, if pparg was activated, microglia were no longer able to induce th differentiation. in conclusion, activation of pparg impairs microglial apc function leading to reduced activation of antigen-specific t cells and, in addition, inhibits the induction of th cells. therefore, activation of pparg in microglia is a promising approach to limit local activation of autoreactive t cells in the cns in cns-autoimmune deseases. bacterial lipopolysaccharide (lps) triggers monocytes and macrophages to produce several inflammatory cytokines and mediators. however, once exposed to lps, they become hyporesponsive to a subsequent endotoxin challenge. this phenomenon is defined as lps desensitization or tolerance. previous studies have identified some components of the biochemical pathways involved in negative modulation of lps responses. in particular, it has been shown that the il- receptor-related protein st could be implicated in lps tolerance. the natural ligand of st was recently identified as interleukin- (il- ), a new member of the il- family. in this study, we investigated whether il- triggering of st was able to induce lps desensitization of mouse macrophages. we found that il- actually enhances the lps response of macrophages and does not induce lps desensitization. we demonstrate that this il- enhancing effect of lps response is mediated by the st receptor since it is not found in st ko mice. the biochemical consequences of il- pretreatment of mouse macrophages were investigated. our results show that il- increases the expression of the lps receptor components myeloid differentiation protein- (md ), cd and tlr- and the myeloid differentiation factor (myd ) adaptor molecule. in addition, il- pretreatment of macrophages enhances the cytokine response to tlr- but not to tlr- ligands. thus, il- treatment preferentially affects the myd -dependent pathway activated by the tlr. c. klotz , b. lenz , r. lucius , s. hartmann humboldt-university berlin, molecular parasitology, berlin, germany chronic helminth infections are shown to be negatively associated with allergic disorders in humans and animal models and parasite cysteine protease inhibitors (cystatins) have been identified as a major class of modulators from filarial parasites. recently we showed that recombinant parasite cystatin (avcystatin), derived from the model parasite acanthocheilanema viteae, effectively abolished ova-induced allergic airway responsiveness in a mouse model of asthma (schnoeller et al., ) . the cystatin effect was blocked by the application of anti-il- receptor antibodies and by depletion of macrophages using clodronate liposomes. we hypothesize that parasite cystatin induced regulatory macrophages characterized by secretion of immune suppressive interleukin- (il- ). the aim of the present study was to elucidate the molecular mechanisms by which avcystatin induces il- in primary macrophages. in vitro experiments with peritoneal macrophages from balb/c mice confirmed specific and concentration dependent il- production after avcystatin stimulation. application of specific inhibitors revealed that the il- induction was p and erk dependent, and inhibitor titration indicated a higher sensitivity towards p . western blotting experiments confirmed the phosphorylation of p and erk in macrophages after avcystatin stimulation. in addition, by using specific inhibitor and western blotting, we showed that avcystatin induced il- is also regulated by the phosphatidylinositol- -kinase (pi k) -proteine kinase b (akt) pathway. further analysis indicated a hierarchical signalling pattern and cross regulation of the identified pathways. hence, we conclude that avcystatin renders macrophages into a regulatory state by addressing a broad range of signalling cascades that ultimately lead to the expression of il- and possibly other regulatory markers. in general, revealing fundamental knowledge about induction of regulatory macrophages by helminth immunomodulators will help to design new strategies for the treatment of inflammatory disorders. we screened approximately half the (putative) human kinome to identify novel candidates interfering with macrophage activation in response to endotoxin. this screen revealed the impact of several novel kinases as well as kinases with previously established function. one of the top candidates identified to block endotoxininduced tnf-a secretion was carkl, a gene with no previously described function. subsequent biochemical analyses unequivocally revealed that carkl is a phosphotransferase protein using sedoheptulose as a phosphate acceptor and atp as a donor. sedoheptulose is a monosaccharide consisting of seven carbon atoms and a functional ketone group. the product sedoheptulose- -phosphate (s- p) is also an intermediate metabolite of the pentose phosphate pathway (ppp) and so far was only known to be produced by condensation of ribose- p and xylulose- p via a transketolase reaction. to identify the molecular mechanism by which carkl modulates the immune response, we investigated its endogenous regulation and function in the course of macrophage activation. so far, our data favor a model where post-stimulatory downregulation, i. e. loss of carkl is essential for the activation of macrophages by various pro-inflammatory stimuli. disentangling the signaling pathways responsible for the rapid regulation of carkl unearthed nf-kb and p /jnk but not erk as driving forces. counterbalancing endotoxin induced loss of carkl by over-expression led to an impaired cytokine response and a concomitant block of free radical production. comparison of wild type and catalyticinactive forms of carkl unveiled that most of the effects of carkl on the inflammatory response were due to its phosphotransferase activity. expression profiling using gene chip analysis further supported the concept that carkl may represent a new key modulator of inflammatory processes. taken together, detailed analyses to study the molecular function of carkl should ultimately lead to a more profound understanding of cellular metabolism and especially clarify new mechanisms involved in the regulation of inflammation. in addition, connecting the ppp and its impact on the cellular redox state with inflammatory disease models might reveal new therapeutic targets. in this context, the sedoheptulose kinase carkl and its product s- p may provide a novel basis for interfering with adverse immune responses. t. bosschaerts , y. morias , p. de baetselier , a. beschin vib, cmim, vub brussels, brussels, belgium the development of classically activated monocytic cells (m ) is a prerequisite for effective elimination of parasites, including african trypanosomes. however, persistent m activation causes pathogenic damage including liver injury during infection, resulting in death of the host. we aim to identify mechanisms involved in regulation of m activity in order to dampen their pathogenicity and increase the resistance of the host to parasitic diseases. methods: we have scrutinized the phenotype and cellular origin of liver m in trypanosoma brucei infected by facs analysis and bone marrow transfer experiments. the contribution of different signaling pathways, including myd , ifng, il- , ccr and nf-kb to the development and/or recruitment of pathogenic m in the liver was investigated using knock-out mice or by delivering il- in infected mice. results: we established that cd b+ly c+cd c+ tnf and inos producing dcs (tip-dcs) represent the major m liver subpopulation. tip-dcs differentiated in an ifng/myd -dependent manner from cd b+ly c+ inflammatory monocytes in the liver of infected mice. ccr promoted the egression of inflammatory monocytes from bone marrow to blood but not their entry, differentiation and maturation to tip-dcs in the inflamed liver. as a consequence, ccr ko mice experienced reduced pathogenic symptoms. on the other hand, the absence of il- enhanced the recruitment of inflammatory monocytes as well as their differentiation and maturation to tip-dcs, resulting in exacerbated pathogenicity and early death of the host. in addition, the therapeutic liver-specific delivery of il- in t.brucei infected mice efficiently limited the differentiation and maturation to tip-dcs, hereby limiting disease-associated pathogenicity. finally, the absence of the nf-kb member p was associated with increased tissue injury associated with increased production of pathogenic tnf and no by inflammatory monocytes, but not by tip-dcs. conclusion: our data demonstrate that nf-kb p and il- play a role in preventing infection-associated pathogenicity in hosts confronted with a chronic inflammatory situation by limiting the activity of pathogenic m , in particular tip-dcs. the inflammatory activity of liver m is controlled by il- and/or p nf-kb at different levels, including recruitment of inflammatory monocytes to the liver, their differentiation to pathogenic tip-dcs, or their production of tnf and no. a. popov , j. driesen , z. abdullah , a. niño castro , t. chakraborty , m. krönke , o. utermöhlen , c. wickenhauser , j.l. schultze limes institute, laboratory for genomics and immunoregulation, university of bonn, bonn, germany, institute of molecular medicine and experimental immunology, bonn, germany, institute of medical microbiology, university of giessen, giessen, germany, institute for medical microbiology, immunology and hygiene, university of cologne, cologne, germany, institute for pathology, university clinic leipzig, leipzig, germany dendritic cells (dc) and macrophages play an important role in pathogen sensing and antimicrobial defense. here we report on a new role for the myeloid antigen presenting cells (apc) in granulomatous infections. infection of myeloid dc and macrophages with listeria monocytogenes results in a distinct regulatory phenotype characterized by expression of multiple inhibitory molecules, including indoleamine , -dioxygenase, cyclooxygenase- and cd and production of prostaglandin e (pge ) and interleukin . all these molecules are strictly dependent on autocrine tnf, released during infection, and are in concert suppressing t-cell responses; cd , expressed by regulatory myeloid cells, acts as an il- scavenger. importantly, myeloid cells with regulatory phenotype are characterized by increased resistance to infection and demonstrate significantly improved bactericidal activity against intracellular bacteria. furthermore, infected cells can transfer the regulatory phenotype to the uninfected ones in a cell-cell contact independent manner, thereby extending the pool of infection-resistant myeloid cells. induction of regulatory and protective phenotype in macrophages and dc require at least two signals provided by tnf and either pge or tlr ligands. transcriptional changes in human macrophages, infected by mycobacterium tuberculosis, resemble the ones induced in dc during infection with l.monocytogenes. in fact, granuloma in patients with tuberculosis and listeriosis are enriched for cd + ido + cox- + regulatory myeloid cells, whereas most effector cell populations, such as t cells, b cells and nk cells, are expelled from the granuloma. of note, in tuberculosis granuloma consist mostly of macrophages, whereas in listeriosis dendritic cells predominate. altogether, our studies provide strong evidence that intracellular pathogens such as m.tuberculosis and l.monocytogenes induce a specific polarization of myeloid dc and macrophages characterized by a functional preponderance of inhibitory mechanisms. we postulate that these regulatory myeloid cells play a dual role during life-threatening granulomatous infections. on one hand, they promote pathogen containment by efficiently killing intracellular bacteria; on the other hand, these myeloid cells inhibit granuloma-associated t cells and thereby might be involved in the retention of granuloma integrity protecting the host from granuloma break-down and pathogen dissemination. the interferon-gamma (ifn-g) component of the immune response plays an important and essential role in infectious and non-infectious diseases. induction of ifn-g secretion by human t and nk cells through synergistic co-stimulation with interleukin (il- ) and il- in the adaptive immune responses against pathogens is well known, whereas a similar activity by macrophages is still controversial, largely due to criticisms based on the contamination of macrophages with nk or t cells in the relevant experiments. the possible contribution of macrophages to the interferon response is, however, an important factor relevant to the pathogenesis of many diseases. to resolve this issue, we have determined the production of ifn-g at a single cell level by inmunohistochemistry and by enzyme-linked immunosorbent spot (elispot) analysis and have unequivocally demonstrated that human macrophages derived from monocytes in vitro through the combined stimulation of il- and il- or with macrophage-colony stimulating factor (m-csf) were able to produce ifn-g when further stimulated with a combination of il- and il- . in addition, naturally activated alveolar macrophages immediately secreted ifn-g upon treatment with il- and il- . therefore, human macrophages in addition to lymphoid cells contribute to the ifn-g response, providing another link between the innate and acquired immune response. a. j. denzel , m. rodriguez gomez , m. niedermeier , y. talke , n. göbel , k. schmidbauer , m. mack unversity hospital regensburg, internal medicine ii, regensburg, germany, university hospital regensburg, regensburg, germany we have shown previously that basophils recognize and react to free antigen during a memory immune response in vivo and release large amounts of il- and il- . activation of basophils is dependent on the presence of free antigen, antigen specific immunoglobulins and expression of immunoglobulin fc-receptors. we now have analysed in more detail the binding of antigen to basophils, the recruitment of basophils to lymphoid organs and the basophil dependent migration of other leukocytes during the first days of a memory immune response. following restimulation with soluble antigen only antigen specific basophils but not basophils from naïve mice migrate from bone marrow and spleen to the site of restimulation (e.g. the peritoneum) and the draining lymph nodes. peripheral blood basophils are markedly reduced during the first hours after restimulation. in the blood, spleen lymph nodes and bone marrow basophils can bind intact antigen on their surface for up to h, with basophils in the bone marrow binding the lowest amount of antigen. depletion of basophils also affects the recruitment of various other leukocyte subsets in immunized mice. our datas show that basophils are recruited to draining lymph nodes during a memory response. tnf-a is a pro-inflammatory cytokine that mediates inflammation in response to various pathogens, including mycobacterium tuberculosis. it is also a key factor in the pathogenesis of autoimmune diseases like rheumatoid arthritis. three tnf-a-blocking drugs have been approved to treat selected autoimmune diseases; two are monoclonal antibodies against tnf-a (adalimumab and infliximab); the other is a soluble tnf receptor/fc fusion protein (etanercept) . tnf-a-blockers have been shown to increase the risk of reactivation of latent tuberculosis and this risk appears to be higher in patients treated with the monoclonal antibodies. we studied the effects of tnf-a blockers on the maturation of mycobacteria-containing phagosomes in human macrophages. all three drugs had an inhibitory effect on ifn-g-induced phagosome maturation in pma-differentiated human thp- cells infected with m. bovis bcg, the avirulent m. tuberculosis h ra strain and the virulent m. tuberculosis h rv strain. adalimumab and infliximab, but not etanercept, suppressed phagosome maturation in primary human peripheral blood monocyte-derived macrophages (mdm) in the presence or absence of ifn-g. macrophages secreted tnf-a in response to infection with mycobacteria and this response was enhanced by activation of the cells with ifn-g. treatment of infected macrophages with tnf-a increased maturation of mycobacteria-containing phagosomes. these results suggest a role for tnf-a in activating phagosome maturation and highlight a novel mechanism through which tnf-a blockade can affect the host innate immune response to mycobacteria. z. g. dobreva , l.d. miteva , s.a. stanilova trakia university, faculty of medicine, molecular biology, immunology and genetics, stara zagora, bulgaria il- is a heterodimeric cytokine composed of a p subunit associated with the il- / p subunit. like il- , il- is expressed by the activated antigenpresenting cells and both cytokines induce ifn-gamma secretion by different t cell subsets. the proper balance between il- p -related cytokines controls the appearance of normal th and pathological th mediated immune responses. in this study, we examined the dynamics of inducible il- p and il- p mrna expression and protein production in purified human monocytes and how jnk and p mapks inhibitors influenced il- p and il- production. the cytokines' quantity determination was performed by elisa. quantitative real-time polymerase chain reaction (qrt-pcr) was performed for mrna transcripts detection. results were calculated in fold increase compared with gene expression in nonstimulated monocytes. il- p gene expression was higher than those observed for il- p gene at all time-points. the level of il- p mrna increased after th h and reaching a maximum level at th h ( . fold for c bgp and . fold for lps). c bgp and lps triggered il- p gene transcription were almost equal at the rd h ( . and . fold) and at th h ( . and . fold, respectively) after stimulation. the higher level of il- p gene expression was detected at th h in lps compared to c bgp stimulated monocytes ( . vs. . fold). however, il- p and il- protein production was increased in the highest level after c bgp stimulation. the inhibition of p led to the statistical significant augmentation of c bgp stimulated il- p production. the inhibition of the same map kinase enhanced lps stimulated il- p production without significant difference. the inhibition of jnk and p mapks significantly decreased c bgp stimulated il- production from human monocytic cells.in summary, the present study demonstrates the different time-course and ability of c bgp and lps to induce the expression of il- p and il- p mrnas in purified human monocytes. we showed that inhibition of p mapk down regulated il- and upregulated il- p production in stimulated monocytes. we concluded that in human monocytes p map kinase activation has an opposite effect on the il- p and il- p expression. neutrophils represent key components of the innate immune system with the ability not only to phagocytose and killing invading pathogens, but also to produce a variety of proteins, including cytokines and chemokines, with important consequences on the recruitment and activation of other immune cells, such as monocytes, dendritic cells, t and b cells. for instance, it has been shown that neutrophils can directly interact with, and induce functional maturation of, immature monocyte-derived dendritic cells (modc). indeed, upon interaction with neutrophils, modc up-regulate the expression of costimulatory molecules, such as cd , cd and cd , and secrete il- , thus acquiring the ability to induce proliferation and th polarization of naï ve t cells. in order to extend these findings, the present study was designed to address whether human neutrophils interact with peripheral blood-derived dendritic cells and the pathological consequences that such interaction could eventually produce. in human peripheral blood, dendritic cells can be divided in plasmacytoid dendritic cells (pdc) and myeloid dendritic cells (mdc), the latter further divided in three different subsets based on the expression of cd c, bdca- , and cd . by analyzing different chronic inflammatory pathologies, such as crohn's disease, psoriasis and sweet's syndrome, we found that neutrophils co-localize with a subtype of myeloid dendritic cells (mdc) with characteristics resembling the cd + subset of mdc. in order to characterize the interaction between the two cell types, autologous neutrophils, highly purified by an in-house built immunonegative selection protocol, and cd + dc were isolated from healthy donors and analyzed in a co-culture system under different stimulatory conditions. here we show that neutrophils modulate different effector functions of cd + dc, including their survival and their ability to produce il- p . besides providing the basis for a better understanding of the cellular interactions that occur in pathological conditions, our results further emphasize the importance of neutrophils in the modulation of the inflammatory response. chitin is a linear polymer of n-acetyl-d-glucosamine (glcnac) residues present in human pathogenic fungi or nematodes. chitotriosidases (cht) and acetic mammalian chitinase (amcase) have been identified as the only functional chitinases in mammalians. the expression of both chitinases appears to be strongly species dependent, indicating distinct physiological functions. amcase is considered as predominant chitinases in mice while cht is regarded as major chitinases in humans. interestingly, cht is constitutively expressed by human phagocytes at high levels while it is absent in mice phagocytes. although, amcase received increased attention as modulator of the innate immune response against chitin in mice, the physiological function of cht in humans is virtually unknown. to evaluate the physiological function of cht we have characterised the substrate specificity of human cht and the mode of substrate cleavage by analysing chtproduced fragments of chitosan, a close but water soluble derivate of chitin. degradation products of chitosan have been investigated by gel electrophoresis and maldi-tof mass spectroscopy. moreover, the application of a computer-based model of cht activity revealed the mode of substrate cleavage. we found that cht is a processive endo-cleaving chitinase resulting in the production of only small diffusible chitin/chitosan fragments. in further studies we could show that those cht-produced small chitin/chitosan fragments exhibit a strong ability to induce a pro-inflammatory response in human blood derived monocytes/macrophages as indicated by an increased release of the pro-inflammatory cytokines tnf-a, il- , il- and mcp- involving the transcription factor nfxb. moreover, these stimulated monocytes/macrophages revealed an increase of cht expression indicating an autocrine positive feed-back regulation. our data suggest that human cht is involved in the early recognition of chitin/chitosan containing human pathogens due to the generation of immuno-stimulatory chitin/chitosan fragments. m. hasenberg , s. wolke , a. brakhage , m. gunzer otto-von-guericke universität, institut für molekulare und klinische immunologie, magdeburg, germany, hans-knöll-institut, abteilung für molekulare und angewandte mikrobiologie, jena, germany since their discovery in nucleic extracellular traps (nets) released by certain cell types including neutrophil and eosinophil granulocytes were shown to play a crucial role in mediating innate immune responses towards different bacterial und fungal pathogens. recently it was found by us and others that neutrophil granulocytes release nets also upon contact to the filamentous fungus aspergillus fumigatus. in the present study we aimed to characterize this process in more detail focusing on the kinetics of net-formation as well as clarifying the responsible cell-biological mechanisms. by the use of several microscopic techniques (scanning electron microscopy, fluorescence widefield microscopy, confocal-and -photon microscopy) we initially demonstrated the generation of net like structures after coincubation of a. fumigatus germlings and freshly isolated murine or human pmn. the analysis of our time lapse video microscopy data allowed us to examine the exact time course from initial contact to the fungal surface to explosive release of nets up to hours later. moreover, we investigated the dependency of this phenomenon on the induction of an oxidative burst. therefore we added the nadph-oxidase inhibitor dpi to the cell coincubation and found clearly reduced net formation. by fluorescence staining of reactive oxygen species we could demonstrate that ros are released prior to net detection. interestingly, our data currently suggest that in contrast to other pathogens investigated so far, nets are not directly toxic to fungal elements. whether and how nets control growth of a. fumigatus currently remains open. to summarize our data, we found rapid net formation as a commonly observed immune response of neutrophil granulocytes contacting a. fumigatus. consistent with studies on different pathogens this mechanism seems to be ros-dependent, however not toxic for the fungus. thus, in the future we will have to clarify whether net-formation really occurs in vivo and how nets can control the outgrowth of a. fumigatus at sites of infection. production of type i interferons (ifn-i, mainly ifn-a and ifn-b) is a hallmark of innate immune responses to all classes of pathogens. when viral infection spreads to lymphoid organs, the majority of systemic ifn-i is produced by a specialized 'interferon-producing cell' (ipc) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pdc). it is unclear whether production of systemic ifn-i is generally attributable to pdc irrespective of the nature of the infecting pathogen. we have addressed this question by studying infections of mice with the intracellular bacterium listeria monocytogenes. protective innate immunity against this pathogen is weakened by ifn-i activity. in mice infected with l. monocytogenes systemic ifn-i was amplified via ifn-b, the ifn-i receptor (ifnar) and transcription factor interferon regulatory factor (irf ), a molecular circuitry usually characterisitic of non-pdc producers. synthesis of serum ifn-i did not require tlr . in contrast, in vitro differentiated pdc infected with l. monocytogenes needed tlr to transcribe ifn-i mrna. consistent with the assumption that pdc are not the producers of systemic ifn-i, conditional ablation of the ifn-i receptor in mice showed that most systemic ifn-i is produced by myeloid cells. furthermore, results obtained with facs-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pdc is responsible for bulk ifn-i synthesis. the amount of ifn-i produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. based on these data we propose that the engagement of pdc, the mode of ifn-i mobilization, as well as the shaping of the antimicrobial innate immune response by ifn-i differ between intracellular pathogens. t. naessens , s. vander beken , p. bogaert , j. grooten ghent university, biomedical molecular biology, zwijnaarde (ghent), belgium introduction: although the effector and modulator functions of activated macrophages in innate and adaptive immunity are well documented, their exact role in the initiation and propagation of immune pathologies is still not fully understood. recent insights in monocyte and macrophage heterogeneity render the picture even more complex. in addition, it is unclear to what extend resident and elicited macrophages differ functionally and hereby differentially contribute to immune pathologies. in this study we focused on the dynamics and function of resident alveolar macrophages (ram) during and after allergic bronchial inflammation. strategy: we used an ovalbumin (ova)-alum based mouse model of allergic asthma and an ova-complete freund's adjuvant (cfa) based mouse model of hypersensitivity pneumonitis, constituting a th -driven immunological counterpart of the th -driven experimental asthma. ram were distinguished by prior in situ labelling with fluorescent polystyrene microspheres. as complementary approach, ram and elicited alveolar macrophages (eam) were distinguished using cd bone marrow chimeric mice. combined with flow cytometry and fluorescence activated cell sorting, both approaches allowed us to trace resident and elicited am populations in the course of th -and th -driven allergic airway inflammation. results: during the acute phase of the allergic response, isolated ram and eam showed distinct gene expression signatures, reflecting a possible functional heterogeneity between these two macrophage subsets. in both types of allergic inflammation, microsphere-tagged cd . + ram remained constant in cell number for the first days of chronic ova-exposure and then dropped sharply, having nearly completely disappeared from the alveoli by day of ova-exposure. as a consequence, following the clearance of inflammation, inflammation-experienced ram replaced the initial ram population. strikingly, in both types of allergic inflammation, this secondary ram population showed a markedly altered responsiveness to lps stimulation. this involved macrophage activation markers and nf-kb inducible inflammatory genes. however, especially genes induced by ifn-beta showed strongly increased expression in secondary ram as opposed to their near lack of induction in primary ram. this switch from an ifn-beta deficient to an ifn-beta adequate phenotype may increase the inflammatory sensitivity of allergic inflammationexperienced lungs as also observed in asthmatic patients, showing an increased sensitivity to bacterial infection. e. schlecker , a. stojanovic , a. cerwenka german cancer research center, innate immunity, heidelberg, germany myeloid-derived suppressor cells (mdsc) are a heterogeneous population of cells that expand during cancer, inflammation and infection. these cells play a critical role in suppressing t cell responses. the exact nature and function of mdsc remain unclear. here we show that a subpopulation of mdsc (gr- + cd b + f / + ) isolated from rma-s tumor-bearing mice did not suppress but rather activated nk cells to produce ifn-g. additionally, nk cells eliminated this subpopulation both in vitro and in vivo. in order to identify molecules and pathways that might be involved in mdsc accumulation in tumor bearing mice and their suppressive/activatory function, gene expression profiling of mdsc subpopulations was performed using whole genome microarrays. understanding the reciprocal interaction of mdsc with nk cell could improve the efficiency of cancer immunotherapy. g. solinas , f. marchesi , m. fabbri , s. schiarea , c. chiabrando , a. mantovani , , p. allavena istituto clinico humanitas, rozzano, italy, istituto mario negri, milano, italy, università di milano, milano, italy experimental and clinical evidence has highlighted that tumor-associated macrophages (tam) represent the principal component of the leukocyte infiltrate and are usually associated with tumour growth, progression and metastasis. macrophage population is generally divided into two distinct subsets: m and m . m macrophages act as a first line of defence against pathogens whereas m cells participate in wound repair and maintenance of tissue integrity. in the tumour micro-environment tam interactions with the extracellular matrix, neighboring cells, and soluble stimuli largely influence their gene expression and behavior. to investigate the role of the tumor micro-environment on macrophage differentiation, we cultured freshly isolated human monocytes with pancreatic cancer cell line supernatants, in the absence of exogenous cytokine addition.. in selected cultures, about % of the monocytes differentiated after days into macrophages. the phenotype analysis of tumor-conditioned macrophages (tc-macro) demonstrated high expression of the mannose receptor, cd , cd and low levels of mhc class ii. tc-macro produced il- , il- , tnf but not il- , even after lps stimulation. moreover, tc-macro produced a panel of chemokines including ccl , cxcl , ccl and cxcl . the transcriptional profile of tc-macro revealed that several genes in line with an m polarization are highly expressed. the nature of the tumor-derived factors inducing macrophage differentiation is currently under investigation; biochemical analysis indicated that the biological activity is excluded from exosomes and have a high molecular weight ( g . kda). il- and il- were not detectable in tumor supernatants whereas m-csf was present at low levels. by mass spectrometric techniques, we surprisingly found that the tumor-derived m-csf had peculiar migration patterns which were different from those expected for the common human homodimeric glycosilated protein, suggesting an interesting structural differences for the tumor-secreted isoforms of this primary regulator of mononuclear phagocyte. the characterization of tumor-derived factors inducing macrophage differentiation could better clarify the intricate cross-talk between tumor cells and macrophages and thus might aid in the process of devising novel anti-tumor treatments. genomic effects of glucocorticoid hormone (gc) are exerted by glucocorticoid receptor (gr)-mediated changes of gene expression. this is relatively timeconsuming process, needing hours to develop. in contrast, non-genomic effects may occur within minutes. gcs are used for a long time for the therapy of anaphylactic reactions, where mast cells play crucial role. moreover, many cells and cell lines of haemopoetic origin are sensitive to gc-induced apoptosis. recent findings indicate, that non-genomic gc effects mediated by mitochondrial gr may have important function in generating pro-apoptotic signals. we aimed the investigation of non-genomic gc effects on in vitro cultured rbl- h rat mast cell line. we demonstrate that gr nuclear translocation begins within minutes and completes after minutes in dx treated rbl- h cells. since genomic effects occur in the nucleus through gene expression changes, we considered gc effects within minutes as non-genomic. studying gc-caused apoptosis, rbl- h cells proved to be gc-resistant and no mitochondrial gr translocation neither impaired mitochondrial function could be observed upon gc treatment. in further experiments we used rbl- h cells sensitized with anti-dnp (dinitrophenyl) ige and dnp-conjugated bovine serum albumin was used for stimulation. minutes of dx treatment inhibited ca + -signaling in antigen stimulated rbl- h cells in the concentration range of nm - mm. moreover, minutes of dx treatment altered the tyrosine phosphorylation pattern of rbl- h cells. dx treatment alone caused slight increase in tyrosine phosphorylation, while dx treatment of activated cells caused also an increase in tyrosine phosphorylation compared to the solvent-treated controls. the tyrosine kinase syk plays indispensable role in regulating mast cell activation through the fc[epsilon] receptor i. our immunoprecipitation studies show, that dx treatment results in decreased syk phosphorylation in both resting and activated cells. this finding raises the possibility, that syk phosphorylation thus kinase activity may be directly or indirectly regulated by gcs via non-genomic pathway. taken together, our experiments along with the clinical experiences suggest that gcs rapidly influence mast cell activation via a non-genomic pathway, too. the elucidation of the exact signal transduction mechanisms behind rapid gc effects need further experiments. high mobility group box (hmgb ) is a non-histone nuclear protein that binds chromatin and has transcriptional and architectural functions. notably, hmgb is highly mobile in the nucleus and is passively released by necrotic cells, while it is bound firmly to apoptotic chromatin ( ) . extracellular hmgb can act as a cytokine and a chemoattractant, mediating inflammatory responses. interestingly, hmgb exerts antibacterial functions in human adenoid and testis ( ) . recent investigations have revealed that neutrophils eliminate microbes not only by intracellular phagocytosis but also by trapping them in three-dimensional structures called neutrophil extracelluar traps (nets), made of dna fibers, nuclear proteins (histones) and granule proteins. it has been shown that histones on nets have an anti-microbial activity ( ). we asked whether hmgb from neutrophils is a component of nets and whether it has a function in nets. we purified human primary neutrophils from peripheral blood of healthy volunteers on ficoll gradients. to induce net formation, we stimulated cells for or minutes with nm phorbol ester (pma), ng/ml interleukin (il- ), or ng/ml lps. the presence of nets was assessed by immunofluorescence using antibodies directed against the granule protein myeloperoxidase (mpo) and against a dna-histone h a-histone h b complex. dna was stained with hoechst. using a polyclonal antibody we found hmgb in the euchromatin of polylobulated nuclei of resting neutrophils and on the filamentous structure of nets induced by all stimuli. elisa assays revealed that hmgb is not present in the supernatants of activated neutrophils, confirming its binding to nets. in conclusion, we found that hmgb localizes on nets. we hypothesize that net-bound hmgb might exert a direct antimicrobial function, or that nets might concentrate hmgb locally to recruit macrophages to the site of infection. these receptors were present on the mast cell surface. incubation ( °c, h) of hlmc with vegf-a, vegf-b, vegf-c, vegf-d and placental growth factor- induced concentration-dependent chemotaxis that was blocked by a combination of anti-vegfr- and anti-vegfr- antibodies. these data indicate that human mast cells represent both a source and a target of vegfs and therefore may play a role in inflammatory and neoplastic angiogenesis through the expression of proangiogenic factors and their receptors. macrophages are important effector cells in immunity to intracellular pathogens and at the same time are exploited as host cells by a number of microorganisms such as mycobacterium tuberculosis. a very important mechanism of intracellular killing is delivery of invading microbes to phagolysosomes. whilst mycobacteria can block phagosome maturation in resting macrophages, and hence survive and replicate inside the host cell, the ifn-g activated macrophage utilizes a diversity of defense mechanisms to eliminate the invader. these include putative killing by antibacterial peptides/proteins and overcoming phagosome maturation block, possibly by induction of autophagy, production of reactive nitrogen or oxygen intermediates and deprivation from nutrients such as iron. mycobacteria are not eliminated even upon onset of protective immunity rather leading to persistence. we hypothesize that the very early steps of pulmonary infection directs the outcome of disease. therefore, we investigate initially infected lung cells and their role in infection in the lung with respect to their anti-microbial mechanisms against mycobacteria in vitro as well as in vivo. preliminary data show that m. tuberculosis is able to persist in the alveolar space for several weeks and bacterial numbers do barely drop even after very low dose infection, indication that bacterial killing is inefficient from the very beginning. cells harboring mycobacteria are found during early and late stages of infection. both, autophagy and nitric oxide production seems to contribute to growth restriction of mycobacteria by macrophages. neutrophils, although recruited in vast numbers to infected lungs, are not able to reduce bacterial numbers in the absence of il- . altogether, the initial response in the barrier organ lung executed by resident and immigrating cells restricted by the local environment can determine the outcome of infection. human cd molecules are dedicated to lipid presentation to t cells and are implicated in inflammatory and auto-immune responses. the cd a protein is almost exclusively expressed at the cell surface of dendritic cells and is dedicated in surveying extracellular environment. our previous studies have demonstrated that ii associated with cd a and cholesterol-dependent lipid rafts impact on cd a surface expression and cd a-restricted t cell response. bacterial infections can induce an increase in self glycolipid synthesis in dendritic cells and such activated dcs acquire the ability to stimulate cd -restricted autoreactive t cells. this mechanism of self recognition induced by bacterial infection is believed to be involved in the development of auto-immune disorders. sulfatide, which is a major component of the myelin sheath, is also the only known self-antigen presented by cd group i molecules. the functional role of these molecules has not been investigated in auto-immune diseases and we propose that regulation of glycolipid presentation by cd a molecules could impact in such pathologies. we have thus conducted a preliminary study to understand the implication of cd molecules in multiple sclerosis. we first analyzed cd expression on monocytes from ms patients and the influence of sera and plasma from these patients on dendritic cell differentiation from healthy donors. results obtained in this preliminary study demonstrate that cd a was not expressed on ms patient monocytes, while the other members of the cd family were expressed. moreover ms sera and plasma induced an earlier and more rapid dendritic cell differentiation than ab sera. these preliminary results confirm our hypothesis that cd molecule expression is modified in ms and also reveal that serum from patients with ms modifies lipid-antigen presenting cells. further studies should contribute to define precise mechanisms involved in lipid presentation by cd molecules in this context. c. ohnmacht , d. voehringer ludwig-maximilians-universität munich, institute for immunology, munich, germany basophils are effector cells of the innate immune system which are associated with allergic inflammation and infections with helminth parasites. however, their development and in vivo functions are largely unknown. here, we characterize basophil turnover, tissue localization and effector functions during infection with the gastrointestinal helminth nippostrongylus brasiliensis. for this purpose, brdu incorporation experiments and in situ fluorescence microscopy of il- reporter ( get) mice as well as in vivo depletion of basophils are used to uncover their role during type immune responses. our results demonstrate that under homeostatic conditions basophils have a lifespan of about h. n. brasiliensis induced basophilia is caused by increased de novo production of basophils in the bone marrow. basophils are found near the marginal zone in the red pulp of the spleen, in the lamina propria of the small intestine and in the lung parenchyma. activated basophils promote systemic eosinophilia, were associated with differentiation of alternatively activated macrophages in the lung and contributed to efficient worm expulsion of n. brasiliensis in the absence of th cells. these results demonstrate that basophils play a crucial role as effector cells in type immune responses which might hold great potential for the treatment of helminth infections and allergic diseases. during acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. neutrophils show pro-inflammatory activity and may contribute to tissue damage. in pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. this damage may be due to excessive neutrophil activity. we here show that transgenic expression of bcl- in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. the persistence of neutrophil brain infiltrates was accompanied by high levels of il- beta and g-csf as well as reduced levels of anti-inflammatory tgf-beta. significantly, bcl- -transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. in vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. the inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. in wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. these results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils. objectives: to investigate the existence of systemic inflammatory response to subchronic oral warfarin (wf) consumation in rats. methods: dark agouti (da) rats were treated with warfarin in drinking water ( mg and mg daily) for days. oxidative activity (cytochemical nbt reduction) and myeloperoxidase (mpo) intracellular content of peripheral blood neutrophils, plasma levels of il- and tnf-a (elisa) and superoxide dismutase (sod) activity (red blood cell lysates) were analyzed as inflammatory parameters in rats following warfarin consumation. changes in prothrombin time (pt), as basic biological warfarin activity was determined as well. results: significantly increased pt was noted at the lower wf dose, with tremendous rise after the higher dose. increase of pma-stimulated neutrophil nbt reduction capacity (neutrophil priming) was noted at both wf doses, while increase in mpo intracellular content was noted at the higher wf dose solely. warfarin consumation resulted in no changes in plasma levels of il- and tnf-a. significant decrease in the sod activity was detected in red blood cell lysates at both wf doses, suggesting systemic oxidative activity. conclusion: increased neutrophil priming as well as prooxidant activity in peripheral blood of rats following subchronic warfarin consumation imply proinflammatory effects of oral warfarin administration. absence of the rise in inflammatory cytokines in circulation, suggest low-grade inflammation in these rats. this work is funded by serbian ministry of science and technological development (grant ). objectives: although many different macrophage receptors and serum proteins have been shown to play a role in phagocytosis of apoptotic cells, the unique microenvironment of an inflammatory site will have considerable influence upon the molecular pathways which are utilized in apoptotic cell removal. we have recently reported that immune complexes (ic) are able to specifically bind to the surface of human apoptotic neutrophils which may have profound implications for their physiological clearance. in disease situations where immune complexes are present neutrophils undergoing apoptosis would be predicted to become coated with ic. here we address the consequences of ic opsonisation of apoptotic cells upon phagocytosis and cytokine response by macrophages that would be expected to be present at the earliest stages of inflammatory responses (type- macrophages, mph ), and during resolution of inflammation (type- macrophages, mph ). methods: mph / were generated by culturing cd + human monocytes for days in the presence of gm-csf or m-csf, respectively. phagocytosis by mph / of ic opsonised and unopsonised neutrophils was assessed by flow cytometry. after phagocytosis mph / were stimulated with lps and secreted il- , il- , il- , il- p and tnf were quantified by elisa. results: mph are relatively efficient phagocytes for apoptotic neutrophils whereas mph are only poorly phagocytic. opsonisation with ic leads to enhanced neutrophil uptake by both mph and mph which is specifically inhibited in the presence of a blocking mab for macrophage fcyrii. uptake of ic opsonised neutrophils causes a shift towards an anti-inflammatory cytokine profile. in both macrophage subsets il- , il- and tnf production is suppressed while il- secretion is increased. in contrast, engagement of macrophage fcyr with ic alone induces the release of pro-inflammatory cytokines. conclusion: our data demonstrate that ic opsonisation of apoptotic neutrophils increases the proportion of macrophages capable of phagocytosis and that apoptotic cell recognition interactions provide a dominant anti-inflammatory signal, suppressing macrophage responses, even in the presence of ic opsonisation. we suggest that ic present in the inflammatory milieu would opsonise apoptotic neutrophils, enhance macrophage phagocytosis and thereby facilitate the process of resolution of inflammation. excessive production of reactive oxygen species (ros) produced by neutrophils is known to be a factor accelerating ageing because of damaging effect on cells. on the other hand, intracellular heat shock proteins (hsp) are involved in protecting cells from the damaging effects, and provide cell resistance to stress. in this work, correlation analysis was applied to analyze relationship between ros production and intracellular hsp in neutrophils of elderly people. neutrophils were isolated from peripheral blood of donors of years old and older (long-livers). intracellular ros and hsp levels were registered by flow cytofluorimetry upon labeling with ', '-dichlorofluorescin diacetate (invitrogen) and anti-hsp antibody (brm- , sigma), respectively. intracellular level of hsp was also estimated in neutrophils after heat shock (hs) performed at °c for min. extracellular ros production from zymosan-activated neutrophils was detected by luminol-dependent chemiluminescence. a positive correlation was determined for intracellular ros level and zymosan-mediated extracellular ros release although the dynamics of ros release at - min time range varied within the group. the correlation was unaffected by hs of neutrophils performed for min at °c, although this short heat treatment decreased significantly ros release. there was no correlation between basal intracellular hsp (hsp basal ) and ros level, both intracellular and extracellular. at the same time increased hsp level immediately after hs (hsp ( min)) correlated negatively with intracellular ros (initial and after hs). the hsp increase value (hsp ( min) -hsp basal ) correlated negatively also with intracellular ros and extracellular ros release in response to zymosan; and the correlation with ros level became lower when hsp increase was registered in min after hs (hsp ( min) -hsp basal ). thus it was found that within this age group the alteration in hsp induced by hs in neutrophils but not basal hsp itself is the parameter associated negatively with both spontaneous ros level and ros production in response to activating action of zymosan. this work is supported by istc grant # . d. goyeneche-patiño , z. orinska , f. mirghomizadeh , s. bulfone-paus forschungszentrum borstel, borstel, germany several studies have shown different roles of mast cells (mc) in innate and adaptative immune responses. in fact, crosstalk between cd + t cells and mc has shown to induce multiple genes implicated in the signaling of specific programs such as type ifn. two novel genes, receptor transporter protein (rtp ) and virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (viperin) are ifn inducible and were found to be over-expressed in chip analysis. the aim of this study is to characterize the expression and protein production of rtp and viperin in mast cells after tlr ligand stimulation in mice lacking of ifnra and the adapter proteins myd and trif. bone marrow derived mast cells (bmmc) from wt, ifnra -/-, mydd -/and trif -/mice, were exposed to tlr ligands (lps, pic, cpg, p(da-dt) and new castle disease virus (ndv)) during and h. mrna and protein extraction were performed for further qrt-pcr and sds page analysis for rtp and viperin. intracellular stimulation of tlr was performed transfecting cells with nucleic acids using lipofectamine . stimulation of wt cells with pic, pda-dt and ndv showed an increased expression of viperin and rtp in comparison to control cells (untreated). the same trend was observed for mc from the trif and myd knockout mice. in contrast, in the ifnra deficient mice, expression of genes and protein production was abrogated to the same levels of wt untreated cells. lipofectamine stimulation does not increase the expression/production of the genes. direct stimulation of the well recognized viral sensors tlr and , as well as, infection of mast cells with ndv (rna virus) induce the expression of rtp and viperin. the findings suggest that activation of mc with the ulterior expression of genes is type i ifn dependent. in contrast, the adaptor proteins myd and trif and the pathways that they represent are not relevant in the expression of rtp and viperin. these findings provide bases for performing further studies focused to elucidate the functions of these proteins and show an alternative role of mc in innate immune responses. in recent years, it has been suggested that the phenomenon of "myc-dependent cell competition" described in drosophila melanogaster, could be a critical step when a cell initiates nascent tumour field. we have taken a step forward and applied the phenomenon of cellular competition to the human macrophage system: inflammatory macrophages theoretically have the ability to eradicate cancer due to their tumoricidal capability and, at the same time, acting as antigen-presenting cells (apcs) to activate lymphocytes; but inflammatory macrophages do not express c-myc and, within the tumour, they encounter two powerful rivals: tumoral cells and alternative tumour-associated macrophages which express c-myc. we studied some phenomenons suggested to be myc-dependent such as the ability to feed, the ability to survive in a competitive medium, the ability to proliferate and the ability to eliminate competitors and we observed that alternative macrophages have more resources to survive in a tumoral microenvironment and could be involved in tumour growth by collaborating with tumour cells in transforming inflammatory macrophages into anergic cells which enter into apoptosis and are then phagocyted. finally, using lentiviral vectors, we over-expressed exogenous c-myc in inflammatory macrophages in an attempt to increase their chances of survival in the tumour microenvironment, in vitro and in vivo, and to determine whether it can be utilized as a potential anti-tumoral cell therapy. g. germano , e. erba , r. frapolli , m. d'incalci , a. anselmo , s. pesce , p. allavena , a. mantovani , humanitas clinical institute, rozzano, italy, mario negri institute, milan, italy, institute of general pathology, university of milan, milan, italy several lines of evidence suggest a strong association between chronic inflammation and tumor progression; therefore the use of anti-inflammatory drugs may be beneficial in anti-tumor therapies. inflammatory mediators (e. g. cytokines, chemokines) are produced at the tumor site both by tumor-associated macrophages (tam) as well as tumor cells, and are attractive target of novel anti-tumor therapies. trabectedin (et- ) is a natural product derived from the marine tunicate ectenascidia turbinate, it binds the minor groove of dna, affects transcriptional factor activity and blocks cell cycle. this novel anti-tumor agent is currently used in phase ii studies in patients with sarcoma, ovarian and breast cancer, with clinical regressions. we previously demonstrated that trabectedin is selectively cytotoxic, in vitro, to monocytes/macrophages, being active at concentrations that spared lymphocytes suggesting a possible alternative target for the anti-tumoral role of this drug. we tested the effect of trabectedin on primary cultures and liposarcoma cell lines showing that at sub-cytotoxic concentrations the production of some inflammatory mediators were down-modulated. trabectedin significantly reduces ccl , cxcl and the inflammatory protein pentraxin (ptx ) either at transcriptional and protein level, especially after tnfa/il b stimulation. down-regulation of ccl , cxcl , ptx and also of il and vegf were confirmed in primary cultures of liposarcoma. according to the previous in vitro data we now show in a mouse model , using the fibrosarcoma mnmcai , that trabectedin treatment selectively affects monocytes in the blood and bone marrow. moreover trabectedin treatment strongly reduces the number of macrophages and of cd + vessels in the tumor microenvironment, in line with its selective activity on monocytes/macrophages. overall these results suggest a possible triple role of trabectedin. besides its direct cytotoxic effect on tumor cells, trabectedin also affects tumor associated macrophages and at low dose the transcriptional activity of inflammatory genes involved in the tumor-microenvironment cross-talk. as the local expression of inflammatory mediators may play a role in tumor progression, this newly recognized effect of trabectedin makes it an attractive candidate in inflammation-associated tumors. interleukin- (il- ) is a key cytokine of the t helper cell response. il- has been found to be a major regulator of immunoglobulin class switching to ige and has important functions in the regulation of allergic diseases. here, the onset of the il- production after birth was investigated in equine neonates. the form of equine placentation does not support the transfer of cytokines or immunoglobulins in utero and maternal immunity is exclusively transferred to the neonate with the colostrum after birth. il- producing cells were measured in peripheral blood mononuclear cells (pbmc) of neonates and foals by flow cytometric analysis. at day - after birth, a small population of il- producing cells was observed in the absence of any stimuli. the il- + population was not detectable at or weeks of age. other cytokine producing cells (ifn-g, il- ) were not detected using these conditions. the stimulation of neonatal pbmc with pma and ionomycin did not alter the il- + cell population. phenotyping of the neonatal il- + cells showed that they were ige + /mhcii -/cd cells. the occurrence of cd + il- producing cells after pma stimulation increased slowly with age and did not reach adult levels by weeks after birth. magnetic cell sorting of the ige + /mhciicells identified them as basophils. previous work has shown that foals do not produce endogenous ige for at least six months of life. ige bound to the surface of neonatal basophils was found to be of maternal origin and transferred with the colostrum after birth. here, the stimulation of neonatal pbmc with anti-ige induced the secretion of il- at day after birth. neonatal pbmc collected before colostrum uptake did not produce il- in response to anti-ige. in summary, equine neonates provide a model to investigate ige mediated il- responses after birth. the transfer of maternal ige from allergic individuals could potentially provide a direct mechanism for the early induction of an allergen-specific neonatal il- response mediated by the mare's accumulated acquired immunity to allergens. s. schmechel , d. voehringer ludwig-maximilians-university munich, institute for immunology, munich, germany macrophages display broad phenotypic heterogeneity depending on their microenvironment. the initial inflammatory response to th cytokines is predominantly mediated by classically activated macrophages whereas macrophages undergo alternative activation in a stat -dependent manner when stimulated with the th cytokines il- or il- . alternatively activated macrophages (aam) are implicated in diverse disease pathologies such as host response to parasitic infection and asthma. furthermore, it has been shown that aam suppress the proliferation of t cells by a yet to be determined mechanism. currently there is still very limited information about the phenotype, migration and function of aam. we began to elucidate whether macrophage turnover and recruitment to inflammatory sites is regulated in a stat -dependent manner. to this end we generated mixed bone marrow chimeras with bone marrow from congenic wild-type and stat -deficient mice and infected these chimeras with the helminth nippostrongylus brasiliensis to determine whether lack of stat in macrophages affects their turnover and recruitment to the lung side-by-side in the same animal. the highest turnover of macrophages was found in the peritoneum, irrespective of stat expression. no major differences in tissue distribution and turnover were observed between both populations suggesting that macrophage proliferation and recruitment during parasite infection is not dependent on stat expression in macrophages. we could further confirm that in vitro generated aam from wild-type but not from stat -deficient mice have a strong inhibitory effect on t cell proliferation. we are now trying to identify the mechanism(s) by which t cell proliferation is inhibited. furthermore, we work on the cellular cross-talk between eosinophils and macrophages and try to determine the plasticity of macrophage differentiation. we have previously shown that aam can recruit eosinophils to inflammatory sites and we now try to clarify which chemotactic factor are involved in this process. to identify potential aam-derived eosinophil chemotactic factors we currently compare the gene expression profile of il- exposed macrophages from wild-type and stat -deficient mice. candidate genes will be expressed using retroviral transfections of stat -deficient macrophages and supernatants from these cells will then be used to induce eosinophil recruitment in transwell assays. macrophages are an essential component of leukocytes infiltration in the tumor. they are identified as tumor associated macrophages (tams). these cells are also present in pleural effusions which appear as a consequence of spreading of neoplasm in the pleural cavity. the aim of the study was to assess the influence of the pleural macrophages on cells from human malignant cell lines. we tested the dynamics of growth of the malignant cells, their apoptosis and expression of proteins regulating this process under the influence of conditioned media from cultures of macrophages isolated from pleural effusion. we have also attempted to interpret our results by assessing the expression of a variety of immune modulating factors, their receptors on the malignant cells surface as well as the transcription factors. in the study we used macrophages isolated from a total of pleural effusions, including malignant and nonmalignant tumors. the following human malignant cell lines were tested: a , ht , hct , sw , mcf , mda-mb , jurkat and hl . results: our results suggest that the conditioned media isolated from the cultures of pleural macrophages can up-regulate the proliferative activity of the human malignant cell lines. macrophages from pleural effusions can act as a factor promoting or inhibiting apoptosis of malignant cells. down-regulation of apoptosis may depend on modulation of expression and activity of proteins regulating this process. macrophages can affect the apoptosis regulatory proteins and their activity through the immune-modulatory molecules, e. g. cytokines, chemokines, and growth factors. the up-or down-regulation of transcription factors expression may control the expression of pro-and anti-apoptotic proteins. the results indicate that macrophages from malignant and non-malignant pleural effusions differ from each other insignificantly; however the macrophages isolated from the non-malignant tumors show a pattern comparable to m , and the tams isolated from the malignant effusions similar to m . among the alternative stimuli, glucocorticoids are the most effective stimulus up-regulating ms a a and ms a a: highest trascriptional level after h of stimulation with - m dexamethasone. ms a murine genes are differently expressed respect to the human counterpart and only the homologs of ms a a (ms a b, c and d) have a similar regulation. finally, egfp-tagged ms a a, ms a a, and ms a expressed in cho cells showed that all molecules traffic to the cell membrane. though the biological functions of these ms a proteins has not jet been defined, their membrane localization and the structural relationship with other better characterized ms a members suggest a potential involvement in signal transduction, either as components of multimeric receptor complexes or as components of ligand-gated ion channels. during inflammatory reactions endogenously produced cytokines and chemokines act in a network and interact with hormones and neurotransmittors to regulate host immune responses. these signaling circuitries are even more interfaced during infections in which microbial agonists activate toll-like (tlr), rig-like (rlr) and nod-like (nlr) receptors. on the basis of the discovery of synergy between chemokines for neutrophil attraction, we here extended this phenomenon between the chemokine monocyte chemotactic protein- (mcp- )/ccl and the gpcr ligand fmlp or the tlr agonist lipopolysaccharide (lps) on monocytes. in fact, the bacterial tripeptide fmlp, but not the cytokines il- b or ifn-g, significantly and dose-dependently synergized with ccl in monocyte chemotaxis. furthermore, lps rapidly induced the expression of interleukin- /cxcl , but not of the ccl receptor ccr in monocytic cells. in turn, the induced cxcl synergized with ccl for mononuclear cell chemotaxis and the chemotactic effect was mediated by cxcr /cxcr , because cxcl receptor antagonists or antibodies were capable of blocking the synergy, while keeping the responsiveness to ccl intact. these data recapitulate in vitro the complexity of innate immune regulation, provide a novel mechanism of enhancing monocyte chemotaxis during bacterial infections with gram-negative bacteria and demonstrate the importance of local contexts in inflammatory and infectious insults. objectives: in recent years the existence and effects of cell-derived vesicles (e. g. exosomes, microparticles) have been revealed in several physiological functions, such as antigen presentation, hemostasis or receptor transfer to innocent cells. most data were collected on endothelial cells and on thrombocytes. however, there are only few data on vesicles derived of neutrophilic granulocytes (pmn), and most of these investigations applied only pharmacological agents. our aim is to investigate pmn-derived cell-free particles and their possible role in bacterial killing methods: preparation of pmn and investigation of bacterial killing by our semi-automatic method was described by rada et al. (blood, ) . cell-free vesicles were prepared after co-incubation of human pmns with different activating agents for min at °c with gentle shaking, followed by spinning down of pmns for min, at °c and g. the supernatant was sedimented at g for min, °c, and we used the sedimented fraction for our investigations. formation of particles was followed by fluorescent and electron microscopic assays. the amount of particles was estimated with flow cytometer and by their protein content. we observed that upon co-incubation of pmns with s. aureus, opsonized by mixed human serum, pmns produce a well detectable amount of vesicles. omitting opsonization or opsonizing with heat inactivated serum caused a minimal amount of particles. production of particles could be inhibited with diphenyl-iodonium (dpi), cytochalasin b (cb) or with azide treatment. treating pmns with dnase or withdrawing glucose during co-incubation had no effect on vesicle formation. in killing assays we detected remarkable antibacterial effect, which correlated well with the protein content of the used fraction. this antibacterial activity could be inhibited by dpi, cb, azide treatment or by withdrawing glucose from the medium during the killing assay. however, treatment of the microvesicles with dnase had no effect on their antibacterial capacity. for long, cd has been used for the detection and identification of natural killer (nk) cells. recently, the presence of a minor subset of cd low cd + blood monocytes (mo) in healthy individuals and the increase in cd +cd + blood mo in patients with inflammation has been reported. the functional activity of human cd + blood mo has been studied in vitro but not tested ex vivo so far. healthy people living permanently in malaria endemic areas are exposed to plasmodium infection, and we hypothesized that blood mo of these individuals could be activated and display increased cd expression. we tested if this phenotypic expression was associated with detectable changes in the mo anti-parasitic activity. the mo phenotype of healthy malaria naï ve and malaria exposed individuals was determined by three-color flow cytometry. myeloid cell markers included cd and activation markers such as hla-dr and trem- . percentages of blood mo involved in phagocytosis activity either with or without immune sera were then identified by flowcytometry, and the potential association between a given mo phenotype and phagocytosis activity was then looked for, using spss ® and statview ® softwares. our results showed that, compared with malaria naï ve individuals, there was a . fold increase (p x . ) in the total number of circulating cd low mo present in the blood samples of healthy malaria exposed asian individuals living in thailand. according to the density of surface antigen determined by fluorescence intensity (fi), the decrease in cd and the concomitant increase in hla-dr expressions indicated that in this malaria endemic area, blood mo were mature and highly activated by comparison with surface markers of mo from malaria naï ve donors. the relative levels of cd + blood mo were associated with the percentages of membrane-bound ifn-g present at the mo surface. in conclusion, (i)-a subset of cd + blood mo expressed increased levels of cd on mo of healthy malaria exposed individuals; (ii)-blood mo with activated (hla-dr+) and mature (trem- +) phenotypes were present in these healthy individuals; (iii)-increased expression of hla-dr and cd on cd high mo was associated with a high phagocytosis activity. introduction: adipokines, initially described for their function within metabolism, have been characterized to exert a regulatory role on the immune response. for instance the appetite-regulating hormone leptin has been identified to modulate the response of the innate as well as the acquired immune system. the present work focuses on the effects of leptin on the reactivity of m -and m -polarized human macrophages. methods: monocytes were isolated from the peripheral blood by magnetic cell sorting. polarization to m and m macrophages was induced by culture in the presence of mcsf or gmcsf respectively. polarized cells were characterized by flow cytometry, stimulated with lps and response assessed by characterization of cytokine profiles via cytometric bead array (cba). results: culture of monocytes in the presence of mcsf or gmcsf induced two different phenotypes. cells cultured in the presence of gmcsf represented the m type and were cd negative but cd and mhcii positive and produced high levels of il- , tnfalpha and il- following lps stimulation. culture in the presence of mcsf resulted in induction of the m phenotype. these cells were cd positive with intermediate expression of cd and mhcii expression and produced high levels of il- , il- and il- following lps stimulation. interestingly, already baseline il- production was high in these cells. stimulation with leptin alone increased cytokine production in both cell types as compared to cells cultured in medium alone. however, if leptin was present in cultures stimulated with lps, the induction of cytokine production was significantly reduced in both, m -and m -polarized cells as compared to cells stimulated with lps alone. summary: whereas presence of leptin enhances baseline cytokine production in polarized macrophages, it reduces the cytokine production in response to stimulation with the tlr ligand lps. thus, abundant leptin levels like present in obesity or in the hypertrophied fat as present in crohn's disease patients might exert modulating effects on macrophage response to bacterial antigens. methods: hl cell line was used as a model of leukemic myeloid cell differentiation cultured in suspension or on fibronectin matrix prior to pma ( ng/ml) treatment for h. morphological evaluation was performed with conventional microscopy and electron microscopy. immunephenotype and phagocytic activity of the cells were determined by flow cytometry and immunocytochemistry. a colorimetric nitro-blue-tetrazolium reduction assay was performed to assess the production of reactive oxygen species (ros). results : besides their distinctive macrophage morphology and ultrastructure with spindle cell-like features and high granularity, the pma-treated fibronectinadherent hl cells expressed antigen receptors cd , tlr , tlr and cd , and displayed enhanced phagocytic activity and production of ros. expression of cd , cd and cd was also maintained however the cells were hla-dr and cd a negative. conclusion: we describe the enhanced ability of fibronectin-adherent hl cells to differentiate into macrophages in response to pma. hl may provide a functional model for macrophage differentiation. above all, this finding may stimulate further research on myeloid leukemia biology and potential adjuvant therapies. a. aporta , n. ferrer , a. gómez , j. gonzalo , a. arbués , a. anel , c. martín , j. pardo , apoptosis, immunity and cancer university of zaragoza, molecular and cellular biochemistry and biology, zaragoza, spain, university of zaragoza, mycobacterium genetics, zaragoza, spain mycobacterium tuberculosis is an intracellular pathogen that uses alveolar macrophages as its preferred habitat, being capable of produce both a progressive disease and an asymptomatic latent infection. it has been postulated that infected macrophage apoptosis may contribute to host defence against this intracellular infection by, firstly, eliminating supportive environment for bacterial growth and, secondly, by leading to the formation and release of apoptotic vesicles containing mycobacterial antigens. it has been proposed that m. tuberculosis inhibits host cell apoptosis thus interfering with the immune system response. however the biological relevance of this process is not clear. our group has generated so , a m. tuberculosis phop mutant strain that was shown (perez et al ) to be more attenuated than the present attenuated vaccine strain bcg and conferred protective immunity against m. tuberculosis infection in mice and guinea pigs (martin et al ) . in the present study, we compare the time course and phenotype of cell death induced by so , bcg and wild type m. tuberculosis on the murine macrophage cell line j and on bone marrow derived mouse macrophages. our results indicate that wild type m. tuberculosis induces macrophage cell death analysed by a clonogeneic assay much faster than the attenuated bacteria. of note cell death presented apoptotic features like caspase- activity and nuclear condensation. in order to analyse the consequences of this apoptotis-like cell death, it has been invetigated whether dead cells translocate phosphatydilserine to the outer part of the plasma membrane and if this traslocation is enough to promote phagocytosis by fresh macrophages. experiments are ongoing with macrophages derived from trl x deficient mice and wt animals in order to study the role and implication of those receptor on the susceptibility to infection and death induced by the virulent and attenuated phop m. tuberculosis strain. objectives: vip is a potent anti-inflammatory peptide, mainly acting as endogenous macrophage deactivating factor. type receptor for vip (vipr ) gene is highly conserved through species and, in humans, is highly polymorphic. vipr has been reported to be down-modulated in cells of the immune system after activation. an association of some snps with some autoimmune diseases has also been reported. in this study we have investigated the correlation between these snps and gene expression in monocytes exposed to lps. methods: monocytes from blood donors were separated from pbmc and stimulated with lps. total rna was reverse transcribed and the level of vipr in untreated or lps-stimulated monocytes was measured by real-time rt-pcr and protein expression. protein level was measured by western blot and densitometric analysis. the kinetic of expression of vipr after , , and h of exposure to lps was firstly analysed in monocytes from five individuals. there were two kinetics: one in which a reasonable high levels ( g %) of mrna was maintained trough time and a second one in which the decrease of mrna was pronounced. the experiments were repeated using monocytes from donors that had been typed for the relevant vipr snps. the down-regulation of vipr correlates with the presence of a t at rs mapping in the '-end of the gene (p= . ). the vipr protein level was decreased about % in monocytes of subjects typed as t/t at rs whereas subjects typed as c/c at rs maintained a high level of expression after h of lps treatment. the data show that different haplotypes of the vipr gene correlate with a different kinetics of gene expression in activated monocytes. a possible consequence of these data is that the anti-inflammatory properties of vip governed by the vipr vary in different individuals and can eventually contribute to the genetic predisposition to some autoimmune diseases. j. oujezdska , t. vavrochova , d. filipp , immunobiology institute of molecular genetics as cr, immunobiology, prague, czech republic phagocytes which appear in early mouse development (e . - . ) represent a unique embryonic macrophage lineage that differs from adult macrophages phenotypically, biochemically and by their origin. recent studies suggested that there are at least three waves of macrophages populating an early embryo: a maternallyderived one and two waves of extraembryonal, ys-derived phagocytes. in addition, the occurence of early embryonic phagocytes of undetermined origin in the anterior head mesoderm in several invertebrate and vertebrate species is well documented. this origin-related heterogeneity among early embryonic phagocyte subpopulations coupled with the lack of their specific surface markers makes it difficult to distinguish them phenotypically and study their potentially distinct physiological roles in early development. the aim of this study is to identify a set of surface markers expressed on embryonal phagocytes suitable for phenotypic distinction among embryonic phagocyte subpopulations. here we report the temporal and spatial expression of toll-like receptors (tlrs) and cd in the early mouse embryo (me). facs analysis of cell suspension prepared from . day me showed that about . - % of cells were positive for cd b. these cells exclusively were also positive for cd , tlr , and cd antigens. using qpcr and flow cytometry we show that tlrs and other tir domain-containing signaling molecules are expressed in the embryo through embryonic days , - , . reciprocal matings between wild type and mhcii-egfp knock-in mice revealed that while maternallyderived mhcii + macrophages are present in the embryo from early developmental stages (e , ), embryo-derived mhcii + macrophages start to appear in the embryo around day . multicolor facs analysis of cd b, cd , cd , f / , tlr , tlr , c-kit and mhcii surface markers revealed differential expression of tlr and c-kit on embryonal phagocyte subpopulations. moreover, the microarray analysis of cd b + tlr + cells isolated from the e , embryos has revealed significantly upregulated expression of several novel genes in comparison to their expression in murine peritoneal macrophages. these molecules are currently being tested for their use as embryonic phagocyte specific-lineage markers. these results are first to characterize the regulated expression of tlrs on early embryonal phagocytes and demonstate their potential to serve as novel markers for their detection and isolation. humans may be exposed to a variety of mycobacteria ranging from environmental or bcg vaccine to more pathogenic mycobacteria. only a minority of individuals exposed develop disease, this susceptibility may result in part from variability of host immune responses genes through simple (mendelien disease) and complex (polymorphisms with milder effect) inheritance mechanisms. interestingly, key elements of inflammatory pathways are particularly involved in this susceptibility to mycobacteria. il /il -dependent ifng pathway of macrophage activation plays a central role in inflammation and cell-mediated immune responses to mycobacteria. due to the high rate of consanguineous marriages in the north african countries, recessive genetic disorders including primary immunodeficiencies occur with a relatively high prevalence. in tunisia, among patients affected with primary immunodeficiencies presented with disseminated bcg infection (bcg-osis). among them, five have an underlying well-defined primary immunodeficiency either a severe combined immunodeficiency or a chronic granulomatous disease and have a mendelien susceptibility to mycobacterial disease. using a candidate gene strategy, we have identified in out of these patients mutations in several ifng pathway genes, other candidate genes are being investigated for the other patients. in the general population, common polymorphisms with milder effect on the risk of tuberculosis have been identified including mhc and nramp . we did focus on the study of genes which are considered as important pathogen recognition receptors of the innate immune system: tlr is the principal mediator of macrophage activation in response to mycobacteria through nfkb pro-inflammatory signaling pathway and dc-sign is the major receptor of m. tuberculosis on human dendritic cells and in contrast induces anti-inflammatory il- cytokine. using a case/household-contact cohort we did investigate polymorphisms of these genes in tunisian patients affected with active pulmonary tuberculosis and have shown specific patterns of snp and microsatellite polymorphisms associated with susceptibility/resistance to tuberculosis. host inflammatory responses play a major role in granuloma formation and control of the infection. unraveling these pathways might be crucial in order to identify new therapeutic targets and strategies including immunotherapy e. g. ifng therapy for tuberculosis, particularly in this era of emergence of multi-drug and extensively-drug resistant m. tuberculosis strains. francisella tularensis is a gram negative bacterium that is the causative agent of tularemia. research into francisella has expanded over recent years due to its designation as a potential biological warfare agent. several species of francisella exist and have varying degrees of pathogenicity. f. tularensis live vaccine strain (lvs) is an attenuated strain of the holarctica subspecies and has been shown to be an effective vaccine in humans. however, it is pathogenic in mice which can, therefore, act as a useful model of human tularemia. f. tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. therefore, if phagocytosis could be disrupted via the addition of inhibitors, uptake of f. tularensis would decrease and antibiotic treatment may be more effective. a flow cytometric assay was developed to measure bacterial uptake. this method used a fitc labelled anti-f. tularensis antibody in conjunction with antibodies to cell surface markers to determine specific cell phenotypes that were positive for bacteria. a series of phagocytic inhibitors have been tested in vitro on an alveolar macrophage derived cell line (mhs) and on ex-vivo mouse lung tissue to determine whether uptake of f. tularensis lvs could be altered. the presented data shows that several inhibitors work efficiently to reduce lvs uptake by up to - % in both the in vitro and ex vivo assays. however, cytotoxicity of some of the inhibitors was high and, therefore, it was essential to concentrate on inhibitors with low cytotoxicity for further assessment. in addition, bacteriological data suggests that the combination of inhibitors with antibiotics may be a useful therapeutic against f. tularensis. it may also work against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.ã crown copyright. dstl, . hsp are intracellular proteins but it is known that these proteins can be expressed on cell surface and contained in extracellular medium, in particular in peripheral blood serum. it is also known that extracellular hsp have pronounced immunomodulatory properties. to study the pathways of the protein modulating action on immune system we investigated effect of exogenous and cell surface hsp on reactive oxygen species (ros) release from phagocytes, namely human neutrophils, during process of phagocytosis (respiratory burst). neutrophils were isolated from human peripheral blood by using a standard protocol. respiratory burst induced by opsonized zymosan was measured by method of luminol dependent chemiluminescence. for the experiments human recombinant hsp (low endotoxin) and paraformaldehyde fixed mouse thymocytes exposed surface hsp were used. exogenous hsp was used in concentration - ug/ml, fixed thymocytes were added to neutrophil samples in quantitative ratio : and : directly before the measuring. as the control we registered amplitude of oxidative burst in samples supplemented with pbs or live mouse thymocytes having no hsp on their surface. results demonstrating effect of exogenous hsp on phagocytosis-induced ros release from human peripheral blood neutrophils have been obtained. it was demonstrated marked dose-dependent inhibiting action of exogenous hsp on amplitude of respiratory burst. the cells expressing surface hsp impacted on ros production in this model similarly. the results of chemiluminescence analysis demonstrated that zymosan induced ros production was essentially decreased under action of fixed thymocytes, and was decreased slightly in presence of live thymocytes in the neutrophil samples. the effect was more pronounced for increased amount of thymocytes added to the samples. thus, immunomodulatory effects of exogenous hsp might be caused by influence of the protein on ros release from phagocytes. we suppose that the registered effects are connected with ability of hsp to inhibit activity of nadp-oxidase -the key enzyme for ros production during respiratory burst. results: we recruited pts, with so far five complete pathological remission, five partial responses and five no responses. no substantial changes were detectable in the number of circulating monocytes. in contrast we observed a clear expansion of cd /cd and cd /cd double positive subsets. this event was transient; it abated at the later time point suggesting a causal relationship to the treatment. it correlated with sensitivity to the treatment. in fact we observed that in the responder patients the expansion of the cd / subset was clear in the first weeks of treatment and decreased there after. in contrast in non-responder patients it was already expanded before the neo-adjuvant therapy. all the patients had an initial expansion of the cd / subset. in the responder patients this population was still present at the time of surgery. the immunohistochemical study revealed a massive tumoral infiltration by macrophages that displayed clear features of alternative m polarization. conclusion: these data suggest that neo-adjuvant therapy modulates the cellular components of innate immune responses that could represent valuable predictive factors. m. dimitrijević , i. pilipović , s. stanojević , k. mitić , k. radojević , v. pešić , g. leposavić , institute of virology, vaccines and sera "torlak", immunology research centre "branislav janković", belgrade, serbia, faculty of pharmacy, university of belgrade, department of physiology, belgrade, serbia the primary aim of our current study was to ascertain whether rat resident peritoneal macrophages synthesized catecholamines and to unmask putative effects of catecholamines on nitric oxide (no) and hydrogen peroxide (h o ) production and phagocytic activity of these cells. in addition, given that chronic administration of b-adrenoceptor antagonist increases the density of b-adrenoceptors on both non-immune and immune cells and thereby affects their sensitivity to catecholamine action, we hypothesized that such treatment could also affect macrophage responsiveness. to address our proposition, we determined adrenoceptor expression on peritoneal macrophages from rats subjected to -day-long propranolol treatment and measured both no and h o production and phagocytic activity of these cells. using both immunocytochemical and flow cytometric analyses of rat peritoneal exudate cells constitutive expression of tyrosine hydroxylase and both b -and a -adrenoceptors on macrophages was revealed. furthermore, according to the characteristic assemblage of tyrosine hydroxylase and adrenoceptor subtype expression different macrophage subsets were identified. in vitro treatment of macrophages with the non-selective a,b-adrenoceptor agonist arterenol and/or the b-adrenoceptor antagonist propranolol indicated that b-adrenoceptors potentiated no production and suggested a-adrenoceptor-mediated suppression of hydrogen peroxide h o production. an increase in h o production in the presence of the a -adrenoceptor antagonist ebrantil provided support for this. chronic propranolol treatment in vivo led to increased no and h o production by peritoneal macrophages. furthermore, this treatment resulted in opposing effects on the expression of b -and a -adrenoceptors on peritoneal macrophages (a stimulatory effect on b -adrenoceptors and a suppressive effect on a -adrenoceptors). in conclusion, a subset of resident peritoneal macrophages synthesizes catecholamines, which may exert differential effects on h objectives: monocytes display great phenotypical and functional heterogeneity and are divided into two major subsets: cd ++ cd -('classical') and cd + cd + ('pro-inflammatory') monocytes. a central monocyte function is cytokine production in response to toll-like receptor (tlr) ligation. the cd + cd + monocytes display higher tlr and - expression, produce higher levels of pro-inflammatory cytokines and have increased potency for antigen presentation than the cd ++ cd monocytes, suggesting that the two subsets could play different roles in antimicrobial responses. newborns are vulnerable to infections and an immaturity of both adaptive and innate immunity has been described. studies of neonatal monocyte antimicrobial responses show contrasting results and much remains to be learned, especially regarding monocyte subpopulations. thus we aimed to compare monocytes from newborns and adults, focusing on monocyte subpopulations and responses following tlr stimulation. methods: cord blood (n= ) and peripheral-blood (n= ) mononuclear cells were stimulated in vitro for hrs with peptidoglycan and subsequently analysed for cd and cd and intracellular il- p and tnf expression. the mann-whitney u-test was used to evaluate differences between groups. results: a significantly higher percentage of neonatal monocytes were positive for il- p , both unstimulated and after peptidoglycan stimulation, as compared to adults. geomfi of il- p was low and similar between groups, although significantly higher in newborns after stimulation. in both newborns and adults, il- p (% positive cells and geomfi) was significantly higher for cd + cd + cells than for cd ++ cd cells, unstimulated and stimulated. regarding tnf, neonatal and adult monocytes did not differ in unstimulated cultures, however geomfi of tnf was significantly higher in neonatal monocytes after stimulation. whereas the tnf response following stimulation was similar between the adult monocyte subsets, in newborns the cd ++ cd cells were positive for tnf to a significantly higher extent than the cd + cd + cells. in particular the tnf response to tlr stimulation differed between newborns and adults, with neonatal monocytes having a higher per cell production of the cytokine. notably, in newborns the cd ++ cd monocytes were positive to a higher extent for tnf following stimulation pointing towards a functional immaturity of neonatal monocyte subset responses. objective: chronic granulomatous disease (cgd) is an uncommon congenital phagocyte disorder characterized by recurrent life-threatening infections. cgd generally present with recurrent suppurative infections; however, intracranial fungal abscess complicating cgd may cause a diagnostic problem to anyone who is unfamiliar with its clinical and radiological features. we report a -year-old boy who admitted with complaints of seizures during the previous months. there was a history of axillary and perianal suppurative skin infections and cavitary pneumonia. the family history was unremarkable, and the parents were unconsanguineous. physical examination was only remarkable for oral moniliasis and skin scars at axillary and perianal region. a large frontol mass with diffuse peripheral vasogenic edema was discovered on mri. subfalcine herniation was noted secondary to mass effect. cgd was suspected and the analysis with flow cytometric dihydrorhodamine assay (dhr assay), for functional analysis of neutrophils was compatible with the diagnosis of cgd and no bimodal histogram pattern spesific for x-cgd was found in the mother and sister. after the diagnosis of cgd, neurosurgical removal of the abscess cavity was performed due to peri-lesional edema and herniation risk. aspergillus fumigates grew from the culture; liposomal amphotericin b and voriconazole were started; which were found to be sensitive to the cultured species. in addition, interferon-g ( mgr/m /day, subcutaneously every other day) was started. after months, control mri showed regression of the lesion, and the anti-fungal treatment was continued for months. the screening of the other family members with dhr assay demonstrated that one of his sisters had also cgd and phenotype was autosomal recessive. mutaton analysis in "hot spot" in ncf gene concerns the well-known gt deletion in the second exon of ncf gene both at the patient and his sister. results: this was an atypical clinical presentation of cgd in an adolescent boy with cerebral aspergillosis, mimicking intra-cranial tumor. we documented a good response to the combination of ifn-g, liposomal amphotericin b and voriconazole after surgery. conclusion: cgd should be considered in the differential diagnosis for all children presenting with invasive fungal infections particularly, those involving the central nervous system. recent data suggest that ubiquitin has anti-inflammatory properties and therapeutic potential after severe trauma and brain injuries. therefore, we hypothesized that ubiquitin treatment can modulate the local inflammatory response triggered after brain injury. to test this hypothesis, a focal cortical contusion was induced using a controlled cortical impact (cci) model in sprague-dawley rats. animals (n = ) subjected to moderate brain injury were randomized, and received either . mg/kg ubiquitin or vehicle (placebo) intravenously within min after cci. levels of tnf-a, il- b, il- , il- and il- receptor antagonist were analyzed in brain tissue using real time rt-pcr at and hours after treatment. immune cell infiltration was studied by immunostaining for neutrophils and macrophages/ microglia at h and days. data were analyzed with the mann-whitney u test and a two-tailed p x . was considered significant. all cytokines were highly up-regulated hours after cci but no differences between the groups were observed at this time point. three days after trauma the levels of il- were significantly lower in the ubiquitin treated animals, whereas the levels of il- and tnf-a were higher when compared to the placebo group. interestingly, macrophages/ activated microglia were significantly increased in the pericontusional cortex after ubiquitin treatment at day . the infiltration of neutropils was not affected by ubiquitin treatment. here, we could demonstrate for the first time that a single injection of ubiquitin immediately after brain trauma is able to modulate the inflammatory response triggered after brain injury at the cellular as well as at the cytokine level. macrophage activation and oxidative metabolic changes are commonly implicated in pulmonary tuberculosis (ptb) patients. efficient plasma antioxidant activities are needed to neutralize high free radical load in pulmonary tuberculosis (ptb) patients. there is limited information about the plasma levels of neopterin (a marker of macrophage activation) and oxidative stress indices such as total plasma peroxide (tpp), total antioxidant activity (taa), malondialdehyde (mda), and oxidative stress index (osi) in ptb patients during chemotherapy with or without micronutrient supplementation. the present study was designed to assess the levels of neopterin, tpp, taa, mda, and osi during chemotherapy with (c+m) or without (c-m) micronutrient supplementation using elisa and spectrophotometric methods. thirty-eight ( ) newly diagnosed ptb patients and forty non-ptb apparently healthy subjects volunteered to participate in this study. twenty of the ptb patients were on anti-tuberculosis drugs supplemented with micronutrients (c+m) while were treated with anti-tuberculosis drug alone (c-m) for a period of four weeks. the levels of neopterin (p= . ), tpp (p= . ), osi (p = . ), mda (p = . ) were significantly raised but taa (p = . ) was significantly reduced in ptb patients compared with controls. the levels of mda (p = . ), neopterin (p= . ) and tpp (p= . ) were significantly reduced in c+m after two weeks of treatment compared with baseline values before commencement of treatment. the levels of tpp (p= . ), mda (p= . ), neopterin (p= . ), osi (p= . ) were significantly reduced while taa (p= . ) was significantly raised in c+m after weeks of treatment compared with the baseline concentrations. in c-m, only mda showed significant decreased after weeks of treatment when compared with the baseline values. plasma level of neopterin, tpp, osi and mda declined faster in c+m than c-m. therefore, micronutrient supplementation of ptb drugs with synthetic antioxidants or naturally occurring ones (fruits and vegetables) should be attempted. this will improve deranged macrophage activation and reduce oxidative stress indices in ptb patients. a. p. aguas , e.m. cunha , m.j. oliveira icbas, university of porto, anatomy, porto, portugal the acute in vivo intake of mercury (hg) microparticles ( nm in diameter) by neutrophils and macrophages was studied with the use of in situ detection of hg by scanning electron microscopy coupled with x-ray elemental microanalysis (sem-xem). the intracellular distribution of hg particles was compared, at high resolution, between macrophages and neutrophils, and between activated and non-activated phagocytes. balb/c mice were injected intraperitoneally (ip) or in a subcutaneous air-pouch with mercury chloride, and the animals were sacrificed up to minutes after the injection. in some mice, before the hg injection, peritoneal phagocytes were activacted by ip injection of bsa. pre-injections with a selenium (se) salt were also performed in order to study the putative modulatory role of se on hg intake by phagocytes. peritoneal cells were collected by washing of the peritoneal or subcutaneous cavities with pbs, they were cytospinned, fixed with formaldehyde, and processed for observation by sem-xem. five min after the hg injection more than half of the mouse phagocytes were positive for hg. a higher percentage ( %) of macrophages contained the metal particles than neutrophils ( %). phagocyte activation enhanced the number of hg particles seen inside the phagocytes. pre-injection of the peritoneal cavity of mice with se resulted in finding that more than half of the hg intracellular particles were coupled with se. subcellular topography of hg particles showed that they were presented in individual small cytoplasmic vesicles. we conclude that hg microparticles are rapidly ingested by macrophages and neutrophils, a processed that is enhanced by cell activation. hg particles are ingested by pinocytosis and sorted in the cytoplasm of macrophages and neutrophils inside individual small vesicles. this study was supported by a grant from fct, portugal. mast cells play central roles in allergic inflammatory reactions and innate immunity. swap- is a rac-interacting protein expressed in several cells types of the hematopoietic system including mast cells. in b cells and mast cells swap- regulates f-actin cytoskeletal rearrangements, cell polarisation and cell migration. (pearce et al., ; sivalenka and jessberger, ) . swap- -/-bone marrow derived mast cells (bmmc) are specifically impaired in fceri-mediated activation and degranulation and in c-kit-induced activation, migration and cell adhesion (gross et al., ; sivalenka and jessberger, ; sivalenka et al., ) . crucial regulators of these processes are members of the rho family of small gtpases such as rac and rhoa. swap- interacts with rac in vitro and preferentially binds the active gtp-bound rac . swap- supports the increase of active rac in vitro by a yet to be defined mechanism (shinohara et al., ) . in this study, in vitro pull-down assays with purified recombinant proteins were employed to characterize the interaction between swap- and rac . it was found that fulllength swap- preferentially binds to constitutively active rac (rac q l) but not to its dominant negative form (rac t n). binding assays with swap- truncated mutants showed interaction of swap- 's n-terminus with gtpgs rac or rac depleted of guanine nucleotide, whereas swap- central or c-terminal regions do not bind to any form of rac . preliminary competitive-binding assays with overlapping mer peptides, spanning the entire swap- sequence, mapped the rac binding site near the n-terminus of swap- . full-length swap- site-specific mutants will be generated to test the relevance of these interactions in mast cells in terms of adhesion, migration and activation of rho gtpases. elucidating the molecular interactions of swap- with rho gtpases and the relevance of these will shed light on the biology and biochemistry of mast cells and possibly other hematopoietic cells, which express swap- . v. c. barbosa , c. d. polli , m.c. roque-barreira , m.c. jamur , c. oliver , g. pereira-da-silva mast cells are essential cells in ige-associated immune responses. fceri crosslinking induces mast cell degranulation and release of proinflammatory mediators. we have previously shown that the lectin artinm induces mast cell activation but the mechanisms involved in this activity remain unknown. objective: the present study was undertaken to further characterize the ability of artinm to activate mast cells. methods: rbl- h cells were sensitized with ige anti-tnp and stimulated with dnp -hsa or artinm. artinm binding to rbl- h cells was assessed by flow cytometry. mast cell degranulation was determined by measurements of released b-hexosaminidase activity. microplate binding assays were utilized to assess artinm binding to ige. to investigate fceri recognition by the lectin, western blots of cell lysates were stained with biotinylated artinm and be's antibodies specific for fceri b-subunit. intracellular protein phosphorylation was detected by specific antibodies and analyzed by confocal microscopy. mcp- and tgf-b levels released by mast cells were measured by elisa. results: artinm binding to the cell surface was dependent on sugar recognition and resulted in mast cell degranulation in the presence or absence of ige. the release of b-hexosaminidase doubled when cells were sensitized by the immunoglobulin and was abrogated in the presence of d-mannose, suggesting that mast cell degranulation induced by artinm might be the result of interactions between the lectin crds and glycosylated components on the cell surface, like fceri or ige. indeed, it was observed that the lectin bound to ige in a dose-dependent manner and recognized the fceri b subunit in western blot analysis. exposure to artinm resulted also in phosphorylation of intracellular proteins, mcp- release and tgf-b production. significant increases in these activities were observed upon sensitization with ige. conclusions: these results suggest that artinm may bind to glycans of the high affinity ige receptor and/or of the ige (bound to fceri) and that such interactions would be implicated in its ability to activate and degranulate mast cells. in view of the well-established significance of mast cells in allergic inflammation, the participation of sugars as binding receptors on mast cell surface opens new ways of controlling allergic disorders. the adhesion receptor l-selectin is a key player of the innate immune response in the process of leukocyte migration from the blood stream to inflamed tissue. it is expressed on leukocytes and promotes the initial contact to the endothelium resulting in steady rolling and eventually diapedesis. a distinct feature is the exclusive presentation of l-selectin on the tip of finger-like cell membrane protrusions called microvilli which cover the entire leukocyte surface. this topography was shown to facilitate the first transient interactions of the free flowing cell to the static counterreceptor particularly in the context of high dynamic shear. other adhesion molecules such as p-selectin glycoprotein ligand (psgl- ), b and b -integrins also share this special phenotype. taken together, prominent adhesion receptor positioning reflects a widespread biological principle contributing to inflammation as well as hematogenic tumor metastasis. despite the functional relevance and frequent occurrence, however, molecular mechanisms of cell surface receptor compartmentalization remain largely unknown. in this study we identified the highly conserved transmembrane domain of l-selectin to regulate microvillus receptor positioning and adhesion under flow. taking advantage of the inverse surface expression pattern of cd (cell body) compared to l-selectin (microvilli) in a myeloid cell line, we investigated domain swapped chimeric receptors regarding their substructural surface localization and their ability to initiate rolling under flow. transmission electron microscopy showed a crucial impact of the transmembrane domain to target the chimeric receptors to a certain cell surface compartment independent of the intracellular anchorage. in turn, the receptor shift from microvilli to the cell body goes along with a substantial decrease of rolling cells in an in vitro parallel flow chamber assay. thus, contrary to the common view of single membrane spanning domains to simply act as a mechanical anchor, our results attach an important functional component as well and might point out a new general principle for targeting receptors to specific membrane compartments. objectives: macrophages are one of the principal effector cells involved in the innate immunity response. they kill microbes through phagocytosis and upon activation, secrete pro-inflammatory cytokines such as il- b, il- and tnf-a. herpes simplex virus (hsv- ) is an enveloped dna virus that infects mostly oral mucosa and sensory neurons. innate immunity responses activated by hsv infection consist of: activation of macrophages; activation of the complement cascade, and production and secretion of a variety of cytokines and chemokines. il- and tnf-a are cytokines produced by macrophages that contain known anti-hsv properties. the objective of this study was to characterise the secretome of human primary macrophages infected with hsv . methods: human monocytes were purified from the peripheral blood mononuclear cells of healthy blood donors and differentiated in vitro into macrophages. macrophages were left untreated or primed with poly(i:c) ( ug/ml), a mimetic of double-stranded rna, after which cells were left uninfected or infected with hsv- for h. after this, cell culture supernatants were collected, concentrated and proteins purified. the secreted proteins were digested into peptides, identified and quantified using itraq (isotope tagged relative and absolute quantitation) -labelling of the peptides followed by peptide fractionation by cation exchange chromatography and analysis by nanolc-ms/ms. the raw ms/ms data was analysed using proteinpilot . software. results: in the first itraq experiment over human proteins were identified in the hsv infected cell supernatants. from these proteins had at least fold increase after poly(i:c) + hsv infection compared to the uninfected cells. hsv infected cells had clearly more proteins in their cell supernatants after infection compared to the uninfected cells: itraq labelling showed a total of . fold increase in the protein amount in the poly(i:c) + hsv infected cell supernatant and a . fold increase in the hsv infected cell supernatant when compared to the uninfected cell supernatant. amongst the upregulated proteins there were known inflammatory proteins: chemokine (c-x-c motif) ligand , il- , tnf-a induced protein , complement factor b, galectin- and mxa. at present, further experiments are on-going for more detailed analysis of the hsv infected macrophage secretome. h. p. prakash , german cancer research centre, translational immunology, heidelberg, germany, max planck institute for infection biology, molecular biology, berlin, germany chlamydophila pneumoniae are the major etiological factors for worldwide pneumonia, chd and copd. chlamydia lives and multiplies inside their host epithelial cells where they confer resistance for apoptosis by inducing expression and stability of anti-apoptotic proteins called inhibitor of apoptosis proteins (iaps). the significance of cellular inhibitor of apoptosis protein- (ciap- ) and x-linked inhibitor of apoptosis proteins ( xiap) in chlamydia pneumoniae pulmonary infection and innate immune response of macrophages was investigated in ciap- and xiap knockout (ko) mice using a novel non-invasive intra-tracheal infection method. in contrast to wildtype, iap knockout mice failed to clear the infection from their lung. wildtype mice responded to infection with a strong inflammatory response in the lung. in contrast, the recruitment of monocytes and macrophages was reduced in iap ko mice compared to wildtype mice. the concentration of interferon gamma (ifn-g) was increased whereastumor necrosis factor (tnfa) was dysregulated in the lungs of infected iap ko mice compared to infected wildtype mice. ex vivo experiments on mouse peritoneal macrophages and splenocytes revealed that iaps are required for innate immune responses of these cells. our findings thus suggest a new immunoregulatory role of iaps in c.pneumonaie pulmaonry infections. methods: human monocytes were purified from venous blood of normal volunteers by ficoll density gradient centrifugation. hrgal- ( mg/ml) binding to monocytes, in the presence or absence of mm lactose or sacarose, was assessed by flow cytometry and confocal microscopy. in transwell systems, assays were performed using hrgal , laminin or fibronectin immobilized or not on the filters. these were added to wells containing soluble hrgal or rpmi and monocytes ( x ) were added into each insert. when necessary, hrgal was pre-incubated with mm lactose or sacarose. mcp- ( ng/ml) was used as positive control. we observed that hrgal- binds to the surface of human monocytes through its crd, since this interaction can be inhibited by lactose. we corroborated some data of literature that hrgal- is able to induce monocyte migration in a dose-dependent manner, resulting in a bell-shaped curve as seem with other known attractants. when we evaluated the participation of the ecm laminin and fibronectin in monocyte migration induced by hrgal- , we observed that the association between these glycoproteins and hrgal- resulted in a % increase in the number of migrating cells. both n-and c-terminal domains of hrgal- are involved in the association between laminin or fibronectin and hrgal- , since the presence of lactose resulted in % and % inhibition of monocyte migration induced by the lectin, respectively conclusions: our results showed that hrgal- induces monocyte migration by haptotaxis, through the interactions established between both n-and c-terminal domains of the lectin and ecm glycoproteins, laminin and fibronectin. in a vertebrate embryo, macrophages develop in two sites (yolk sac and liver) and constitute the primary mechanism of host defense. their phagocytic function may be required during the earliest stages of development both for survival and for organogenesis. recent studies have shown that monocyte heterogeneity is conserved in humans and mice. the different monocyte subsets seem to reflect developmental stages with distinct physiological roles but nothing is known whether the macrophage diversity arises in early ontogeny. in order to study the ontogeny of the monocyte-macrophage lineage, we developed a new culture technique using human embryonic stem cells (hesc).culturing of embryoid bodies for weeks in the presence of bmp ,vegf and a mixture of hematopoietic cytokines resulted in a generation of a significant cell population of cd +cd + cells. the sorted cd +cd + cells were further cultured for - days in the presence of m-csf and gave rise to a homogenous population of adherent mature macrophages. embryonic stem cells derived macrophages were identified by several criteria including morphology and ultrastructural features observed by microscopy and by expression of nonspecific esterase and myeloperoxidase by histochemical staining. while virtually all embryonic-derived macrophages expressed the lps-receptor cd , m-csf receptor cd and the scavenger-receptor cd , we characterized two distinct subpopulations of macrophage based on their difference in size and density and the expression of the cd and cd (fcgammariii) : the cd lowcd -and cd + cd +. trancscriptional, phenotypic and functional assays suggest the alternative (m ) polarization of cd +cd + embryonic stem cell-derived macrophages.(anti-inflammatory cytokines secretion, active phagocytosis, m -related gene expression).the exact chemokine receptor expression pattern, phenotype and transcriptional activity of their foetal counterparts are currently under investigation. collectively, our data provide insight into alternative macrophage polarization in humans and and adds further data to the growing body of evidence that establishment of macrophage heterogeneity is related to early ontogeny. b.-s. choi , p. kropf imperial college london, immunology department, london, united kingdom the balance between t helper (th) and th cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized th or th responses are not sufficient to account for healing or nonhealing. we have recently shown that arginase-induced l-arginine depletion results in local suppression of antigen-specific t cell responses in nonhealing leishmaniasis. healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. moreover, supplementation with l-arginine restored t cell effector functions and resulted in reduced lesions size and parasite load. however, despite the efficient production of ifn-g by cd + t cells at the site of infection and despite the reduced pathology, the mice did not heal. we hypothesised that arginase-expressing macrophages contribute to persistent disease and become refractory to ifn-g mediated signals. to test this hypothesis, we used a well-defined model of bone marrow derived macrophages and determined whether the differentiation state of parasitized arginase-expressing macrophages could be altered. in addition, we also tested whether alternatively activated macrophages can be induced to switch off arginase and upregulate inducible nitric oxide synthase (inos) to kill the intracellular parasites. vg vd t lymphocyte are activated following recognition of non-peptidic phosphorylated metabolites. the phosphoantigen isopentenyl pyrophosphate (ipp) is overproduced by tumors following hyperactivation of the mevalonate pathway of isoprenoid synthesis. previous work has shown that a molecular complex homologous to mitochondrial atp synthase (ecto-f -atpase) is expressed on many cell types and is a possible specific ligand for the vg vd tcr. the present study aims at understanding the role of f -atpase in antigen regognition. using video microscopy calcium imaging in single vg vd t lymphocytes, we can now show that the t cell response to ipp requires contact with bystander cells of variable tissue origin but that this requirement is not fulfilled by a cell line deprived of surface f -atpase. purified f -atpase immobilized on polystyrene beads can partly replace the need for cell-cell contact. ipp in soluble form is highly sensitive to terminal phosphatases and addition of these enzymes in t cell activation assays clearly shows that it is not recognized as such on tumors. however, we could detect nucleotide derivatives of phosphoantigens which are resistant to terminal phosphatases in the cell lysates of stimulatory tumors. one of these, a derivative of ipp, is barely able to stimulate vg vd cells in the absence of apcs, as opposed to the non-nucleotidic antigen ipp. however it can bind stably to f -atpase. thus the f -atpase complex acts as a presenting structure for nucleotide phosphoantigens. altogether, our data suggest that vg vd t cells are dedicated to the recognition of phosphoantigens in the form of nucleotide derivatives, on the surface of tissue cells and that antigen recognition involves multiple antigen modification steps, in including final cleavage by a nucleotide pyrophosphatase activity. surface plasmon resonance was used to analyse the molecular interaction between tcr and f -atpase. by using purified f -atpase and peptides derived from vg vd tcr sequences, interaction sites between f -atpase and tcr were identified on both ligands. based on these findings a generalized model for vg vd t cell activation is proposed. ligands for the cytotoxic lymphocyte activating receptor nkg d are highly expressed on cells stressed by numerous agents including genotoxic damage, thereby contributing to the elimination of transformed cells by nkg d(+) lymphocytes. a key question is whether this represents a primary inductive means of immune surveillance, or merely enhances responses initiated by dendritic cells and antigen-specific t cells. a second key issue is the scope and scale of events that follow nkg d activation in vivo. by transiently overexpressing the nkg d ligand rae- -beta in the skin of transgenic mice, we showed that this alone provoked rapid, coincident and reversible changes in the organization, morphology and activation state of tissue-resident vgamma vdelta gamma-delta t cells and langerhans cells (lc), that were swiftly followed by epithelial infiltration of unconventional alpha-beta t cells. these data indicate a novel primary immune surveillance pathway whereby epithelial upregulation of nkg d ligands is sufficient to provoke a series of multicomponent immunological changes. the effects on lc, which lack nkg d and presumably respond to changes initiated by local gamma-delta t cells, are particularly interesting. ongoing microarray and co-culture experiments are now providing a molecular definition of the immume surveillance response to nkg d ligands in vivo. to assess the scope of this response, ovalbumin was applied to the skin concomitant with rae induction. the primary systemic th response is increased by concomitant responses to a stress antigen. we will now resolve whether this increased response contributes to the adaptive memory pool, or whether it is a primary, regulatory response that may limit adaptive responses to auto-antigens exposed during stress. in addition, the many ligands available to the nkg d receptor suggest that different ones may play unique roles. a novel nkg d-ligand, h c, is uniquely expressed in mouse skin. when the expression of this was further increased in a novel transgenic system, there was again an overt alteration in the local immune compartment, but with features that are seemingly distinct from the action of rae- induction. such studies may help resolve a long-standing puzzle over the pleiotropy of nkg d ligands, and dissect immune surveillance of changes in gene expression levels rather than absolute levels. a.-s. invariant natural killer t (inkt) cells are a distinct lineage of t lymphocytes that co-express a highly conserved ab t cell receptor (tcr) along with typical surface receptors for natural killer (nk) cells. these lymphocytes recognize glycolipid antigens presented by the non-classical class i molecule cd d. inkt cells are characterized by their capacity to produce rapidly large amounts of both th (ifn-g, tnf) and th (il- , il- ) cytokines, which enables them to play a role in the regulation of many different types of immune responses, ranging from self-tolerance to responses against pathogens and tumors. converging studies in mouse models suggest that inkt cells can prevent the development of type diabetes. the frequency of inkt cells is lower in non-obese diabetic mice (nod mice). manipulation of inkt cells, either by increasing their frequency or by stimulating them with agonists such as a-galcer, inhibits diabetes onset in nod mice. recently, a new population of cd -nk . -inkt cells producing high levels of the pro-inflammatory cytokine il- has been identified (inkt cells). given that this cytokine has been implicated in several pathologies including autoimmune diseases, we investigated the role of inkt cells in type diabetes. interestingly, nod mice exhibit a higher frequency of inkt cells producing il- as compared to c bl/ mice. this increased frequency was observed in the thymus as well as in peripheral lymphoid tissues. as previously described in normal mice, inkt cells present in nod mice were mainly cd -nk . -, express the ror-g transcription factor and il- receptor, both molecules being usually associated with th commitment. we are currently analyzing, using co-transfer experiments, whether these inkt cells play a beneficial, a deleterious, or any role in the development of type diabetes in nod mice. j. s. dodd , r. muir , s.s. affendi , p.j. openshaw imperial college london, respiratory medicine, london, united kingdom natural killer t (nkt) cells are a heterogeneous population of innate t cells that have attracted interest because of their potential to regulate immune responses to a variety of pathogens. upon activation with their cognate glycolipid antigen presented by cd d molecules, activated nkt cells produce copious and numerous cytokines which endow these cells with potent immunoregulatory properties. consequently, nkt cells have become the focus for the development of vaccine adjuvants, cancer immunotherapeutics and modulators for autoimmune and inflammatory conditions. respiratory syncytial virus (rsv) is a common cold virus of the family paramyxoviridae. it is the most frequent viral cause of serious lower respiratory tract infection in infants and children worldwide and a significant contributor to winter deaths in the elderly. despite its global impact, there is still no safe and effective vaccine and our understanding of the immunological mechanisms that regulate protection and pathology is incomplete. it is known that cd d-deficient mice with poor nkt cell responses have inefficient induction of cd t cells and reduced clearance of rsv, perhaps because of ifn-g release by activated nkt cells. we now show that activation of lung nkt cells with intranasal agalcer during rsv infection of mice boosts th immunity (increasing il- and il- ), promoting pulmonary eosinophilia and ablating cd t cell recruitment. by contrast, intraperitonal injection of agalcer enhances nk cell recruitment and boosts pulmonary cd t cell activity (as measured by cd expression), increasing ifn-g production in the airway and lung and inhibiting viral replication. effects on illness (as measured by weight loss) were similarly distinct: intranasal agalcer induced early (d ) weight loss independent of conventional t cells, whereas intraperitonal agalcer enhanced late (d ) weight loss by a cd t cell dependent mechanism. therefore, nkt cells stimulated by agalcer administered via different routes induce distinct types of immune response to viral infection in the lung with the intraperitonal route leading to optimal viral clearance. in general, neonatal conventional t cells, especially cd + ab t cells, are regarded as immature or t h biased. vg + vd + t cells are unconventional lymphocytes: they are mhc-unrestricted and can react rapidly upon activation with pyrophosphates (e. g. (e)- -hydroxy- -methyl-but- -enyl pyrophosphate (hmb-pp)) or aminobisphosphonates (e. g. zoledronate) in adults. until now, little is known on the functional reactivity of neonatal vg + vd + t cells towards these activators. because il- is preferentially secreted by neonatal dendritic cells (dc) upon tlr stimulation, we investigated the potential costimulatory effect of this cytokine on hmb-pp and zoledronate-treated neonatal vg + vd + t cells. herein, we observed that zoledronate induced neonatal vg + vd + t cell proliferation and ifn-g production in cord blood mononuclear cells (cbmc) cultures. other t h -like cytokines like tnf-a and gm-csf were also produced upon this stimulation, but less than ifn-g, while t h -like cytokines such as il- and il- were not induced. addition of il- to zoledronate selectively costimulated ifn-g production from neonatal vg + vd + t cells. furthermore, zoledronate/il- treatment resulted in neonatal vg + vd + t cells expressing high levels of the cytotoxic mediators perforin and granzyme a. zoledronate induced the expression of the receptor for il- (il- r) and the transcription factor t-bet, which is known to be important for the production of ifn-g in gd t cells. in addition, costimulation with il- resulted in a further increase of t-bet expression in neonatal vg + vd + t cells. these changes in the expression of il- r and t-bet likely contribute to the observed selective ifn-g response towards zoledronate/il- treatment. of note, in contrast to adult peripheral blood vg + vd + t cells, hmb-pp had no or only a minor effect on the functional reactivity of neonatal vg + vd + t cells. altogether, these observations show that neonatal vg + vd + t cells are functionally active and that this t cell population might play a role in protective immune responses to infections with intracellular pathogens in early life, in particular when dc-derived il- is produced in response to microbial stimuli. the evasion of antigen presentation is a feature common to herpesviruses. one of the strategies employed to inhibit antigen presenting molecules is ubiquitination, internalisation and lysosomal breakdown by viral e ligases such as hhv encoded k , k or mhv encoded mk . these viral genes represent homologues of the march family of cellular genes whose function is the regulation of cell-surface antigen presentation and reduction of the lifetime of loaded antigen complexes. ubiquitination targets surface molecules to the lysosome via the multivesicular body (mvb), a structure which also has an important role in the budding of many viruses. we investigated the existence of alternative fates for antigen presenting molecules post-ubiquitination, and how viral e ligases manipulate them. we discovered that both the cellular march and viral e ligases ubiquitinate cd molecules. however, whereas viral molecules inhibit cd -antigen presentation, the march molecules are essential for the recirculation and function of the long-lived and lysosome-resistant cd molecules. in contrast mhc class ii was only targeted by cellular and not by viral e ligases. furthermore cd molecules could be found in viral particles as a result of ubiquitination, presumably via the mvb. thus, virally expressed and cellular e ligases have opposite effects, despite their homology. how this is achieved is a matter of active investigation. gamma delta (gd) t cells recognize stress-induced auto-antigens and contribute to immunity against infections and cancer. our previous study revealed that vd negative ( neg ) gd t lymphocytes isolated from transplant recipients infected by cytomegalovirus (cmv) killed both cmv-infected cells and ht colon cancer cells in vitro. in order to investigate the anti-tumor effects of vd neg clones in vivo, we generated hypodermal ht tumors in immunodeficient mice. concomitant injections of vd neg clones, in contrast to vd + cells, prevented the development of ht tumors. vd neg clones expressed chemokine c-c motif receptor (ccr ) and migrated in vitro in response to chemokines secreted by ht cells, among which were the ccr ligands macrophage inflammatory protein (mip)- d and monocyte chemoattractant protein (mcp)- . more importantly, a systemic intraperitoneal (i. p.) treatment with vd neg clones delayed the growth of ht subcutaneous (s. c.) tumors. the effect of in vivo gd t cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-ccr antibody. gd t cell passive immunotherapy was dependent upon the cytotoxic activity of the gd effectors towards their targets since vd neg clones were not able to inhibit the growth of a hypodermal tumors. our findings suggest that cmv-specific vd neg cells could target in vivo cancer cells, making them an attractive candidate for anti-tumor immunotherapy. more recently, we generated ht cells expressing the luciferase and realized orthotopic injection of ht -luc cells. progressive tumor development and regression following « gd treatment » will be observed in vivo using bioluminescent imaging. intraepithelial lymphocytes (iel) compose large, oligoclonal, tissue-associated repertoires of non-mhc-restricted t cells that play key roles in immunosurveillance. it is commonly considered that the characteristic iel repertoires are positively selected by thymic epithelial molecules that are also stress-induced in specific tissues, thereby activating iel function. however, no such molecules have been identified. here we characterise skint , currently the only known determinant of a canonical iel compartment, that is selectively required for vg vd + dendritic epidermal t cell (detc) development. we show that both peripheral and thymic skint expression is essential for full detc development. its effects are highly specific since even substantial and ubiquitous over-expression neither negatively selects detc, nor affects any other t cells. unexpectedly, however, skint genes are not expressed by cell lines and are downregulated rather than activated by carcinogenesis. mouse genetic models allow powerful insight into skint function; for example, we demonstrate that the constitutive expression of wild-type skint fully restores detc development in a skint mutant mouse, but does not rescue normal detc function. thus, skint provides a novel perspective into how epithelia regulate the development and function of specific tissue-associated t cell compartments, and how normal versus dysregulated tissues may be demarcated. marginal zone (mz) b cells are strategically localized in the mz of the spleen. since most of the blood reaching the spleen is passing through this region such localization favors contact with blood born antigens and pathogens. besides being able to rapidly secrete antibodies, mz b cells may also act as professional antigen presenting cells (apcs). they are known to express high levels of cd d which is the presenting molecule for nkt cells which are also located in the mz. therefore we hypothesised that mz b cells may be efficient activators of nkt cells. to test this hypothesis, we used freshly sorted splenic mz b cells (cd + cd hi cd lo cd c -) and splenic conventional dendritic cells (cdcs) (cd c hi cd a +/-cd b +/-b -) from wt and cd d -/mice as apcs for nkt cells from va -ja transgenic or wt mice. the apcs were treated with agalactosylceramide (agalcer) or heat killed (hk) listeria monocytogenes or salmonella typhimurium. both mz b cells and cdcs proved to be highly efficient apcs for priming of nkt cells and induced robust proliferation. in contrast, other populations of b cells failed to activate nkt cells. we showed, using cd d -/mice as well as blocking antibodies to icosl, that proliferation of nkt cells depends on tcr/cd d and in case of mz b cells, also on icos/icosl interactions. importantly, apcs primed with hk bacteria were not able to induce nkt cell proliferation. interestingly, mz b cells exclusively induced production of il- by nkt cells. in contrast, cdcs mostly induced production of ifn-g and il- producing cells were scarce under these conditions. cytokine production by nkt cells proved to be independent of tcr signalling, but dependent on icos/icosl interactions when mz b cells were used as apcs, and gitr-dependent when cdcs were used. taken together, our data suggest that both mz b cells as well as cdc act as professional apcs for nkt cells. notably, the nature of apcs appears to be critical for polarization of the immune response: mz b-cell-primed nkt cells induce cytokine milieu fostering a t h response, whereas cdc-primed nkt cells rather favor a t h response. objectives: il- is an innate cytokine present in elevated levels in sera from patients suffering from autoimmunity (eg. sle and ra) and the allergic disease atopic eczema. in mice, injections of il- give rise to an early polyclonal isotype switched antibody response which is absent in inkt cell deficient (cd d -/-) mice. we set out to investigate the activated b cells in il- injected mice and how these are regulated by inkt cells. methods: mice received daily i. p. injections of il- ( mg) for days and the antibody response in serum was monitored using elisa. the b cell activation in the spleen at day was evaluated by flow cytometry and immunohistology. results: mice injected with il- developed self reactive (anti-pc and anti-dna) antibodies in the serum, in line with the autoreactive antibodies in patients with e. g. sle and atopic eczema. the antibody producing cells formed cd + cell clusters in the red pulp of the spleen, a typical feature of extrafollicular activation frequently associated with autoreactive responses. surprisingly, the antibody response induced by il- was increased in inkt cell deficient (cd d -/-) mice, in contrast to published data. an increased response to il- was also observed in ja -/mice, which lack the a-chain of the tcr used by inkt cells, and thus our data suggest that inkt cells inhibit antibody producing cells in il- induced antibody responses. further characterization of the recruitment of b cells in il- injected mice revealed a marked expansion of the marginal zone b cell (mzb) population in the spleen, suggesting an important role for mzbs in the il- induced autoreactive antibody response. mzbs are innate-type b cells that express high levels of cd d, are prone to autoantibody production and often involved in early immune responses. the il- induced antibody response in mzb deficient (cd -/-) mice was either decreased (igg) or delayed (ige), supporting the importance of mzbs in il- induced antibody responses. we conclude that the role for inkt cells in il- induced antibody responses is to inhibit the production of autoreactive antibodes from mzbs in extrafollicular foci. objectives: amoebiasis is a widespread human parasitic disease caused by the intestinal protozoan entamoeba histolytica. there are two major clinical manifestations of the disease, amoebic colitis and amoebic liver abscess (ala). interestingly, only a small proportion of e. histolytica-infected individuals develop invasive disease, whereas the majority harbors the parasite within the gut without clinical symptoms. so far, cells of the innate immune system have been described to constitute the main host defense mechanism for the control of amoebiasis, relying largely on the early production of interferon-g (ifn-g). however, information is lacking about the sources of early ifn-g production as well as the amoeba antigens involved in this activation process. methods: using a recently developed c bl/ mouse model for ala, the contribution of natural killer t (nkt) cells for protection against amoebic disease was investigated. applying nkt cells and dendritic cells as antigen-presenting cells from various ko-mice, the signaling pathways implicated in recognition of amoebic antigens and activation of cytokine-secretion by nkt cells was analysed. results: nkt cells were found to play a key role in the defense against ala. specific activation of nkt cells by a-galactosylceramide (a-galcer) induced significant protection, whereas jalpha -/-and cd d-/-mice lacking inkt as well as dnkt cells suffered from more severe abscess formation. a lipopeptidophosphoglycan, which is present in large quantities on the surfcae of e. histolytica trophozoites (ehlppg), was identified as a major amoeba antigen that activates nkt cells resulting in the production of ifn-g, but not of il- . moreover, ifn-g production required the presentation of ehlppg by cd d and signaling through the tlr receptor cascade in combination with a simultaneous secretion of il- . similar to a-galcer application, treatment of mice with purified ehlppg significantly reduced the severity of ala in amoeba-infected mice. our study provides a mechanism for the innate control of amoeba invasion that might explain why the majority of e. histolytica-infected individuals do not develop amoebic disease. a few years ago, we have observed a significant expansion of circulating effector gamma delta t cells following cytomegalovirus (cmv) infection in kidney transplant recipients (ktr). these unconventional t cells display tcr dependent cytotoxicity against both cmv-infected cells and carcinoma cells. in the present study, an extensive phenotyping of gamma-delta t cells allowed us to demonstrate an over-expression of cd in cmv-infected individuals. cd is the fcgammariiia, a natural killer cell marker usually absent on conventional t cells. we found that . ± . % of gamma-delta t cells from cmv-infected ktr expressed cd , when compared with only . ± . % in non cmv-infected ktr (p x . ). similarly, . ± . % of gamma-delta t cells from cmv-seropositive blood donors expressed cd compared to . ± . % in cmv-seronegative donors (p x . ). cd + gamma-delta t cell lines generated from cmv-infected individuals were able to produce ifn-g (a potent anti-viral cytokine) in a cd -dependent manner when activated by cmv/igg immune complexes. this production greatly increased in the presence of il- and ifn-alpha, two cytokines highly produced during cmv-infection. the supernatants of gamma-delta t cells activated with agonist anti-cd mab inhibited cmv replication in vitro and this effect was abrogated in the presence of a blocking anti-ifn-g antibody. cmv/igg immune complexes were also able to induce the expression of the cytotoxicity marker cd a on cd + gamma-delta t cell lines. cd is well-known to mediate antibody-dependant cellular cytotoxicity (adcc), especially in natural killer cells. accordingly, we demonstrated that cd + gamma delta t cell lines could make adcc against the daudi lymphoma cell line and the a skin carcinoma cell line pre-incubated either with rituximab (anti-cd ) or cetuximab (anti-egfr), respectively. in contrast, no addc could be observed against cmv-infected fibroblasts pre-incubated with polyclonal anti-cmv igg (cytogam), probably because cytogam weakly stained infected cells. these data reveal a new cd -dependent anti-cmv function of gamma-delta t cells through recognition of immune complexes and secretion of ifng. moreover, they demonstrate that these cells are able to kill through adcc lymphoma and skin carcinoma cells, two tumour types frequently encountered in ktr. dendritic epidermal t cells are a prototypic population of intraepithelial gd t cells in the mouse skin. found in the basal layer of epidermis and in close contact with langerhan's cells and keratinocytes detc facilitate vital immunological and physiological processes e. g. wound healing, homeostasis, tumor surveillance and regulation of inflammation. gd t cells respond rapidly to non-peptidic microbial and stress induced self antigens in a non-mhc restricted manner and are therefore proposed to bridge the gap between innate and adaptive immunity. by using gd t cell knock-out mice tcrd-/-, ovalbumin transgenic k mova mice and a skin grafting model we aimed to elucidate the role of gd-detc in adaptive immune responses associated with elimination of foreign antigen presented in the skin.we show that in the absence of gd t cells in the skin there is a decrease in rejection of ovalbumin expressing skin grafts compared to wildtype mice. we show that optimal regimens of antigen delivered subcutaneously in conjunction with adjuvant elicits comparable responses in wildtype and knockout mice. however frequency of primed host animals is reduced in tcrd-/-mice when antigen is delivered epidermally via skin grafting; suggesting detc enhance cross presentation of classical mhc bound antigens in the skin. considering the incapability of gd t cells to recognize peptide antigens in the context of mhc we plan to dissect the relationship between detc and professional antigen presenting cells in the skin. understanding the underlying mechanisms of this relationship will expand our knowledge of enhancing professional apc function in skin by detc and potentially other epithelia by intraepithelial gd t cells and can be useful in designing therapies to epithelial infections and malignancies. we demonstrate a rapid and hmb-pp-dependent crosstalk between gd t cells and autologous monocytes that resulted in the production of inflammatory mediators including il- , ifn-g, tnf-a, osm, ccl , cxcl , cxcl , and trail. moreover, under these co-culture conditions monocytes showed enhanced survival and differentiated overnight into inflammatory dcs with antigen-presenting functions. these cells expressed cd , cd , hla-dr, and dc-sign, and lost cd , ccr , ccr , and cxcr . addition of further microbial stimuli (lps, peptidoglycan) induced ccr and enabled these inflammatory dcs to trigger antigenspecific cd + effector ab t cells expressing ifn-g and/or il- . importantly, our in vitro model replicated the responsiveness to microbes of effluent cells from pd patients and translated directly to episodes of acute pd-associated bacterial peritonitis, where vg /vd t cell numbers and soluble inflammatory mediators were elevated in patients infected with hmb-pp-producing pathogens. conclusion: our findings suggest a direct link between invading pathogens, microbe-responsive gd t cells, and monocytes in the inflammatory infiltrate, which plays a crucial role in the early response and the generation of microbe-specific immunity. the mechanism(s) responsible for their dichotomous behaviour are poorly understood, and the outcome of nkt cell manipulation remains unpredictable. there is growing evidence that the nkt cell pool is composed of functionally distinct subsets, but such a possibility has not yet been investigated in a model of nkt cellmediated immunosuppression. we examined the differential ability of nkt cell subsets from the thymus and liver to prevent type i diabetes when transferred into prediabetic nod mice. the transfer of abtcr+dn thymocytes (a population enriched for nkt cells) has previously provided robust protection against tid development; however it has not been formally shown that nkt cells are solely responsible for the protection. our study found that while the transfer of thymic dn nkt cells can prevent tid and severe insulitis in nod mice, not all nkt cell subsets show the same tolerogenic capabilities. these findings both formally demonstrate the disease-preventing effects of nkt cell transfer in nod mice and provide further evidence that nkt cells are a functionally heterogeneous population. objective: vg /vd t cells constitute a minor t cell population in human blood that expands specifically and rapidly in response to the microbial metabolite hmb-pp. our previous microarray studies showed that vg /vd t cells stimulated with hmb-pp in the presence of il- express markers associated with a possible follicular b cell helper function. we therefore investigated in more detail whether and how hmb-pp and il- regulate expression of the b cell attracting chemokine cxcl /bca- , its receptor cxcr , and co-stimulatory molecules involved in b cell help. purified peripheral vg /vd t cells were co-cultured with autologous monocytes or b cells (as feeder cells) for up to days with and without hmb-pp, in the absence or presence of il- or il- , or in medium alone. cells were analysed by flow cytometry and immunofluorescence microscopy. results: high levels of cxcl protein were detected in co-culture supernatants only when both il- and hmb-pp were provided, implying an il- -dependent and tcr-dependent expression. vg /vd t cells were confirmed as producers of cxcl by flow cytometry and immunofluorescence. under the same conditions, activated vg /vd t cells expressed cd , cd , cd l, cd , icos and ox . in contrast, neither cxcr nor ccr changed markedly by il- stimulation of peripheral vg /vd t cells. conclusion: our findings confirm on the protein level that stimulation of vg /vd t cells with hmb-pp and il- induces markers typically associated with follicular b helper t (t fh ) cells. these data suggest that gd t cells contribute to humoral immune responses and play a role in germinal centre formation and production of high-affinity antibodies in microbial infection. ongoing analyses of gd t cells in inflamed and non-inflamed lymphoid tissues (tonsils, appendices) aim at demonstrating the physiological relevance of our findings. y. emoto , m. emoto gunma university school of health sciences, department of laboratory sciences, maebashi, japan invariant (i) natural killer (nk)t cells become undetectable after stimulation with a-galactosylceramide (a-galcer) or interleukin (il)- . although downmodulation of surface t cell receptor (tcr)/nkr-p c (nk . ) expression has been shown convincingly after a-galcer stimulation, it is unclear whether this holds true for il- stimulation. to determine whether failure to detect inkt cells after il- stimulation is caused by dissociation/internalization of tcr and/or nkr-p c or by block of de-novo synthesis of these molecules, and to examine the role of il- in disappearance of inkt cells after a-galcer stimulation, surface (s)/ cytoplasmic (c) protein expression as well as mrna expression of tcr/nkr-p c by inkt cells after stimulation with a-galcer or il- , and influence of il- neutralization on down-modulation of stcr/snkr-p c expression by inkt cells after a-galcer stimulation were examined. the s/ctcr + s/cnkr-p c + inkt cells became undetectable after in-vivo administration of a-galcer, which was partially prevented by il- neutralization. whereas s/cnkr-p c + inkt cells became undetectable after in-vivo administration of il- , s/ctcr + inkt cells were only marginally affected. mrna expression of tcr/nkr-p c remained unaffected by a-galcer or il- treatment, despite the down-modulation of ctcr and/or cnkr-p c protein expression. in contrast, ctcr + cnkr-p c + stcr -snkr-p c -inkt cells and cnkr-p c + snkr-p c -inkt cells were detectable after in-vitro stimulation with a-galcer and il- , respectively. our results indicate that tcr and nkr-p c expression by inkt cells is differentially regulated by signaling through tcr and il- r. they also suggest that il- participates, in part, in the disappearance of inkt cells after a-galcer stimulation by down-modulating not only snkr-p c but also stcr. the fetus and infant are highly susceptible to viral infections. a number of viruses, including human cytomegalovirus (cmv), cause more severe disease in early life compared to later life. it is generally accepted that this higher susceptibility to viral infections is due to the immaturity of the immune system. gd t cells are unconventional t cells that can react rapidly upon activation and show mhc-unrestricted activity. herein, we show that upon cmv infection in utero, fetal gd t cells expand and become differentiated. the response was restricted to vg -gd t cells, irrespective of their vd chain expression. differentiated gd t cells expressed high levels of ifn-g, transcription factors t-bet and eomes, natural killer receptors and cytotoxic mediators including perforin and granzymes. in addition, congenital cmv-infection induced a highly restricted complementary-determining region d (cdr d ) and cdr d repertoire, with a striking enrichment in a specific germline-encoded cdr d sequence. differentiated gd t cells and the enriched cdr d sequence were detected as early as after weeks of gestation. our results indicate that functional fetal gd t cell responses can be generated during development in utero and suggest that this t cell subset could participate in anti-viral defense in early life. results: spectratyping showed only in-frame selection for vd -jd and vgi-jg . / . rearrangements in tcrgd thymocytes and to a lesser extent in tcrgd cb cells. in contrast, clear in-frame vd -jd and vg -jg . selection was seen in pb tcrgd cells. detailed analysis of the cdr motifs revealed selection determinants in both vg -jg . (canonical length and cdr motif) and vd -jd (minimal cdr length in combination with an invariant t nucleotide) rearrangements. upon evaluation of the replication history we found a clear increase in the number of cell divisions from naïve tcrgd thymocytes (˚ ) and tcrgd cb cells ( - ) to tcrgd pb cells (˚ or more). no increase was seen between cb and pb tcrgd t cells within the first year of life, suggesting that peripheral proliferation occurs later in life. our results indicate that the human peripheral tcrgd repertoire is shaped by (antigenic) selection and proliferation processes. moreover, the ontogenetic changes in the gd repertoire between the central and peripheral immune systems are clearly influenced by proliferation. background: natural killer (nk) t cells have been implied in the regulation of disease in the non obese diabetic (nod) mouse model of type diabetes (t d). we have previously shown that transgenic expression of a cd d-restricted, va . -vb tcr in nod mice lead to an increase in cd d-restricted type ii nkt cells ( abnkt cells), and prevention of the development of t d in the transgenic mice. in this study we have investigated the requirements and underlying mechanism of disease protection by type ii nkt cells in a disease transfer model. to investigate the mode of regulation by abnkt cells, we explored a disease transfer model into nod.scid mice using transgenic diabetogenic bdc . cd + t cells, in the presence or absence of selected cells from abnkt cell transgenic mice. results: in ab transgenic mice a high frequency of activated transgenic nkt cells was found in the pancreas of the protected mice. in this organ, abnkt cells expressed a high level of cxcr and a low level of ccr and cd l, a pattern similar to that observed in t cells homing to inflammatory tissues. adoptive transfer of cd + bdc . t cells into nod.scid recipients rapidly induced onset of diabetes. using this model, we found that co-transfer of spleen cells from ab transgenic mice with bdc . cd + cells resulted in the prevention of diabetes development. the protection from disease required a minor cd + subset of ab+ nkt cells, but was independent of cd + t regulatory cells. analogs of alpha galactosylceramide (a-galcer) that may modulate the strong activation of inkt and at the same time prolong their effect upon in vivo administration are a long standing goal of research in this area due to their putative immunotherapeutical applications. a new class of non glycosidic analogues bearing an aminocyclitol ring as galactose surrogate have been synthesized and assayed in their capacity to be presented by cd d and recognized by inkt. the structural novelty of these compounds resides in the presence of a cyclohexane that substitutes the sugar moeity and the substitution of the o glycosidic linkeage with the ceramide by a n. in this basic structure, substitutions in the cyclohexane ring with oh in different conformations mimicking different sugars, differences in the length of the sphingosine lipid and differences in the orientation of the n linkeage conform a series of analogs that have been analyzed in their capacity to stimulate inkt cells. proliferation assays in bulk splenocyte cultures and cytokine secretion determinations show that inkt cells are specifically stimulated by some of the analogs tested. in particular, the active compound hs , induces in vitro inkt cell expansion and ifng and il- secretion in a similar fashion but less potently than a-galcer. dose response assays show a bias towards a th profile response after recognition by nkt cells, more similar to the response induced by och. the degree of structural similarity of the cyclitol ceramides with a-galcer parallels their cellular activities. these data open the way towards the development of a new class of a-galcer lipid analogues having charged amino substituted polar heads resistant to glycosidase degradation, thus enhancing their in vivo biodisponibility, and expands the range of potential inkt cell sphingolipid agonists that can modulate the immune response due nkt cell activation. objectives: invariant natural killer (ink) t cells represent an innate lymphocyte subset with important modulatory functions. in the presence of pathogens or tumors, inkt cells play an adjuvant function that boosts t cell immunity through cytokine secretion and dc maturation. in steady-state conditions, i.e. in the absence of pathogens, inkt cells acquire a regulatory function that promotes t cell tolerance and prevents autoimmune disease. our aim was to assess the mechanism of action of inkt cells in the steady state and, specifically, to test the hypothesis that inkt cells promote immune tolerance through modulation of dcs. methods: to assess the direct influence of regulatory inkt cells on dc maturation in resting conditions, we derived murine inkt cell lines in vitro and, after staining with agalcer-loaded cd d tetramers and magnetic purification, we tested their capacity to modulate bone marrow-derived myeloid dcs in the absence of any other maturation signals. we analyze the transcriptional profile (microarray analysis) as well as maturation, cytokine expression profile and pro-tolerogenic antigen-presenting function of inkt cell-modulated dcs (inkt-dcs). the cell-cell interaction with inkt cells provoked dramatic phenotypical changes on immature dcs that acquired the cardinal features of tolerogenic dcs such as intermediate levels of mhc class ii and co-stimulatory molecules expression and high secretion of il- with no release of pro-inflammatory cytokines. most importantly, inkt-dcs acquired tolerogenic antigen-presenting function inducing the differentiation of regulatory tr cells and immune tolerance in vivo. dcs, simultaneously stimulated with inkt cells and through toll-like receptor (lps) completely lost the pro-tolerogenic phenotype and acquired a proinflammatory cytokine profile. conclusion: it is still mysterious how inkt cells can play a dual role and either boost t cell immunity or promote immune tolerance. our results suggest that the same mechanism could underlie both inkt cell functions. in the presence of pathogen-driven maturation signals, the inkt cell-modulation of dcs favors their acquisition of a pro-inflammatory phenotype and function. on the contrary, if inkt cells are activated in the absence of pathogens, e. g. during autoimmune conditions, their interaction with immature dcs promotes their tolerogenic maturation to maintain peripheral tolerance and counter-regulate autoimmune diseases. th -type immune responses have been reported to fight extracellular bacterial infection, but as well to cause autoimmune diseases and allergy. the th immune response is characterized by the secretion of il- a and il- f. the il- locus encodes the highly conserved il- a and il- f cytokines that are syntenic in kb distance to each other. besides cd + th and nkt cells, approximately % of the il a producers are gd t-cells. like cd + th cells, il- producing gd tcells have recently been implicated to play a major role in the immune response to infections with extra-and intracellular bacteria. our findings show a difference between the il- production of gd t cells in the peripheral system and mucosal epithelia. mucosal gd t-cells generally do not produce th cytokines. in the periphery, we define novel subsets of gd t-cells that can produce either il- or ifn-g. combined with the well known classification of il- producing gd t-cells along the markers cd and cd , our data point at specialized functions of the different gd t cell subsets depending on their location and origin. functional studies are currently carried out in order to address the role of the different gd t-cell subsets for th -type immune responses in vivo. in this context, the potential redundancy of il- a and il- f may complicate the analysis. so far, most studies were carried out with il- a single-deficient or il- f single-deficient mice. to further clarify these issues, we will have to address the above mentioned findings in il- a and il- f double-deficient mice. several subsets of gd tregs have been described and intensively studied, but the potential regulatory role of innate t cells in controlling immune responses remains unclear. lymphocytes expressing gd tcr are involved in both innate and adaptive immune responses. vg vgd t cells, which represent a major peripheral blood gd t-lymphocyte subpopulation in humans, display a broad reactivity against microbial agents and tumors.here we report that tgf-b and il- differentiate in vitro a subset of gd t lymphocytes with regulatory functions (vd tregs) in the presence of specific antigen stimulation. these cells express the forkhead/winged helix transcription factor (foxp ) and, similarly to ab tregs, suppress the proliferation of anti-cd /anti-cd stimulated-pbmc. detailed knowledge about the phenotype and functionality of vd tregs will improve our understanding of the role of gd t cells in the pathogenesis and regulation of autoimmune, infectious and cancer diseases. a-galactosylceramide (a-galcer) has the potential to activate invariant (i) nkt cells, which in turn release a wide variety of cytokines that stimulate immunocompetent cells. although this rapid and vigorous cytokine release appears critical for regulation of various immune responses, it remains elusive whether protection against intracellular bacteria can be induced by a-galcer. here we show that treatment with a-galcer ameliorates murine listeriosis, and inhibits inflammation in the liver and spleen following listeria monocytogenes infection. liver infiltration of granulocytes and g/d t cells was accelerated by a-galcer treatment. granulocyte and g/d t cell depletion exacerbated listeriosis in a-galcer-treated mice, and this effect was more pronounced in granulocyte than in g/d t cell depletion. although secretion of gm-csf and il- was detected among the nkt cell population in the liver and bone marrow immediately after a-galcer treatment, infiltration of granulocytes into the liver was not prevented by neutralizing mab. yet, in parallel to the numerical increase of granulocytes expressing cd b in the liver following a-galcer treatment, numbers of cells lacking cd b diminished in the bone marrow. in addition, respiratory burst in granulocytes was enhanced by a-galcer treatment. our results indicate that a-galcer-induced antibacterial immunity is caused, in part, by accelerated infiltration of inflammatory cells, in particular granulocytes and to a lesser degree g/d t cells, into the liver. we also suggest that the infiltration of granulocytes is caused by an accelerated supply of granulocytes from the bone marrow, rather than by accelerated granulopoiesis. objectives: the aim of this work is to evaluate whether phenotypic and functional features of vgamma /vdelta t cells are influenced by the activity of mevalonate pathway in tumor cells and contribute to determine disease aggressiveness in cll. methods: eighty seven previously untreated cll patients were evaluated for in vitro vgamma /vdelta t cells expansion upon stimulation with zoledronic acid (za) and interleukin- (il- ). gammadelta t cells subset distribution and natural killer receptors profile were evaluated by multicolor flowcytometry. the mutational status of the tumor immunoglobulin heavy chain variable region (igvh) was analyzed by dna sequencing. the activity of the mev pathway was determined by ) the bioinformatic analysis of gene expression profiling data ) the quantification of mev pathway metabolites. results: proliferation of gammadelta t cells was observed in patients ( %) (responders, r), whereas patients ( %) were non-responders (nr). vgamma /vdelta t-cell subset distribution was well balanced in r patients, whereas effectors subsets [i. e., effector memory (tem), and terminally differentiated effector memory (temra)] were largely predominant in nr patients. temra of nr patients mainly expressed the inhibitory receptor ilt , whereas temra of r patients had an higher expression of the costimulatory molecule nkg d. the proliferative response of vgamma /vdelta t cells was significantly associated with igvh mutational status, which is a well known prognostic factor in cll. indeed, % of r patients were m, whereas % of um patients were nr (p x . ). given this association, we evaluated the activity of the mev pathway in tumor cells of m and um patients. the pathway was more active in tumor cells of um than m patients, suggesting that the former can more easily engage gammadelta t cells and drive their differentiation into functionally exhausted t emra . given the association between the r/nr status and the igvh mutational status we also analyzed the independent prognostic impact of r/nr status in multivariate cox analysis. nr patients had a significantly shorter time to first treatment thus pointing to r/nr status as an independent prognostic factor. conclusion: these data define a novel mechanism of immune escape which can contribute to determine disease aggressiveness in cll patients. the studies reported here were undertaken to ascertain and delineate the ability of kupffer cells to regulate the response of inkt cells to biliary obstruction. methods: c bl/ mice were not treated or rendered kupffer cell-depleted by intravenous inoculation of liposome-encapsulated dichloromethylene diphosphonate. to clarify the factors that elicit inkt cell activity, additional mice were administered anti-il- p (clone r - f ; atcc) or anti-cd- d (clone b ) monoclonal antibody (mab) prior to surgery. midline laparotomies were performed; the common bile duct was ligated twice and divided. sham-operated animals served as controls. blood and liver samples were collected at periodic intervals post-surgery. the hepatic lymphoid population was purified and characterized by flow cytometry. the nkt cell population was increased significantly in the livers of control, but not kupffer cell-depleted, mice at hours post-bdl. the response of inkt cells was diminished in mice pretreated with mab specific for il- p , a component of both il- and il- ; pretreatment with anti-cd d mab had no effect. il- rb-deficient mice also exhibited a marked increase in hepatic inkt cells following bdl suggesting that il- was not a critical factor. this suggestion is supported by the increased expression of il- p and il- p (but not il- p ) mrnas by kupffer cells purified from the livers of bdl animals. these findings imply that il- production by kupffer cells promotes the response of hepatic inkt cells to biliary obstruction. objectives: p-glycoprotein (pgp or abcb ) is a member of the abc family of transporter proteins which are characterized by their ability to pump molecules across membranes in an atp-dependent manner. although pgp was first identified for its ability to confer resistance to chemotherapeutic agents in tumor cells, it has now also been described in cells of the immune system. our work primarily focuses on gd t cells that complement and regulate the activities of ab t cells, particularly in tissues. we have recently described functional subsets of gd cells based on cd expression. gd + cells secrete interferon-g, while gd cells are capable of producing il- . this study investigates the role of pgp in gd cells with specific reference to these recently-identified cd -defined subsets. methods: pgp activity was measured based on the expulsion of rhodamine . cells were incubated with rho followed by a period in the presence or absence of the pgp inhibitor cyclosporine-a. cell populations were identified using monoclonal antibodies and flow cytometry. percentages of subpopulations were compared by anova, statistical results are shown as p values that were calculated using a newman-keuls multiple comparison post-hoc test. results: up to % of intraepithelial lymphocytes (iels) from the small intestine are tcrgd + . of these, virtually all displayed pgp activity. indeed, pgp activity was generally higher in tcrgd + than tcrab + iels. in the thymus, pgp activity was observed in only˚ % of gd + cells but not at all in gd cells. by contrast, in peripheral lymph nodes, mesenteric lymph nodes and peyer's patches, - % of gd + cells were positive for pgp activity, although their gd counterparts remained largely negative (p x . ). conclusion: this study demonstrates that subsets of gd cells display different levels of pgp activity depending on their location in the body and their expression of the newly identified functional marker cd . as pgp activity may play a role in cytokine release, cytotoxicity and protection from harmful toxins, it confirms our hypothesis that gd + and gd cells have very different roles in immune responses and provides insight into the mechanism by which gd cells cope with diverse body locations. objectives: an effective immune response orchestrates different cellular activities of both innate and adaptive immune compartments. in this context, the vgamma vdelta t cell biology presents some critical features for their ability to display a broad antimicrobial activity by directly killing infected cells and by inducing an effective adaptive immune response. the activation of vgamma vdelta t cells by aminobisphosphonate drugs such as zoledronic acid (zol) results in a massive release of cytokines and chemokines that may induce a bystander activation of other immune cells such as dendritic cells (dcs) and b lymphocytes. the aim of this work was to evaluate the ability of activated vgamma vdelta t lymphocytes to orchestrate granulocytes functions in terms of migration capability, phagocytic activity and alpha defensin release. methods: peripheral mononuclear cells (pbmc) and purified vgamma vdelta t cells from healthy donors were stimulated with different compounds (zol, ipp) for hours and supernatants from these cultures were tested for their ability to induce granulocytes activation. briefly, we analysed the migration activity, the phagocytic activity and the degranulation process by perforimg migration assays, flow cytometry and elisa tests. we showed that soluble factors released by zol-stimulated vgamma vdelta t cells activate granulocytes by inducing their chemotaxis, phagocytosis, and alpha-defensins release. proteomic analysis allowed us to identify a number of cytokines and chemokines specifically released by activated vgamma vdelta t cells. moreover, mcp- depletion by neutralizing ab revealed a critical role of this chemokine in induction of granulocyte alpha-defensins release. altogether, these data show a vgamma vdelta -mediated activation of granulocytes through a bystander mechanism, and confirm the wide ability of vgamma vdelta tlymphocytes in orchestrating the immune response. conclusion: an immune modulating strategy targeting vgamma vdelta t cells may represent a key switch to induce an effective and well-coordinated immune response, and can be proposed as a way to strengthen the immune competence during infectious diseases. objectives: the aim of this study was to analyse the activity of vg vd t lymphocytes against glioma cells and to verify the possibility to target these innate cells in new immunotherapeutic approaches. human vg vd t cells recognize and kill several cancer cells presenting a disregulation in mevalonate pathway. interestingly, drugs already in clinical use, such as zoledronic acid, are able to promptly activate vg vd t cells through an indirect mechanism involving the block of farnesyl pyrophosphate synthase of the mevalonate cycle. the vg vd t cell activation by zoledronic acid results cytokines and chemokines synthesis and cytotoxic activity. glioma are tumors arising from glia in the central nervous system. unfortunately, the majority of glioma patients die in less then of a year from diagnosis and new treatment strategies are therefore hardly needed. methods: in order to analyse the activation of vg vd t cells and their effects on the viability of glioma cells, we expanded in vitro vg vd t cells from pbmcs of healthy donors by using phosphoantigen stimulation and tested the ability of vg vd t cell lines to kill three different glioma cell lines (t , u , u ) by cytokinic/cytotoxic mechanism by flow cytometry. results: our results demonstrated that vg vd t cells lines are able to recognize glioma cells, to differentiate in effector memory cells, and to kill glioma cells by releasing perforin. moreover, we analysed whether zoledronic acid treatment could improve the susceptibility of glioma cells to vg vd t lines. we showed that zoledronic acid is able to directly induce cell death on glioma cells and to strongly enhance the cytotoxic activity of vg vd t lines. conclusions: altogether, our results suggest that the induction of a strong antitumor response in vitro of vg vd t cells by using aminobisphosphonates could represent a new interesting immunotherapeutic approach for glioma treatment. viral-induced cancers, such as cervical cancer and liver cancer, contribute to approximately % of all cancers and represent a failure of host immunity to control chronic viral infection. natural killer t (nkt) cells are a population of regulatory t lymphocytes that are pivotal to the outcome of host protection to a range of viral infections and cancers, but their role in controlling host defenses to oncogenic viruses in epithelial and cutaneous tissue is virtually unexplored. using a mouse model of chronic viral infection in the skin, in which human papillomavirus (hpv) oncoproteins are expressed as a transgene in epithelial cells, we investigated the role for nkt cells in abrogating protective immunity to viral antigens in cutaneous tissue. we show that local hpv-e protein expression in the skin attracts a large lymphocytic infiltrate, including a population of cd d-restricted nkt cells. this nkt infiltrate is required to maintain local hpv-e -induced immune suppression and results in graft survival when transplanted onto a naive, immunocompetent host. the local suppressive environment evident in e -expressing transplanted skin is dependent on interactions between populations of cd d-expressing cd c+/f + myeloid cells and nkt cells. removal of either donor-resident or host-infiltrating nkt cells is sufficient to break immune suppression and allow e graft rejection. dissecting the suppressive properties of nkt cells in this novel model of chronic viral antigen presentation in the skin will provide valuable new insight into the potential for clinical manipulation of nkt cell populations to restore chronic anti-viral and anti-tumour immunity in epithelial tissues. nkt cells were expanded from total pbmcs from healthy donors by treatment with il- and a-galcer. expression of cd a, cd d and the costimulatory molecules cd and hla-dr, was established by flow cytometry. rna was quantified by real time-pcr. functional assays were performed by analysis of nkts cytokine production (ifn-g, il- ) and cytotoxicity against treated-idcs. results: idcs stimulated with olive pollen lipids up-regulated cd d expression on the cell surface in comparison with control cells. in contrast cd a expression was decreased. cd and hla-dr slightly increased, indicating certain grade of maturation. the amount of cd d mrna was higher in treated cells than in control cells. by contrast, there was less transcription of cd a, cd b and cd c genes than in control cells. nkt cells efficiently killed treated idcs as "in vitro" cytotoxic killing assays showed. ifn-g producing cells increased slightly in response to treated idcs compared to unstimulated cells, but the number of il- producing cells was not modified. similar results were obtained using monocytes as antigen-presenting cells. conclusions: idcs treated with lipidic extracts from olive pollen up-regulate the expression of cd d on the cell surface. in addition, nkt cells are able to recognize idcs and monocytes treated with lipids from pollen, producing ifn-g and cytotoxicity. all these data suggest that nkt cells may play a role in the control of the immune response to allergens, such as the lipids present in pollen grains. outline: in humans, . - % of circulating lymphocytes express a vg vd t cell receptor, yet strikingly little is known about the function and properties of such unconventional t cells. we performed cdna microarrays to find vg -enriched genes compared to conventional mhc-restricted cd + ab t cells, and found reciprocal enrichment of nectin-like adhesion molecules igsf & crtam in gd and ab t cells respectively. because igsf binds to crtam, the data fuel a hypothesis that this may be a novel axis of communication between the two cell types. interestingly, previous studies show that activated nk, nkt and cd + ab t cells express crtam, and that engagement of igsf on epithelial cells renders the latter targets for enhanced cytolytic and cytokine responses. our data extends this to the prospect of cytolytic immunoregulatory interactions between t cells mediated by igsf /crtam. we therefore sought to answer: . what is the function of igsf /crtam on gd t cells? . how is the igsf -crtam axis regulated in t cells? results and conclusions: flow cytometry showed igsf enrichment on resting gd t cells, with expression also detected on˚ % of ab t cells. the properties of those cells are being examined. however, igsf generally correlates with markers of activation/antigen experience such as cd ro. thus, igsf cells may comprise activated-yet-resting/pseudo-memory unconventional t cells and memory-effector conventional t cells. stimulating vg + t cells in vitro led to rapid crtam induction, resulting in the majority of cells co-expressing both igsf and crtam within hours. however, engagement of igsf by crtam or vice versa is not sufficient to induce cytotoxicity, as stable cho cell transfectants expressing either molecules were not specifically lysed by pbmc in vitro, compared to efficient and parallel targeting of mica + cells. instead, our current experiments address the possibility that crtam-igsf may regulate cytotoxic interactions promoted by other receptor-ligand interactions, such as mica-nkg d. this may explain why cells can tolerate co-expression of both molecules, and would refute the hypothesis that crtam-igsf interactions are sufficient for cd t cells to kill gd t cells and/or vice versa. instead, crtam-igsf interactions may set the threshold for cytotoxic immune-surveillance responses. t cell receptor (tcr) is a multisubunit complex in which the invariant subunit cd z is a kda transmembrane protein indispensable for coupling antigen recognition by tcr to diverse signal transduction pathways. approximately - % of human peripheral blood lymphocytes express the gd tcr and the majority of these cells express the vd tcr variable segment associated with the vg segment, and recognize phosphorylated non-peptidic metabolites from microbial or self origin. these compounds trigger vg vd t cells without antigen presentation. in vitro stimulated vg vd t cells with antigens are able to produce ifn-g and tnf-a and exert a powerful cytotoxic activity against infected cells as hiv-infected cells. however, during hiv infection a marked decrease of vg vd t cells was observed and the remaining cells are unable to respond to their non-peptidic ligands. aim of the present work was to study the mechanisms of vg vd t cell anergy observed in hiv+ patients. to this aim, cd z expression and ifn-g production by vg vd t cells from hiv+ and hiv-subjects were analyzed. we show that vg vd t cells from hiv-infected patients expressed lower level of cd z compared with healthy donors. a direct correlation between cd z expression and ifn-g production capability by vg vd t cell was found. however, pkc activation by pma is able to restore cd z expression and ifn-g production. our findings may contribute to clarify the molecular mechanisms of vg vd t cell anergy found in hiv+ patients and have implication in the design of effective immune-based therapies. l. abeler-dörner , m. swamy , s.l. clarke , a. hayday king's college london, immunobiology, london, united kingdom gut intraepithelial lymphocytes (iel) constitute one of the largest t cell compartments in mice and in man. their functions and their interactions with surrounding epithelium are likely to be crucial to the fine-tuned balance between tolerance to harmless food antigens, immunity to gut-associated pathogens, and overall intestinal immune surveillance. intestinal iel comprise many unconventional t cells including tcrgd cells and tcrab cd aa or cd -cd cells, which have been assigned innate-like immune functions and key roles in surveillance of stressed tissue. unlike conventional t cells, iel might initiate an immune response rather than simply being late effector cells. it is therefore important to elucidate the "immunological information flow" in the gut. to this end, this project characterizes different subsets of iel and their interactions with epithelium in steady state and under immunostimulatory conditions in vitro and in vivo. in the past, it has been notoriously difficult to study iel ex vivo. to solve this problem, we developed a novel culture system that allows us to expand the cells ex vivo and study their responses for up to days. the cells are initially activated by plate-coated acd antibody and a cytokine cocktail and maintained further in medium containing low levels of il- . after a resting period, the cells can be restimulated in vitro. in this new system, we studied responses of different iel subsets to stimulation via tcr, nkg d and cytokine receptors, either alone or in coculture with epithelial cells. as readouts we monitored proliferation, cytokine secretion (ifng, il- ) and expression of activating and costimulatory molecules. reactivation in response to various stimuli could already be observed after hours. the in vitro data set forms the basis for analysing iel responses in vivo to stimulatory molecules ectopically expressed as transgenes in the gut. the characterization of iel responses opens new insights into the nature of gut immune responses and should provide a better understanding of the immunology of inflammatory bowel diseases which still remain a major problem in the clinic today. objective: behcet's disease (bd) is a multisystemic disorder with a possible underlying pathology of immune-mediated vasculitis. increased expression of cd in bd patients suggested that nk receptors may play a pathogenic or regulatory role in the pathogenesis. considering the regulatory functions of nkg molecules in heterodimer with cd , we screened the presence of these receptors on t cell subsets in bd. the expression of nkg a/c/d molecules on gd and cd + t cells were analyzed in active and inactive patients with bd and healthy controls. expression of nkg molecules was evaluated on cd +, gd t and cd + nk cells by using flow-cytometry. results: gd t cells were increased in patients with bd compared to controls ( . vs. . %, p= . ). in addition to the increase of gd t cells, increased expression of activating nkg c molecules was also observed on gd t cells ( % vs. %, p= . ). nkg a expression on gd t cells was found to be higher than nkg c expression in patients and controls; but nkg a expression on the t cells was not statistically different in both groups ( . vs. %). nkg d receptors were present on most of the gd t cells in both groups. however these activating molecules on cd + cells were decreased in patients with bd compared to controls ( revlimid is a therapeutic agent used to treat myelodysplastic syndrome (mds), a group of haematological disorders characterised by ineffective haematopoiesis. the mechanism of action for revlimid is poorly understood, but there has been increasing interest in the strong association reported between mds and defects within the immunoregulatory nkt cell compartment. indeed, some studies now suggest an important outcome of revlimid treatment is the restoration of normal cytokine production by nkt cell levels and an increase in their overall numbers. we have conducted the most thorough study to date of the nkt cell compartment of mds patients treated with revlimid/ancestim and can report that mds patients had normal nkt cell levels prior to treatment, and no significant increase as a result of revlimid/ancestim treatment. furthermore, nkt cells from mds patients produced high levels of th and th cytokines when stimulated with pma/ ionomycin and the proportion of nkt cells capable of cytokine production did not increase significantly after revlimid/ancestim treatment. these are highly significant findings given the recent emphasis on nkt cells as a potential therapeutic target for mds. our study provides an extensive analysis of the impact of revlimid/ ancestim treatment on the nkt cell compartment and sheds new light on the role of nkt cells in mds and the mechanism of revlimid immunomodulation. objectives: human gd t cells are potent killers of a variety of tumour cell lines, and mice lacking gd t cells suffer from high incidence of experimentally-induced tumours. however, the molecular mechanisms mediating tumour cell recognition by gd t lymphocytes remain largely unknown. we aim at identifying potential tumour antigens and co-stimulation molecules expressed in ex vivo tumours and in tumour cell lines that activate human gd t cells for tumour cytolysis. as immune evasion mechanisms that down-regulate tumour antigens may operate in vivo, we have identified candidates from human tumour cell lines of hematopoietic origin that constitute in vitro cytolysis targets for vg /vd + lymphocytes. we have screened a panel of lymphoma and leukaemia cell lines using a conventional in vitro killing assay using vg /vd + cells, and selected two susceptible ("target") cell lines (over % death in the assay) and two vg /vd + resistant ("non-target") cell lines (under % death) for cdna microarray analysis. we compared the differential expression in pairs of tumour cell lines of identical origin: the burkitt's lymphoma cell lines daudi (target) vs raji (non-target), and the pre-b cell leukemia cell lines rch-acv (target) vs (non-target), and validated the results by rt-qpcr quantification. results: we identified commonly up-regulated and commonly down-regulated genes that encode cell membrane-associated proteins in susceptible tumours. ulbp , ifitm and prame, for example, are up-regulated, whereas cd and clec d are down-regulated in target cell lines. as these encode membrane-bound proteins with relevant functions in tumour immunity, they constitute potential ligands for gd lymphocyte recognition of tumour cells. the expression of these candidate genes was studied by rt-qpcr in a broader panel of cell lines and primary biopsies. we are currently testing, in functional assays based on rna interference and overexpression, these and other candidate genes in order to determine whether they provide activating or inhibitory signals to gd t cells. the comparison between the transcriptomes of vg /vd + target versus non-target cell lines allowed the identification of candidate genes, whose individual function we are currently dissecting, that may be involved in tumour cell recognition by human gd t cells. mice and humans are the only species in which phenotype and function of inkt cells have been properly described. our aims are to directly identify this cell population and to investigate cd d, in the rat. mice and rats have very similar cd d and inkt tcr genes, with the exception of the va gene segment, which is a multimember gene family in the rat. novel monoclonal antibodies with nearly identical binding capacities to mouse and rat cd d revealed a very similar pattern of cd d distribution, and could inhibit cytokine production after agalcer stimulation of primary cells in both species. response to agalcer was studied in five different rat strains, showing big inter strain differences. notably, ifn-g and il- production was - fold lower in the best responder rat strain (f ) compared to mouse (c /bl ). since nkrp a (rat homologue of mouse nk . ) and tcr are not appropriate markers for rat inkt, cd d oligomers where tested for binding to inkt-tcr transduced cells. newly generated agalcer loaded rat cd d dimers, recognized rat inkt tcr and, although less efficiently, bound to mouse inkt tcr. however, mouse cd d agalcer dimers did not bind to rat inkt tcr. agalcer loaded rat cd d dimers were then used to stain primary intrahepatic lymphocytes. but, although mouse inkt cells were stained to some extent, the identification of a discrete population in the rat was not possible. the reasons behind could be: that the avidity of the dimers for the tcr is not high enough to stain primary cells and/or that the frequencies are so low that the detection by facs analysis is difficult. in order to clarify these issues we currently produce and test rat cd d tetramers. burkholderia pseudomallei is a highly virulent bacterium which causes the potentially fatal disease melioidosis in humans. this disease is endemic in tropical regions, especially thailand and northern australia, and has a serious outcome for many infected individuals. b. pseudomallei is an intracellular bacterium and many b. pseudomallei strains are resistant to antibiotics so antibiotic treatment is aggressive and relapse of the disease is frequent. in addition to this, no vaccine is currently available to prevent the disease. human g d t cells are involved in the immune response to infection with a number of intracellular pathogens including brucella suis and mycobacterium tuberculosis. g d t cells respond to non-peptidic phosphorylated molecules known as 'phosphoantigens' which are byproducts of essential metabolic pathways in both bacteria and mammals. phosphoantigens cause expansion and activation of g d t cells during infection with intracellular pathogens including fransicella tularensis and m. tuberculosis. analogues of natural phosphoantigens have been developed to manipulate g d t cell responses as a cancer therapeutic and are currently in clinical trials for the treatment of hepatitis c virus. we aimed to determine in vitro whether enhancing gd t cell responses in human blood using the synthetic phosphoantigen picostim could reduce growth of intracellular b. pseudomallei in the human monocytic cell line thp- . a significant (p x . ) reduction in intracellular bacterial numbers was observed (n= ) in the presence of pbmcs cultured with picostim+il- in comparison with pbmcs cultured with il- or media alone. picostim+il- caused significant expansion and activation of gd t cells following culture of pbmcs for - days. purified gd t cells stimulated with picostim were able to reduce intracellular b. pseudomallei numbers -fold. this data demonstrates that pbmcs, stimulated with the synthetic phosphoantigen picostim+il- , reduced growth of intracellular b. pseudomallei in a gd t cell-dependent manner. objectives: vgamma /vdelta (gd) t cells play a major role in innate immunity against microbes, stressed and tumor cells. they represent less than % of peripheral blood lymphocytes (pbl), but can be expanded in vitro by zoledronic acid (za)-treated monocytes or dendritic cells (dc).the purposes of this study are: ) to determine whether dc generated from multiple myeloma (mm) patients are as effective as their normal counterparts in the ability to activate gd t cells; ) to evaluate whether gd t cells can exert immunoadjuvant activity on dc generated from mm patients and primed with tumor-specific antigens (survivin-sv); ) to establish whether the same issues could be solved using a simplified protocol of dc generation. ) dc were generated from cd + cells of healthy donors/mm patients; immaturedc on day were induced to fully mature by incubation for hours with tnfa + il- b + pge in the presence or absence of mm za. after days of co-culture dc:pbl, percentages and total counts of gd t cells were determined by flow cytometry; ) idc generated from cd + cells of hla-a* + healthy donors/patients were pulsed with sv-peptide and stimulated for hours with tnfa + il- b + pge in the presence or absence of mm za; after rounds of autologous t cells stimulation by dc, the frequency of sv-specific cd + t cells was determined by svpentamers staining; ) the same experiments were performed both with dc generated following a standard protocol and a h protocol (dc fast objective: depletion of or deficiency in gd t cells aggravate colitis in different animal models. additionally, reconstitution of mice with syngeneic gd t cells ameliorated chemically-induced colitis indicating a suppressive or regulatory role for murine gd t cells in intestinal inflammation. therefore, we asked whether human gd t cells possess also suppressive or regulatory potential, which could be of therapeutical use in chronical inflammatory diseases such as ulcerative colitis or crohn's disease. hence, the proliferation, suppressive activity, and cytokine profile of human peripheral gd t cells were determined in vitro. methods: human gd t cells were isolated from whole blood of healthy donors by macs technology. the proliferation was determined by [ - h]-thymidine incorporation, while suppression of responder cell proliferation was measured by flow cytometry via cfse fluorescence intensity. the cytokine profile was determined by elisa from culture supernatants as well as by flow cytometry intracellularly. finally, the in vitro characteristics of gd t cells were compared to those of cd + cd + regulatory t cells (treg). human peripheral gd t cells show suppressive activity against responder cell proliferation, though being themselve anergic, that is, they produce negligible amounts of interleukin- on stimulation and proliferate poorly. while the proliferation of gd t cells and treg cells is comparable, the suppression of gd t cells on responder cell proliferation is even stronger than the suppression by treg cells though gd t cells being foxp negative. additionally, gd t cells are strong producers for tgf-b, particularly by the vd subset. conclusion: human peripheral gd t cells possess regulatory potential and could be of therapeutical use in treatment of chronical inflammatory diseases as they are anergic and act suppressive. their suppressive activity is even superior to treg cells and might be due to strong tgf-b secretion. for application of human gd t cells in therapy their expansion under maintenance of their regulatory properties should be elucidated. there are previous descriptions of gamma-delta t lymphocytes (gd) from behçet's disease patients (bd) but, in most of cases, they are incomplete or contradictory. it has been suggested that nkg d on gd is involved in bd lesions through interaction with mica molecules. furthermore gdcd + have been recently proposed as a new regulatory t subset (treg). objectives: to study gd phenotype in bd active (bda) (n= ) and inactive (bdna) (n= ), versus healthy controls (hc) (n= ) and patients with recurrent oral ulcerations (ru) (n= ). to determine gd cytokine profile and surface markers treg-related in bd (n= ) and hc (n= ). methods: we obtained mononuclear cells from peripheral blood (pbmc). we determined by flow cytometry: -surface expression of: gd tcr, vdelta , vdelta , cd alpha, cd beta, nkg d, nkg a and cd . -intracellular expression of ctla- , and foxp . -intracellular expression of il- , il- , ifngamma, il- and tgfbeta after pbmc polyclonal stimulation. we used two tailed test for means comparison (mann-whitney u or student's t test). -vdelta + cells were significantly increased in ru. vdelta + and gdcd + lymphocytes were significantly increased in bd versus ru and hc. -the mean fluorescence intensity of nkg d was slightly increased in gd from bda. -nkg a expression by gdcd + was not different in bd versus hc. -most of gdcd + presented cd alpha-alpha homodimers in bd and hc and were negative for cd , foxp and ctla- . gdcd + and gdcd -subsets were (in bd and hc): -high ifngamma-producers without differences. -low il- -producers: il + cells were lower in gdcd + than in gdcd -. -low il- -producers: il + cells were lower in gdcd + than in gdcd -. -low tgfbeta-producers: tgfbeta+ cells were lower in gdcd + than in gdcd --very low producers of il in most of cases. the hallmark in bd was the increase of gdcd /vdelta +. this subpopulation has recently been described as immunosuppressive in infiltrates of human tumours and its function related to nkg a in intraepithelial intestinal lymphocytes from celiac patients. we did not find a cytokine profile or a phenotype t-reg-related for gdcd +, except a lower percentage of il- + cells than in the gdcd -subset. gdcd + from bd did not show significant differences versus hc. natural killer t (nkt) cells comprise a highly heterogeneous subset of t lymphocytes that co-express a t cell receptor (tcr) and nk cells markers such as cd in humans. a subgroup, the invariant nkt cells (inkt), expresses the va vb tcr rearrangement representing a minority subset in peripheral blood and virtually absent in the newborn. objectives: to establish a method to growth cord blood-derived nkt cells (cd + cd + ), in order to evaluate their phenotypic characteristics and the tcrvb repertoire. methods: mononuclear cells were isolated from healthy umbilical cord blood samples and stimulated with ifn-g ( ng/ml), anti-cd ( ng/ml) and il- ( ui/ml). these cells were cultured for days and the expanded cd + cd + cells were isolated by immunomagnetic methods. surface markers were determined by flow cytometry. total rna was extracted from the purified cd + c + cell suspension using trizol ® reagent and mrna expression of twenty tcrvb gene families was measured by semiquantitative rt-pcr. statistical analyses were performed using mann-whitney u test and one-way anova, a p value of x , was considered significant. results: we could significantly expand cord blood cd + cd + nkt cells from , ± , % to achieve an enrichment of , ± , % (p= , ). table shows the percentage (mean±sd,n= ) of phenotypic markers in cd + cd + cells at baseline (day ) and after days of culture. expression of mrna for the vb families studied was confirmed in each individual cell culture with a significant high expression of vb and vb families (p x , ). conclusion: our results show that cord blood-derived nkt cells are mainly cd + and cd + subsets, similar to peripheral blood nkt cell with a low percent of inkt cells. additionally, we confirm a diverse tcr vb repertoire with a significant expression of the vb and vb families in these cells. l. marischen , d. wesch , p. rosenstiel , a. till , d. kabelitz institute of immunology, kiel, germany, institute of clinical molecular biology, kiel, germany gd t cells account for a minority of t cells in human blood, but represent the majority of intraepithelial t cells in the intestinal tract. due to their ability to respond rapidly and in an mhc-independent fashion to particular antigens by cytokine production, gd t cells are considered as a link between innate and adaptive immunity. in addition, the expression of distinct pattern recognition receptors such as toll-like (tlr) and nod-like receptors (nlr) are characteristic for cells of the innate immune response. recent reports have demonstrated the tlr expression in human and murine gd t cells. here we provide evidence also for a gd t cell responsiveness to muramyl dipeptide (mdp), the putative ligand of the nlr family member nod . peripheral blood mononuclear cells (pbmcs) containing gd t cells as well as freshly isolated gd t cells were stimulated via the gd t cell receptor in the absence or presence of mdp and analyzed for proliferation and ifng-production. while the proliferation of gd t cells within pbmcs was decreased, ifng-production was increased after costimulation with mdp compared to the stimulation with a non-activating dd-stereoisomer of the ligand (mdpi). the enhanced ifng production of pbmcs after costimulation was mediated mainly by gd t cells as shown by intracellular flow cytometric staining. with regard to the ifng-production after co-stimulation with mdp vs. mdpi, freshly isolated gd t cells from different healthy blood donors can be divided into responder and non-responder. responder gd t cells showed a significant increase of the ifng-production due to mdp-stimulation, whereas ifng-production was not influenced in non-responder gd t cells. in further experiments, as first approach to explain the different reactivity patterns of gd t cells, it is planned to analyze the polymorphisms of the nod gene in various donors. taken together, our preliminary data indicate that gd t cells are a major source of ifng-producing cells among pbmcs when challenged with specific antigens plus mdp, and support the role of gd t-cells as an important team player in the early immune response against bacteria. objectives & methods: an increasing of gamma-delta t cells during acute p. vivax infection and convalescent period has been reported. moreover, the activation of gamma-delta t cells leads to the inhibition of blood stage p. falciparum parasites in vitro. to determine the killing mechanisms of p. vivax parasites by gammadelta t cells comparing with what has been found in p. falciparum, the gamma-delta t cells were enriched by isopentenylpyrophosphate (ipp) from naïve pbmc. different number of gamma-delta t cells and normal pbmc were incubated with intact of p. vivax parasites and protein extract of p. vivax parasites, recombinant pvmsp and pvama proteins. gamma-delta t cells was daily determined the cytokine and granzyme intracellular releasing by flow cytometry until day culturing. results: among the enriched gamma-delta t cells, the percentage of cells expressing cd + and cd + was elevated after co-culturing with intact and the proteins of p. vivax parasites. the overall gamma-delta t cells showed proliferation at day after the co-cultivation. moreover, the gamma-delta t cells expressing ifngamma + and cd a + (lysosomal associated membrane proteins: lamp- ) elevated from the first day of pbmc collection after co-culturing with the intact and p. vivax antigens. this level was correlated with the significantly decreasing number of parasites and the increasing percentage of parasite growth inhibition. our results showed the activation of gamma-delta t cells during p. vivax infection in vitro. this suggests that gamma-delta t cells could be stimulated by p. vivax parasites and these actively activated gamma-delta t cells could kill the parasites via mechanism of granzyme and cytokines at the early stage of cell activation. this study provides more understanding in activation of the innate immunity during acute malaria infection which may lead to the selection of appropriate malaria proteins as vaccine candidates in the future. objectives: several evidence suggest that invariant nkt cells (inkt) connect innate and acquired immune system. they are able to produce both th and th cytokines after stimulation. atopic dermatitis (ad) is a chronic inflammatory skin disease. th -like and th -like cytokines have been implicated in the pathogenesis of ad, but there are controversial data on their role in ad. the frequency and absolute number of inkt cells in mononuclear cells (pbmcs) of peripheral blood of patients with atopic dermatitis (ad) (n= ) and healthy controls (n= ) were determined by flow cytometry using anti-cd and monoclonal antibody specific for the cdr loop of the invariant tcr a chain of inkt cells (clone: b ). furthermore, after pma/ionomycin stimulation for hours, intracellular ifng and il- cytokines were detected in cd +cd -, cd -cd -(dn), cd -cd + and cd +cd + subsets of inkt cells by five colour flow cytometry in patients with ad (n= ) and healthy controls (n= ). results: both frequency and absolute number of inkt cells were significantly lower in patients with ad (p x . ) compared to healthy controls. the frequency of dn subpopulation was significantly lower in ad patient (p x . ). there was a positive correlation between the frequency of dn cells and inkt cells both in ad patients (r= . and p x . ) and healthy controls (r= . and p x . ). in the intracellular ifng level there were no significant difference in any of the inkt subsets of ad patients, however the intracellular il- level was significantly higher in dn subpopulation of inkt cells of ad patients compared to healthy controls (p x . ). the frequency, the number of inkt cells and the cytokine producing capacity of the cd /cd inkt subsets are different in peripheral blood obtained from ad patients compared to healthy controls. our result suggest that the dn inkt cell subset can serve as a source of il- that promotes the th differentiation in ad patients and might play a role in the pathogenesis of this disease. introduction: intrahepatic immune cells (ihic) are known to play central roles in immunological responses mediated by the liver, and isolation and phenotypic characterization of these cells is therefore of considerable importance. aims: in the present investigation, we developed a simple procedure for the mechanical disruption of mouse liver that allows efficient isolation and phenotypic characterization of ihic. these cells are compared with the corresponding cells purified from the liver after enzymatic digestion with different concentrations of collagenase and dnase. results: the mechanical disruption yielded viable ihic in considerably greater numbers than those obtained following enzymatic digestion. the ihic isolated employing the mechanical disruption were heterogeneous in composition, consisting of both innate and adaptive immune cells, of which b, t, natural killer (nk), nk t cells, granulocytes and macrophages were the major populations (constituting . %, . %, . %, . %, . % and . % of the total number of cells recovered respectively). the ihic obtained following enzymatic digestion contained markedly lower numbers of nk t cells ( . %) . the b, t and nk t cells among ihic isolated employing mechanical disruption were found to be immunocompetent, i. e. they proliferated in vitro in response to their specific stimuli (lipopolysaccharide, concanavalin a and alpha-galactosylceramide respectively) and produced immunoglobulin m and interferon-gamma. conclusions: thus, the simple procedure for the mechanical disruption of mouse liver described here results in more efficient isolation of functionally competent ihic for various types of investigation. nature killer t cells (nkt) are a special t cell population with co-expresses nk and t cell surface markers. murine nkt cells include cd + nkt and cd -cd -nkt cells. nk . + nkt cells may release large amounts of il- , il- , ifn-g and il- after they are activated. it has been reported that a-galactorsykeramide (a-galcer), a glycolipid, may induce proliferation of nkt cells with the role of immune regulation by stimulating mouse spleen cells. this study demonstrated that superantigen staphylococcal enterotoxin b (seb) , a kind of peptide, can activate the nkt cells with the function of immune tolerance. the response ability of seb-activating effect cells to cona, lps and il- had significantly decreased compared with that of normal lymphocytes. the effect cells exerted an inhibitory effect for the response of normal lymphocytes to cona and il- . there was a significantly increase in the percent of cd + nk . + and tcrvb + nk . + nkt cells identified from the seb-activated cells. based on the cell distribution detected in the upper part of the facs picture, expression of cd molecule existed in . % of the cells from large-scale selection. the percent of cd + nk . + and tcrvb + nk . + nkt cell subsets in the giant lymphocytes were enhanced to . and . folds, respectively. under a light microscope at x magnification, the seb-activating lymphocytes in size were larger than not only the cona-activated cells but also the adherent macrophages with an increase of fold observed under a microscope. there were a few granules seen in cytoplasm. the value of cytoplasm vs nuclei was less than . and they are non-adherent cells. the differentiation pathway of the seb-activating cd + and tcrvb + nkt cells was not relative to a nk source. they were produced directly from t cell population and were considered as a subsets of t lymphocytes. our results suggest that the superantigen seb can act on the cd + nkt cell and tcrvb + nkt cells. and the two nkt cell subsets may play a critical role in seb mediated tolerance. gd t cells in the intestinal intraepithelial compartment (gd iiel) show an intrinsic activated phenotype. we hypothesised that their t cell receptor gd (tcrgd) is implicated in the activation of gd iiel. because the tcr gd ligands in mice are not well described, monoclonal antibodies (mab) directed against the gd tcr, like the clone gl which binds the d subunit of tcr gd, are important tools to specifically activate gd t cells. using cytometric indo- am measurement, we could detect calcium flux of intestinal and peripheral gd t cells from tcrd-h begfp reporter mice. stimulation with anti-gd clone gl or anti-cd clone c elicited activation of gd t cells suggesting that tcr gd and cd molecules in gd t cells are functional and signalling competent. next, using elisa and cytometric bead array, we found that iiel stimulated with plate bound gl in vitro produced ccl , ifng and tnfa. therefore, we were interested whether the ccl production of gd iiel influenced the homing of ccr cells such as lamina propria (lp) cd + foxp + cells (tregs). to test this, wt mice were i. p. treated with gl mab and lp tregs were analysed by cytometry at various time points post inoculation. we found similar frequencies of lp tregs population but a slight decrease in ccr + tregs. however, when we compared wt and tcrd -/mice, we found both lower percentages of total lp tregs and of lp ccr + tregs in tcrd -/mice compared to wt mice. in conclusion, our data suggest that intraepithelial activation of gd t cell may directly or indirectly induce changes in the iiel and lamina propria (lp) lymphocyte compartment and influence the ccr expression and the homeostasis of lp treg. the ability of nkt cells to serve a variety of different immunoregulatory functions in vivo may reflect a diversity in function of different nkt cell subsets. diversity in cytokine production by nkt cell subsets has been observed in murine and human studies, although this analysis has largely been following in vitro restimulation. here, we investigated cytokine production by murine nkt cell subsets in vivo under conditions where minimal manipulation of the cells was required. to this end, we examined il- production in g reporter strains in which dna encoding green fluorescent protein (gfp) was inserted into the first exon of the il- gene. in the absence of any manipulation gfp was expressed from the il- locus in populations of immature thymic nkt cells (predominantly cd +cd lotcrhi cells on a balb/c background, and cd +cd lonk . -on a c bl/ background) and some splenic nkt cells, with overall numbers of gfp+ cells in both tissues decreasing with age. after i. v. administration of the nkt cell ligand a-galactosylceramide, il- production was induced predominantly in cd + nkt cell subsets of the liver and spleen, and after i. n. administration, in cd + nkt cells of the airways. spontaneous and a-galcer-induced expression from the il- locus occurred in the absence of stat signalling, and did not require initial exposure to il- protein from other sources in the host. diversification in cytokine expression by nkt cells subsets therefore occurs early in ontogeny, and is also a significant feature of responses to exogenous activating stimuli. interleukin- (il- ) plays an important role in neutrophil recruitment. herein, we investigated the role of il- receptor signaling in polymicrobial sepsis induced by cecal ligation and puncture (clp). methods: adult c bl/ (wt) and il- receptor gene-deficient (il- r ko) mice were subjected to non severe (ns-clp) sepsis. intraperitoneal neutrophil migration, bacteremia, cytokine, chemokines and liver injury were evaluated hours after surgery. the ability of il- mediate the neutrophil microbiocidal activity in vitro, as well the neutrophil migration in vivo and in vitro were also evaluated. the means of different treatments were compared by analysis of variance (anova), followed by bonferroni's t test and the survival rate by the mantel-cox log rank test. results: it was observed that il- r ko mice, subjected to ns-clp sepsis, show reduced neutrophil recruitment into peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared to wt. as a consequence, the mice showed an increased mortality rate. moreover, il- induced neutrophil migration in vivo and in vitro. besides, we demonstrated that neutrophils harvested from il- r ko mice already show reduced microbiocidal activity, compared with wt, suggesting a physiological role of il- receptor signaling in the microbiocidal activity of neutrophils. furthermore, wt neutrophils treated with il- showed strongly enhancement of microbiocidal activity by a mechanism dependent of nitric oxide. conclusion: during ns-clp besides the importance in recruit neutrophils to focus of infection, il- also enhances the microbiocidal activity of neutrophils. therefore, our results demonstrated that il- receptor signalization plays a critical role on host protection during polymicrobial sepsis. objectives: members of the toll-interleukin- receptor (tir) family are important for host defense, inflammation, and immune regulation. their canonical signaling pathway involves adaptor proteins and il- r associated kinases to activate nfxb and p mitogen-activated protein kinase. the il- -induced signal transduction in mast cells is poorly understood. in this work we studied the signal transduction of il- in different mast cell subsets. methods: different mast cells subsets (hmc- , human cbmcs and murine bmmcs) were stimulated with il- . the resulting signal transduction was investigated by immunoblot for activated signaling molecules (pc-kit, perk / , pakt, pnfxb, p and pjnk). additionally, we studied the signal transduction of il- in il- r transfected hek t cells. results: we found, that a tir family member, il- r, transactivates the receptor tyrosine kinase c-kit in mast cells and that il- -induced cytokine production depends on c-kit transactivation. il- r and il- r accessory protein (il- racp) form a physical complex with c-kit. thereby the complexation is dependent on the activity of c-kit. conclusion: these results show for the first time that the biological function of an il- r family member is dependent on the presence of an activated receptor tyrosine kinase. furthermore, these results reveal that certain il- -induced signaling pathways and effector functions are dependent on activated c-kit and could therefore explain the effects of il- in mast cells in absence of iger activation. ( ) . we now provide a molecular mechanism underlying this pathogenic effect by which free heme sensitizes hepatocytes to undergo tnfmediated programmed cell death. independently of newly gene transcription and/or protein synthesis, free heme cytotoxicity is mediated by the unfettered generation of free radicals in response to tnf, presumably due to the participation in the fenton reaction of the fe atom present in the protoporphyrin ix ring. once exposed in vitro to free heme, a sustained c-jun n-terminal kinase (jnk) activation was observed in hepatocytes in response to tnf, an effect that promotes further free radicals production. pharmacologic or genetic (shrna) inhibition of jnk in hepatocytes avoids free radicals accumulation and caspase- activation, also mimicked by the anti-oxidants n-acetylcystein (nac) or butylated hydroxyanisole (bha). expression of the heme catabolyzing enzyme heme oxygnease- (ho- ) in hepatocytes affords protection against heme sensitization to tnf cytotoxicity. recombinant adenovirus mediated ho- expression in the liver suppresses tnfmediated hepatocyte apoptosis and prevents the lethal outcome of plasmodium infection in mice. in conclusion our data reveals a novel signal transduction pathway via which heme sensitizes hepatocytes to undergo tnf-mediated cytotoxic effect, critically involved in the outcome plasmodium infection. the multi-step leukocyte extravasation process is governed by adhesion molecules and chemotactic factors dynamically interplaying in the presence of shear forces. responsiveness to chemotactic ligands is mediated by g protein-coupled receptors (gpcrs) which are finely regulated by a family of cytosolic proteins, betaarrestin and . recent evidence indicates that, in addition to playing a regulatory role in gpcr desensitization and internalization, beta-arrestins may contribute to gpcr signaling by functioning as scaffolds for the recruitment of signaling proteins into complexes with agonist-occupied receptors. on this basis, we investigated the physiological role of beta-arrestin in chemokine-driven dynamics associated with leukocyte extravasation, with special interest to the activation of the rap small gtpase, recently emerged as pivotal regulator of integrin function. the analysis of kc the (keratinocyte-derived chemokine) rap activation profile in rbl (rat basophilic leukemia) cells expressing mcxcr shows a bimodal kinetic, with the first peak at ''/ ' and the second at ' after stimulation. rna interference-mediated depletion of beta-arrestin specifically inhibits the occurence of the second wave of rap activation, whilst it has no effect on the early pick, thereby suggesting that beta-arrrestin is involved in rap activation and that the oscillations in the formation of rap -gtp are regulated by different molecular mechanisms. in order to elucidate the gefs and gaps involved in the gtpase regulation we are at present down-regulating the expression of c g (rap gef) and spa (rap gap): preliminary results suggest that spa- has probably a role in the early activation peak. since this oscillatory chemokine-induced rap activation is present on other myeloid cell lines (hl , d) and fresh pmn's we are also translating our research to these more appropriated cells. interestingly betaarrestins amino acid sequence and three-dimensional structure reveal a unique and evolutionary conserved proline-rich sequence in beta-arrestin , localized in a solvent exposed loop which may serve as a docking site for migration-associated transducers/adaptors. in order to find sh containing proteins that interact with beta-arrestins, we have performed an overlay screening assay of different sh domains that revealed over putative beta-arrestins putative interactors, some of which isoform specific. granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin (il)- and il- stimulate proliferation, differentiation, survival and functional activation of myeloid cells. the cell surface receptors for these cytokines consist of cytokine-specific a subunits and a common b-receptor (bc), required for the activation of intracellular signaling following cytokine engagement. aberrant signalling, stimulated by these cytokines, has been implicated in the pathogenesis of many diseases, including arthritis, asthma and leukemia. as a result, we have sought to define key molecular determinants of these receptor-cytokine interactions in order to gain a greater understanding of receptor activation. here we present novel insights into the role of the ig-like domain of the gm-csfra in gm-csf binding. deletion of the ig-like domain abolished direct gm-csf binding and we identified specific residues directly involved in ligand binding by site directed mutagenesis and binding studies. the results indicate a previously unrecognized role for the ig-like domain of gm-csfra. furthermore, we address a longstanding controversy in the field of gm-csf, il- and il- receptor biology, by performing a systematic study of the role of n-glycosylation upon on the bc, and related murine b il- , in ligand-binding and receptor activation. these data demonstrate definitively that n-glycosylation does not play a role in mediating ligand-binding or receptor activation. these findings clearly establish that the determined human bc structures lacking glycosylation at asn are biologically relevant conformers of the human bc ectodomain. our results appear to suggest that the potency of receptor signalling can be influenced by the biophysical and structural properties of the extracellular receptorligand interactions and it also addresses important, poorly-understood aspects of mechanisms underlying ligand recognition and activation of the gm-csf: gm-csfra: hbc receptor complex. reference: ( ) micrornas (mirnas) are endogenous small non-coding rna molecules acting as key regulators of immune cell differentiation and innate immune responses. mirna- expression is induced by activation of the toll-like/interleukin- receptor pathway (tirpathway), where it targets essential adaptor and signaling molecules, thus serving as a regulator preventing the cells from an exacerbated pro-inflammatory response. since tnfa also up-regulates the expression of mirna- a, we decided to explore whether this mirna is involved in the regulation of apoptosis. to this end, we used the hela human epithelial cell line as a model system for tnfa signaling. following tnfa and cycloheximide (chx) treatment mirna- a transfected cells showed significantly reduced levels of the active proapoptotic caspases and (casp / ). in line with this, mirna- a conferred enhanced protection against tnfa-induced dna fragmentation and mitochondrial potential drop-down. our results demonstrate that mirna- a is a regulator of receptor-mediated apoptosis. similar to the tir-pathway, mirna- a seems to be part of a negative feedback mechanism of the tnfa signaling cascade. ongoing research focuses on the identification of the specific pro-apoptotic molecules targeted by mirna- a. furthermore, we are exploring the relevance of our observations for the mycobacterial infection of human macrophages, where the regulation of apoptosis is critical. objectives: the aim of this study was to evaluate the role of single nucleotide polymorphisms (snps) located in il- , il- r a-chain and il- r a -chain genes in hiv disease progression. methods: we studied antiretroviral treated patients (progressors) and long term non progressors (ltnp). we analyzed snps in the il- gene, snps in the il- r gene and snps in the il- r gene. in univariate analysis, we found an association between the presence of at least one mutated a allele in il- r aa and a higher possibility of being ltnp ( our study suggests that genetic polymorphisms located in il- r and il- r genes can influence the rate of disease progression in hiv+ patients, especially when a combination of aplotypes is present. mutations in the coding regions might compromise the binding of the cytokines or the intracellular signal transduction pathways, therefore leading to the alteration of cd and cd t cells homeostasis. aims: mono-adp-ribosyltransferases (arts) are gpi-anchored ectoenzymes that covalently modify cell-surface or soluble target proteins by transferring an adpribose moiety from extracellular nad+ to specific arginine residues of target proteins. in this study, we report that human tumor necrosis factor (tnf) is adpribosylated by art , and that adp-ribosylation affects both the release of tnf from cells and its cytolytic action. methods: transcription of art in human leukocytes was analyzed by rt-pcr. adp-ribosylation of tnf was detected by monitoring the incorporation of adpribose from labeled nad. release of tnf from transfected hek cells was monitored by elisa. binding of tnf to tnf receptors was analyzed by biacore. tnf cytotoxicity was monitored by flow cytometry. the adp-ribosylation site on tnf was analyzed by lc/ms mass spectrometry. results: we identified art transcripts by rt-pcr analysis in human blood leukocytes. soluble art , released from the surface of transfected cells by phosphatidylinositol-specific phospholipase c (pi-plc), adp-ribosylated recombinant human tnf in vitro. co-transfection of hek cells with art and tnf resulted in modification of tnf at at least distinct sites, i. e. one within the tnf ectodomain, and one on the stalk that remains connected with the cell membrane after cleavage by tnfa converting enzyme (tace). analysis of modified recombinant tnf by mass spectrometry provided evidence that the tnf ectodomain is adp-ribosylated at r , a site that has previously been implicated in binding to tnfr . binding assays indicated that adp-ribosylation inhibited binding of tnf to its receptors. importantly, modified tnf was less potent at inducing cell death in the human t cell lymphoma line kit than wildtype tnf. furthermore, cell surface adp-ribosylation of hek cells co-transfected with tnf and art resulted in reduced release of tnf into the supernatant. conclusions: adp-ribosylation of tnf or other cell surface proteins interferes with the biology of tnf signals by at least two distinct mechanisms. adpribosylation of tnf blocks binding to its receptors, thereby inhibiting tnf-mediated cytotoxicity. additionally, adp-ribosylation of tnf or another protein on the surface of tnf-producing cells inhibits the proteolytic release of tnf. noninflammatory chronic pelvic pain syndrome : immunological study in ejaculate g. n. drannik , t.v.poroshina institut of urology amsci of ukraine, laboratory immunology, kyiv, ukraine chronic prostatitis (cp) is a disease which likely is associated with abnormalities in local immune responses. secretions of the urinary and reproductive tract mucosa contain various protective effector molecules, produced by mucosal cells, lymphocytes, macrophages and neutrophiles. the aim of this prospective study was to observe local immunophenotypic patterns in patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome for further description and as possible surrogate markers for diagnosis and treatment. methods: patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome (cp/cpps) and control men were assessed for slpi, tnf-alpha, il- and free tgf-b in ejaculate by elisas using stat-fax plus. a to day sexual abstinence period was required from the subjects before semen collection. after liquefaction and centrifugation, seminal plasma samples were kept at - degrees c°until assayed. the materials were processed after the standard programmes for statistical analysis.the study was approved by the local ethics committee. the slpi concentration was elevated in all patients ( . ± . pg/ml, p x . ) in seminal fluid, in comparison with the healthy control subjects. the tnf-alpha concentration was elevated in all patients in seminal fluid ( . ± . pg/ml; p x . ). the il- concentration was elevated in all patients ( . ± . pg/ml; p x . ) in seminal fluid. free tgf-b was present in normal seminal plasma in high concentrations ( . ± . pg/ml), while in ejaculates of patients with noninflammatory cp/cpps tgf-b concentrations were . ± . pg/ml. conclusion: ejaculate's slpi, tnf-alpha, il- and free tgf-b are possible surrogate markers for the diagnosis and treatment of patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome. m. r. marrakchi , e. a. elgaaeid faculté des sciences de tunis, biology, tunisie, tunisia ulcerative colitis (uc) and crohn's disease (cd), collectively referred to the inflammatory bowel disease (ibd), represent a group of multifactorial autoimmune disorders of the gastrointestinal tract sharing many clinical and pathological characteristics, however, differing in histological features and cytokine profiles. the excessive production of either th or th cytokines due to perturbed regulation of immune system activation results in chronic inflammatory processes and loss of immune homeostasis that may be implicated in the genesis of ibd. studies have identified a gene that encodes the nod /card protein, which is involved in the immune system's response to bacterial infection and confirmed to influence susceptibility to cd. indead, it has been suggested that high rates of asca (saccharomyces cerevisiae ) in absence of panca (perinuclear anca: anti-neutrophil cytoplasmic) antibodies were associated with aggressive forms of cd and that the important rise of panca was more frequent at uc . in a sample of tunisian patients, we examined the contribution of nod /card gene in cd. we performed a cases /controls study upon cd patients and healthy controls. this study suggests that in northen tunisian population, insc mutation in nod /card gene is a prevalent mutation leading to the typical crohn's disease including ileal location, stricturing and penetrating clinical types and asca expression. since conflicting results were obtained on il- polymorphisms as risk factor for ibd, the aim of our study was also to explore anti-inflammatory il- cytokine genetic profile in patients with ibd. we examined the contribution of il- gene promoter polymorphisms (- and - ) to crohn's disease (cd) phenotype, and the possible genetic epistasis between these polymorphisms and card /nod gene mutations in cd presentation and location. in tunisian population, the insc insertion in nod /card gene is a marker of susceptibility to cd, while the a allele at position - in the il- promoter increases the risk of cd ileal location and severe disease presentation. a genetic epistasis between il- gene polymorphisms and card /nod gene mutation was suggested. in conclusion, genetic and serologic markers might be useful in defining patien gangliosides were shown to inhibit the il- -dependent proliferation of t-cells, implying that gangliosides interfere with one or more of the il- -driven events. it is known that the major mechanism of inhibition is the direct interaction between ganglioside and the cytokine and, as a result, the capture of il- molecule by ganglioside. but gangliosides apparently can also form complexes with il- r; such complexes influence on the signal transduction through il- r. this effect of gangliosides may lead to the failure of this pathway. unfortunately, the biological and structural aspects of this problem are poorly understood. in this study we propose possible modes of interactions between exogenous gangliosides and il- r subunits. in our work we use il- -dependent cytotoxic t-cell murine line ctll- . two different approaches for study of possible interaction types between exogenous gangliosides and il- r subunits were applied: antibody staining of il- r subunits followed by flow cytometry analysis, and photoaffinity labeling of living cells with i-dcp-gm followed by immunoprecipitation of il- r subunits. the fluorescence intensity of the antibody-labeled il- r a-subunit substantially decreases after the treatment of cells with gangliosides. it has been shown that the fluorescent labeled cell fraction decreases by . % after cells incubation with ganglioside gm , and by . % after incubation with gm . labeling of the cells with antibodies to the il- r b-subunit results in a less significant fluorescence decrease after cells incubation either with gm ( . %) or with gm ( . %). to determine the mode of this impressive masking influence of ganglioside gm , photoaffinity cross-linking has been used. in our modification this method could identify is interaction of gm with subunits of il- r occur with or without incorporation exogenous ganglioside into plasma membrane. electrophoresis followed by immunoprecipitation with appropriate antibodies resulted in appearance of the radioactive band only for b-subunit of il- r, but not for il- r a-subunit. these results demonstrate that exogenous ganglioside gm can interact with a-and bsubunits of il- r in different modes. interaction of il- r b-subunit with ganglioside gm requires incorporation of the ganglioside into plasma membrane, but it is not the case for interaction with a-subunit. a. respa , j. bukur , s. purpose: loss of interferon (ifn)-g inducibility of hla class i antigens has been found in some tumor cell lines, but the underlying molecular mechanisms of such deficiencies have not yet been elucidated in detail. this kind of tumor escape mechanism might lead to an inefficient recognition of tumor cells by the immune system. methods: phospho-specific western blot analyses were performed to verify the functionality of the different ifn-g pathway components, intra-and extracellular flow cytometry experiments were employed to determine the expression of antigen processing components and hla class i cell surface antigens, quantitative real time-pcr experiments to confirm the absence of jak and presence of pathway relevant molecules as well as, genomic pcr and chromosome typing technique to prove the deletion of jak . results: different ifn-responsive phenotypes were defined in human melanoma cell lines varying from loss to low, delayed as well as strong ifn-g inducibility. resistance to ifn-g treatment was associated in one melanoma cell line with the lack of jak expression due to a gene deletion on chromosome , whereas the expression and functionality of other ifn-g signal transduction components like stat and jak were not affected. jak blockade by two jak -specific inhibitors resulted in reduced levels of hla class i surface antigens. conclusion: structural abnormalities of jak leading to a lack of jak expression are associated with loss of ifn-g inducibility as well as reduced constitutive hla class i surface expression. in addition the jak inhibition modulates the expression of the hla class i antigen processing components. of renal cell carcinomas (rccs) are associated with the size, grade, t, n, m, stage and death of rccs patients. the genotypes were compared with those of a random sample of controls of the spanish population. methods: two il polymorphisms located on the il- promoter region, snps - a/c (rs ) and - g/c (rs ) were genotyped using taqman snp genotyping assays. the functional il- gene polymorphisms studied do not influence the susceptibility to rccs in the spanish populations (il- - p= , . il - p= , ) but may contribute to disease onset and aggressiveness: the genotype il- - cc genotype, which is associated with higher il- production, was significantly associated with higher tumor size (p= , ), grade (p= , ), t (p= , ), m (p= , ) and stage (p= , ). the influence of the il- - gg genotype was less relevant, however was correlated with higher tumor size (p= , ), grade (p= , ), t (p= , ) and stage (p= , ). the multivariate analysis with cox proportional hazard model revealed that, in this serie, nuclear grade and stage grouping were independent prognostic factors, whereas il- polimorphism can not be considered as independent prognosis factors. our results suggest that the polymorphism variants from the il- gene (il- - and - ) may be associated with an worse prognosis of rcc. high levels of il- production can play an important role in grow, invasion and metastasis of renal cancer. interleukin- (il- ) is a pleiotropic cytokine that is involved in regulation of both the innate and acquired immune response. the most prominent biologic property of il- is its ability to induce the production of ifn-gamma in presence of il- . moreover, it stimulates the expression of tnf-a and il-l, enhances the differentiation of t cells to the thl and impairs the synthesis of the anti-inflammatory cytokine il-l . then it seems that il- has a crucial role in immunity against brucella infection. since the expression of il- can be affected by polymorphisms in its gene, we decided to investigate any probable relation between six different il- gene polymorph isms and brucellosis. methods: a total of patients with brucellosis and healthy farmer who consumed contaminated row milk and dairy products from animals with brucellosis, were included in this study. all individuals were genotyped for six il- polymorphisms at positions - , - , , + , + and codon / using polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp). the distribution of alleles for il- polymorphisms at position - g/+ t/+ c (correlated with high production of il- ), codon / c and - g/- c (correlated with higher production of il- ) were significantly higher in the healthy controls than the patients (p= . , p= . and p= . , respectively). discussion: as data revealed genotypes that have correlation with higher production of il- are more frequent in the controls than the patients. then it might be deduced that individuals who inherited these genotypes/alleles are able to produce higher level of il- at the onset of infection and it leads to more ifngamma production and control brucella infection before appearing brucellosis. abstract withdrawn by author objectives: a dsyregulated cytokine response has been shown to play a role in inflammatory bowel disease (ibd) pathogenesis. to dissect the influence of these cytokines, specifically interferon gamma (ifng), on the inflammatory response and colitis we have created a cell specific ifng receptor (ifngr ) mouse mutant. we have generated a mouse line carrying a conditional ifngr allele using the loxp -flp recognition target (frt) approach. a targeting vector with loxp sites flanking exons - and frt sites flanking the neomycin resistance cassette was generated. after confirmation of homologous recombination the neomycin resistance cassette was deleted by crossing with flp deleter mice. cell specific deletion is being performed by crossing conditional 'flox/flox' mice with specific cre expressing mouse lines. functional inactivation of the receptor has been demonstrated by performing western blots to detect phosphorylated and total stat following ifng stimulation. results: successful deletion of the gene and conditional mutants without the presence of the neomycin resistant cassette have been generated. functional inactivation of the receptor with no stat activation following ifng stimulation has been demonstrated in the complete knock out mice. furthermore, flox/flox mice retain full responsiveness to ifng. breeding with lysm cre and cd cre mice will been completed to create a cell specific deletion of the ifng receptor in macrophages/granulocytes and t cells respectively. the specificity of this deletion will be confirmed through cell sorting and functional assays. in order to determine the influence of ifng on a specific cell type a conditional gene targeting approach has been utilised. this has allowed the generation of conditional mice mutants with deletion of ifngr on macrophages and t cells. this has generated a very powerful tool for dissecting the role of this cytokine in numerous disease models. moreover, the ability to cross the conditional mice with additional cre lines will enable the analysis of more cell types in the future. a. gonzalez , r. carretero , p. saenz-lopez , j. cantón , j. carretero , f. ruiz-cabello , l.m. torres , cts- hospital universitario virgen de las nieves, departamento de ginecología, granada, spain, hospital universitario virgen de las nieves, departamento de análisis clí nicos, granada, spain objetives: cervical cancer is almost associated with infection by human papillomavirus (hpv). however, only a subset of infected women will ever develop the malignancy. ifng dinucleotide (ca) repeat polymorphism is responsible for genetic differences in interferon-gamma production. our objective was to investigated the relationship between ifgn polymorphism and cervical intraepithelial neoplasia (cin) methods: we have studied nineteen women with cin and normal women control. dna was extracted from blood samples, and was genotyped for functional microsatellite (ca) repeats in the first intron of ifn-gamma gene. results: heterozygosis in (ca) allele of ifgn is significant more frequent in healthy control than in cin patient (p= , ) in contrast homozygosis for (ca) allele did not show significant differences between both population. analysis of relation between this polymorphism and the cin stage showed that both heterozygosis and homozygosis is correlated with advanced stage (p= , and p= , respectively). conclusions: our study suggest that ifng (ca) allele may influence in cin risk and progression. ifng is associated with hpv clearance, but it also plays a role as inflammatory cytokine, promoting cervical malignant progression. further studies of the role of ifng and other cytokines may contribute to the understanding of cin promotions and progression. introduction: macrophages play a fundamental role in controlling of brucella infection, mainly through the secretion of cytokines such ifn-y and tnf-a. interleukin- (il- ), a th -related cytokine, triggers inflammatory cell recruitment and increases the expression of ifn-y. as the cytokine production is under the genetic control, we decided to investigate the association between il- single-nucleotide polymorphisms (snps) and susceptibility to brucellosis in iranian patients. methods: patients with brucellosis and healthy animal husband men who kept animals with brucellosis, were included in this study. all individuals were genotyped for il- (c t, g a, c a and a t) polymorph isms using pcr-rflp. results: at position , the distribution of c allele and cc genotype were significantly lower in the patients than the controls (p= . and p= . , respectively). at position , the distribution of c allele and aa genotype were significantly higher and lower in the patients than the controls, respectively (p= . , p= . ). discussion: as shown the frequency of cc genotype and c allele at position were higher in healthy controls than the patients. hence, our study provides evidence that presences of cc genotype and/or c allele are significantly associated with susceptibility to brucellosis. the frequency of aa genotype at position is lower in patients than the healthy controls and the frequency of c allele at position is higher in patients than the healthy controls. hence, our study provides evidence that presences of c allele and lack of aa genotype are significantly associated with susceptibility to brucellosis. objectives: increasing evidence is emerging regarding the ability of mammary epithelial cells to respond to various cytokines. our aim was to investigate the cytokine receptor phenotypes of two distinctive human mammary cell lines, mcf- and skbr- , as well as their ability to respond to several cytokines and to the g- gpr agonist. the mcf- and skbr- human adenocarcinoma cell lines were investigated by immunocytochemistry and flow cytometry for the expression of the following cytokine receptors: il- ra, il- rg, il- /il- /gm-csfr, il- /il- r, il- ra, il- ra, il- r (gp ), il- ra, il- r, tnfr i, tnfr ii, ifngra, cxcr , cxcr , cxcr , cxcr , cxcr . cells were incubated with il- , ifn-g, tnf-a and tnf-b (for which both cell lines displayed receptors) alone and with the gpr agonist. cytosolic ca + concentrations [ca + ] i were measured by the microfluorimetric technique. results: different cytokine receptors phenotypes emerged for the two cell lines. the less well differentiated skbr- cells were found positive for a larger number of receptors than the mcf- cells. both cell lines displayed an important heterogeneity for each of the investigated molecules, in terms of number of positive cells and expression intensity, with chemokine receptors percentages constantly higher than for the other receptors. pretreatment of mcf- cells with il- reduced the calcium response to g- , while pretreatment with ifn-g and tnf-a potentiated the calcium response to g- . tnf-b had no effect on mcf- g- stimulation. no direct effect on basal [ca + ] i stimulation could be noticed when administering the cytokines alone. in skbr- cells, pretreatment with il- or tnf-b had no effect on basal [ca + ] i and did not significantly alter the calcium response to g- , while pretreatment with ifn-g induced calcium oscillations and potentiated the calcium response to g- . pretreatment with tnf-a produced calcium oscillations and reduced the response to g- . conclusions: mcf- and skbr- cell lines express distinctive cytokine receptor phenotypes. furthermore, their ability to respond to cytokines in terms of modulating the gpr stimulation proved to be different. the susceptibility towards various soluble factors of these cell lines can offer us some insights on the complexity and individuality of each primary tumor and thus the distinctive evolution of each particular patient. results: in initial analyses of the early infection phase we identified splenic pdcs expressing ifnb/yfp. furthermore, we show for the first time in vivo that these ifnb producing pdcs are not directly infected with mcmv and that not only cdcs but also cd a -pdcs expressed gfp as a marker for mcmv infection. we observed that at early time points equal frequencies of cd a -pdcs and cdcs were infected with mcmv, whereas after h p. i. the frequency of infected cdcs wins over that of the pdcs. conclusions: with this experimental system we are able to visualize the ifnb response vs. the infection status of mcmv in vivo on a single-cell level. from our initial results we can conclude that infected cells in the spleen induce ifnb production in noninfected pdcs which initiate the antiviral immune response in this organ. recently, we have developed live-attenuated arenavirus vaccine candidates based on lymphocytic choriomeningitis viruses (lcmv) carrying the vesicular stomatitis virus (vsv) envelope gene (rlcmv/vsv-g). since interferon (ifn) signaling is known to be crucial for adaptive immune responses against wildtype (wt) lcmv and control thereof, we wanted to assess the innate and adaptive immune requirements for containing rlcmv/vsv-g infection. mice lacking both, ifn type i and ii receptors generated potent virus-specific cd t cells and neutralizing antibody responses against rlcmv/vsv-g, even exceeding the respective responses in wild type animals. in further contrast to wt lcmv infection, ifn type i and ii signaling was dispensable for rlcmv/vsv-g control. rlcmv/vsv-g infection of rag -deficient mice (lacking mature t and b cells) resulted in persistent levels of circulating viral rna that was solely detectable by qrt-pcr but not by classical measures of infectivity. overt viremia was only found in mice lacking both rag and ifn type i receptor (ar). thus, viral attenuation for vaccine use was found to considerably relax the ifn-dependence of adaptive immune responses and virus control. this redundancy of ifn and adaptive immune responses in rlcmv/vsv-g control provides a better understanding of the attenuation of this vector. it furthermore suggests that the virulence of a particular virus may influence the interferon-dependence of cd t cell and antibody responses, which may have implications for vaccine development. objectives: the class of type i interferons (ifns) consist of multiple ifnas and a single ifnb subtype. although being important for anti-viral defence they have been shown to be detrimental for the host during bacterial infections. while in general the first ifn to be produced is ifnb, the cell types responsible for its initial production remain unclear. to assess the cellular sources of ifnb and its role for the outcome in bacterial infections we use an ifnb reporter-knockin mouse model, in which yellow fluorescent protein (yfp) is expressed from a bicistronic mrna linked by an internal ribosomal entry site (ires) to the endogenous ifnb mrna. methods: to induce a type i interferon response we intravenously injected the tlr agonists poly(i:c) and cpg , respectively, or infected reporter mice or bone marrow derived macrophages (bmdms) or dendritic cells (bmdcs) with the facultative intracellular pathogen listeria monocytogenes. the spatiotemporal tracking of the ifnb response was done using facs analysis and immunofluorescent microscopy. results: after poly(i:c) injection in vivo a small subpopulation of ifnb + cd a + dendritic cells relocalize from the red pulp via the marginal zone to the t cell areas of the spleen whereas ifnb + activated pdcs were localized in the splenic white pulp at the t/b-cell interface after cpg administration. in vitro infection of bmdms, bmdcs and flt -l derived dcs with listeria monocytogenes resulted in a low frequency of ifnb + cells depending on the listeria strain used and multiplicity of infection with bmdms being the most potent producers of ifnb. in vivo mostly activated f / + macrophages were accountable for the ifnb expression. simultanous visualization of the cellular state of infection shows that ifnb + bmdms carry a higher bacterial load then ifnbcells. the cellular detection of ifnb expression reveals a remarkably low frequency of ifnb-producing cells in response to distinct pamps or the infection with intracellular bacteria. this hints at a superior role of few specialised cells for mounting a significant response against distinct stimuli or bacterial disease. additional data will be presented further resolving the timecourse of the ifnb response vs. the state of infection after bacterial challenge. the spleen is a secondary lymphoid organ that is characterized by highly specialized structures and plays a crucial role in the defense of blood-borne pathogens. we investigated the role of the spleen in the generation of antigen-specific cytotoxic cd t cell (ctl) responses in a systemic infection with recombinant adenovirus expressing ovalbumin (adova). although adenovirus mainly infects the liver, the ctl response requires the spleen, as splenectomized mice were incapable to mount an antigen-specific ctl response upon systemic challenge with adova. additionally, dendritic cells (dc), macrophages and an intact splenic structure were mandatory for the induction of an efficient ctl response. we detected adova specific ctl responses only in splenectomized mice that received splenic autotransplants but not after adoptive transfer of single cell splenocytes. furthermore, we asked how toll-like receptor (tlr) ligands influence adova-specific ctl responses. tlr ligands as "danger signals" are generally known to exert immune stimulatory effects. importantly, we observed that systemic administration of single-stranded rna (ssrna) prior to adova infection inhibited the generation of antigen-specific ctl responses in a tlr and type i interferon (ifn) dependent manner. ssrna injection induced the production of type i ifns as detected in sera and supernatants of splenocytes isolated from wild-type mice. experiments performed in ifnbeta reporter mice revealed that splenic plasmacytoid dcs represented the cellular source of type i ifns. additionally, splenic cd c+ dcs purified from ssrna pretreated mice showed a reduced capacity to cross-prime ova-specific cd t cells upon adova challenge. in vivo pre-activation of endogenous ova-specific cd t cells as well as an adoptive transfer of in vitro activated transgenic ova-specific cd t cells prevented the ssrna mediated suppression of the ctl activity. we assume that dcs preactivated by systemic ssrna were impaired in their ability to activate naive cd t cells in response to an adova infection due to an impaired cd t cell help. taken together, we show that type i ifns cannot only stimulate, but also inhibit the induction of ctl responses in the spleen. within hours after infection many viruses induce early type i interferon (ifn) responses that can confer protection against lethal infection. modified vaccinia virus ankara (mva) is a highly attenuated vaccinia virus (vacv) strain generated by more than passages in chicken embryo fibroblasts. mva stimulates systemic ifn responses, whereas vacv-induced cytokine milieus are dominated by il- . to study the impact of virus-triggered ifn on the induction of t cell responses, we used recombinant mva-gp and vacv-gp expressing the gp epitope of lymphocytic choriomeningitis virus. for adoptive transfer experiments transgenic mice expressing the p t cell receptor recognizing the gp epitope were intercrossed with mice devoid of the ifn receptor (ifnar -/-) to obtain p ifnar -/mice. upon adoptive co-transfer of p ifnar -/and p ifnar wt/wt t cells and subsequent challenge with mva-gp , a massive expansion of p ifnar wt/wt t cells was observed, whereas p ifnar -/-t cells expanded less efficiently. in contrast, challenge with vacv-gp induced a rather similar expansion of ifnar competent and ifnar deficient p t cells. in the absence of ifnar-triggering t cell expansion was associated with reduced proliferation capacity and increased apoptosis. to study the impact of ifnar-signaling on the expansion of endogenous t cells, conditional mice with a t cell-specific ifnar-ablation were infected with mva. interestingly, in those mice the virus-specific t cells showed a reduced expansion compared to t cells of wt mice. additionally, we found that ifnar-triggering of dendritic cells but not of macrophages further supported specific t cell expansion. thus, upon virus infection virus-associated properties affected the overall cytokine milieu that influenced the quality and the quantity of expansion of virus-specific t cells. to delineate h n -specific signaling patterns we used a genome-wide comparative systems biology approach analyzing gene expression in endothelial cells infected with three different human and avian influenza strains of high and low pathogenicity. blocking of specific signaling pathways revealed that h n induced an exceptionally nf-kb dependent antiviral response. irf is essential part of this interferon-response of human endothelia. furthermore, we identified hmga as a novel transcription factor specifically responsible for the overwhelming proinflammatory but not anti-viral response induced by h n . finally, nfatc was found to be a transcriptional regulator for specifically h n -induced genes. we therefore describe for the first time defined signaling patterns specifically activated by h n which, in contrast to low pathogenic influenza viruses, are responsible for an imbalance of overwhelming proinflammatory and impaired anti-viral gene programs. objectives: to study the early events of immune antagonism by influenza virus in vivo and how this process impacts the timing at which adaptive immunity is generated. methods: to dissect the contribution of the unique viral antagonist ns , delta-ns influenza virus (a recombinant influenza virus lacking the ns gene) was compared side by side with wild type influenza virus in vivo. mice infected with influenza virus were sacrificed at different time points after infection. to study the onset of inflammation during infection lung and blood samples were isolated with a selected panel of inflammatory and antiviral proteins that were measured by multiplex elisa and quantitative pcr (qpcr). to determine the bridging between innate lung inflammation and adaptive immunity, the kinetics of lung antigenbearing dendritic cell (dc) migration to the draining lymph nodes was quantitated in infected mice. further, functional in vitro and in vivo assays in infected animals with influenza-ova viruses were used to determine whether antigen-bearing migratory dcs were capable of priming transgenic t cells. to investigate the effect of ns during the onset of immunity in vivo, we systematically studied the early events occurring in the lungs and draining lymph nodes upon infection with influenza virus. strikingly, no sign of innate immunity was detected in the lungs for almost two days after infection when a sudden inflammatory burst including ifns, cytokines, and chemokines occurred. this burst preceded the robust dc migration and t cell activation in the lymph nodes. virus-loaded dcs appear in the lymph node starting days post-infection, reached its maximum at day , and triggered t cell priming in vivo. a direct comparison of delta-ns virus with wild type virus infection demonstrates that virus can only trigger rapid inflammation in vivo when it lacks the ns protein. we demonstrate that the delay in the generation of immediate lung inflammation is mediated by the influenza ns protein. thus, we propose that the virally encoded ns protein establishes a time limited "stealth phase" where replicating influenza virus remains undetected thus preventing the immediate initiation of innate and adaptive immunity. keratinocytes represent the major cell population of human epidermis which provides a first line of defense barrier for the host. in addition, keratinocytes actively participate in immune response. viral double-stranded rna (dsrna) is the most important viral structure involved in activation of innate immune response. intracellular detection of dsrna triggers secretion of soluble signaling molecules, interferons, and activates pro-inflammatory response and programmed cell death, apoptosis. here we have used subcellular proteomics to characterise dsrna-induced human keratinocytes. cells were transfected with a mimetic of dsrna, poly(i:c), after which the cells were fractionated into cytoplasmic and mitochondrial fractions. these proteomes were analysed using two-dimensional electrophoresis in combination with mass spectrometry, immunoblotting and confocal microscopy. we show that several proteins involved in apoptosis, cytoskeleton reorganization and intracellular transport are up-regulated upon dsrna stimulation. in mitochondrial proteomes the expression of structural proteins, especially fragments of cytokeratins, is highly up-regulated. we show that cytokeratin is cleaved during poly(i:c) stimulation and fragments are solely localized in mitochondria. similar degradation of cytokeratin is seen in emcv-and vsv-infected keratinocytes. cytokeratin fragmentation after dsrna stimulation is dependent on caspase activation, which indicates a role for cytokeratins in the regulation of apoptosis during viral infection. in addition, we show that - - proteins are upregulated in both mitochondrial cell fraction and cytoplasm after dsrna-stimulation and during viral infection. viral dsrna also induced transient phosphorylation of - - target proteins. thus, these results suggest that - - proteins have a regulatory role in host defence against viral infections. i. wessels , d. fleischer , l. rink , p. uciechowski institute of immunology, rwth aachen university hospital, aachen, germany objectives: the proinflammatory cytokine interleukin (il)- b mediates the expression of a variety of proteins responsible for acute inflammation and chronic inflammatory diseases. however, the molecular regulation of il- b expression is not elucidated, yet. it is known that the il- b promoter is packaged into a nontranscribed but poised architecture in monocytes, rapidly producing il- b when stimulated. b-cells which are il- b non-producers reveal an inaccessible promoter structure. in this study the chromatin structure of the il- b promoter and the impact of methylation on il- b expression were examined in a cellular monocytic differentiation model. methods: promyeloid hl- cells were differentiated into monocytic cells after dihydroxyvitamine d treatment. the monocytic phenotype was confirmed by flow cytometry. the il- b promoter was analyzed using the chromatin accessibility by real time pcr (chart) assay. to test the influence of methylation, cells were treated with -aza- -deoxycytodine (aza) and changes in il- b expression were measured by pcr, elisa and western blot analyses. results: in contrast to undifferentiated hl- cells, differentiated cells displayed upregulation of cd antigen and acquired the ability to express il- b. by comparing the accessibilities of il- b promoter we detected that the il- b promoter was not accessible in undifferentiated hl- cells but highly accessible in differentiated monocytic cells. the accessibilities of differentiated cells were comparable to that observed in primary monocytes. lps stimulation did not affect promoter accessibility in promyeloic and monocytic hl- cells, demonstrating that the chromatin remodeling of the il- b promoter depends on differentiation but is independent of the transcriptional status of the cell. demethylation via aza led to the induction of il- b expression in both undifferentiated and differentiated cells which could be increased after lps stimulation. conclusion: two independent mechanisms involved in the regulation of il- b expression were found. our data indicate that the il- b promoter is reorganized into an open conformation during monopoiesis and that the established poised structure is a privilege of mature monocytes but not of the entire myeloid lineage. as a second mechanism, il- b expression is regulated by methylation acting independent of the developmental stage of myeloid cells. a. holweg , g. wetzel , h. arnold , a. gessner microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany the p family members of interferon (ifn) inducible gtpases also known as guanylate binding proteins (gbps) are among the most abundantly induced transcripts after stimulation with ifn-g. although the stimulatory capacities of the toll like receptor ligands lps and cpg, cytokines like ifn-b and il- b and the intracellular pathogens listeria monocytogenes and toxoplasma gondii have been described to induce their expression, the function of gbps during bacterial infections is still ill defined. here we report for the first time the massive induction of murine gbps in two independent in vivo models of pneumonia (infection with streptococcus pneumoniae and pseudomonas aeruginosa). a strong and rapid induction of mgbp , , , and mrna after intratracheal infection was detected by realtime pcr analysis of bronchoalveolar lavage (bal) cells. using newly generated antibodies in western blots and fluorescence microscopy, we identified macrophages as the main producers of mgbps in bal samples. although the signaling cascade involved in the upregulation of mgbps after ifn-g stimulation has been extensively studied, the mechanisms responsible for mgbp induction upon bacterial stimuli are unclear. in this study we could show that the induction of mgbps upon infection is abrogated in ifn type i/ type ii receptor double-deficient mice and thus absolutely dependent on endogenous interferon production. in contrast, the tlr adaptor molecule myd was found to be dispensable arguing for a trif-mediated interferon production subsequentially resulting in enhanced gbp expression. based on these findings our future experiments will address the functional role of gbps in innate and adaptive immune responses against extracellular bacteria. ( )). activation of resident microglia cells and infiltrating brain macrophages appeared to play a role in modulating virus replication shortly after infection but also appeared to be responsible for the inflammation in brains of infected mice. clearance of replicating virus from the cns required mcmv specific cd + t lymphocytes whose effectors functions still remain incompletely defined (j. immunol. aug ; ( )). humoral immunity appeared to also play a role in the control of mcmv infection in developing brain. infected newborn mice treated with mcmv-specific antibodies had lower viral titers in the cns, significantly less cns inflammation and improved neuronal migration and increased cerebellar area as compared to control mice (j. virol. dec; ( ) ). conclusion: peripheral inoculation of the virus induces focal infection and inflammation in the developing mouse cns followed by strong innate and specific immune response that could also alter developmental programs required for normal development of mouse brain. passive immunization of infected newborn mice reduces mcmv-related pathology in infected brain suggesting that antiviral antibodies are an important component of immunological responses during cmv infection of developing cns. chronic inflammation is associated with the promotion and enhancement of malignancy and tumor growth. tumors enhance the accumulation of myeloid derived suppressor cells (mdsc), which contribute to tumor escape, immune tolerance and immune suppression. previously, we have shown that tumor-derived inflammatory cytokines, such as il- b in the tumor microenvironment can induce a massive accumulation of mdsc in the spleen of tumor bearing mice and induce t cell suppression. in this work, we describe a novel polymorphnuclear mdsc subpopulation -inflammatory mdsc (infmdsc) which accumulates in the bm and spleen of mice bearing inflammatory t breast cancer cells over expressing il- b ( t /il- b) tumor cells, but rarely in untransfected t cells. secretion of il- b from tumor cells is crucial for infmdsc generation and accumulation. infmdsc have the ability to suppress nk cell activity via reduction of the nk activating receptor nkg d in vivo, and in a cell-cell contact dependent manner in vitro. inflammation up-regulates il- ra expression on the cell surface, which correlates with tumor growth and induction of suppression on nk cells. our data suggest that tumor derived inflammation enhances a specific mdsc subset which has the ability induce suppression of nk cells, and perhaps can serve as a new chemotherapy target. objectives: lps constitutes a main target of innate immune recognition of gram-negative bacteria and other lps carrying pathogens. cytokine production after in vitro stimulation of whole blood with lps is used as an expression of individual lps reactivity. we assess genome-wide data to analysed the association to lps induced cytokine response, and replicated the top findings in two independent cohorts. materials and methods: we used healthy caucasian blood donors as discovery samples, and replicated snps from affymetrix -genome-wide human snp array . having p x - in two independent cohorts each consisting of blood donors using a customized chip from illumina and real-time pcr respectively. in all the three cohorts whole blood samples was stimulated for h and we measured the levels of il- , il- , il- , tnf-alfa and il -ra with r&d ® assays on luminex platform.. the association analysis was performed with wald statistical test assuming an additive model. the discovery sample statistical analysis revealed snps with p x , * - . to identify/replicate the association of cytokine production for these we reanalysed these on a cohort. a combined analysis revealed snps with p x , * - .these results are not genome wide significant. the snps showing nominal association to lps cytokine response are being analysed in a replication cohort conclusion: we find nominal associated snps between lps stimulated cytokines in blood samples of healthy caucasian blood donors. the importance of these snps are to be determined in a replication cohort. adipocytes, so far known for their lipid-storage capacity came into the focus of interest because of their immunoregulatory properties resembling those of innate immune cells. adipocyte-derived pro-inflammatory mediators contribute to the development of chronic inflammation, thereby promoting the progression of insulin-resistance/metabolic-syndrome and diabetes. the physiological signals inducing the secretion of inflammatory mediators by adipocytes are unknown. heat shock proteins, such as hsp have been identified as potent regulators of inflammatory innate immune cell-activities, whereas their influence on adipocyteactivities remained elusive. here, we investigated the regulatory effects of hsp on adipocytes. for the first time we could show a hsp -stimulated release of the proinflammatory cytokines il- , cxcl and mcp- in a time-and concentration-dependent manner from murine t -l adipocytes. analyses of hsp -signalling in these adipocytes revealed that members of the mapk-family (erk / , p ) and the transcription factor nfkb are involved in hsp -mediated induction of the mediators il- , cxcl and mcp- . binding-studies with fluorescence-labelled hsp demonstrated that the interaction of hsp with adipocytes exhibits basic features of a receptor-mediated binding. hsp -binding to adipocytes was saturable and reached its maximum at . mm. binding was inhibitable only by the unlabelled ligand ( %), but not by unrelated proteins, thereby proving the specificity of hsp -binding. further analyses to characterize hsp -receptor structures on adipocytes revealed the presence of toll-like receptor (tlr) on adipocytes. tlr has been found to be expressed on macrophages and to interact with hsp , therefore suggesting tlr as a potential receptor candidate for hsp on adipocytes. in order to identify the responsible binding-epitope of hsp we investigated the effect of specific antibodies directed against different epitopes of the hsp -molecule. incubation with antibodies directed against the n-terminus of hsp (aa - ; - mg/ml) were capable of inhibiting the hsp -binding to adipocytes ( - %) indicating that the n-terminal region of hsp is involved in receptor binding. our experiments demonstrate that hsp stimulates the release of proinflammatory adipocyte mediators. the findings implicate that the hsp -mediated induction of adipocyte mediators contributes to inflammatory processes observed in obesity-associated disorders and could serve as a target for the development of therapeutic strategies for patients suffering from diabetes or diabetes-related disorders. legionella pneumophila, a gram-negative facultative intracellular bacterium, is the causative agent of a severe pneumonia known as legionnaires' disease. classically, type i ifns (ifna/b) have been associated with antiviral immunity. ifna/b signal through the ifna/b receptor (ifnar) leading to the induction of hundreds of ifn-stimulated genes (isgs), many of which have anti-microbial activities. recently, it was demonstrated that ifna/b are also produced in host cells infected with (intracellular) bacteria or stimulated with cytosolic dna. here we show by rnai that l. pneumophila infected host cells produced ifnb dependent on irf . we observed enhanced l. pneumophila replication in mouse macrophages lacking ifnar and human cells after irf knock-down, suggesting that endogenously produced ifnb activates a cell-autonomous defence against legionella. moreover, ifnb treatment restricts legionella replication in human and murine host cells. ifna/b impacts formation of large replication vacuoles, but appears not to influence the recruitment of er markers nor fusion of the legionella-containing vacuole with the lysosome. moreover, ifna/b-mediated cellautonomous defence was independent of autophagy and pyroptosis. we thus hypothesize the crucial involvement of antibacterially acting isgs. ongoing studies focus on the role of ifn-induced immunity-related gtpases (irgs). mesenchymal stem cells (mscs) are identified by their capacity to differentiate into connective tissue cell types. mesenchymal stem cells also show a high plasticity and account for a potential therapeutic efficacy. several authors have reported the expression of alfa-smooth muscle actin (a-sm actin) by msc. this protein has been considered a marker for the myofibroblast, has the capacity to polymerize into the cytoplasm and contribute to the cell contractility. this activity may be crucial for the changes of the cell shape, for cell-cell interactions and may therefore be relevant for the physiology of msc. in this work, we study the presence of alfa-sm actin in human msc by flow cytometry and immunoflurescence. we also study the contractility of these cells under the effect of different cytokines. human bone marrow samples were obtained from bone marrow aspirates. bone marrow mononuclear cells were isolated by density gradient centrifugation and cultured in opti-mem culture medium with % of fetal calf serum at °c and % co . bone marrow nonadherent cells were removed after h, and culture medium was refreshed twice per week thereafter. cells grew adherent, with a fibroblastic morphology and expressed cd , cd , cd , cd and stro- and were negative for cd . intracellular staining was performed for alfa-sm actin. cell contractility was measured with the collagen gel contraction assay. alfa-smooth muscle actin was detected in almost % of msc. interleukin- , ifn-gamma and tnf (th cytokines) increased msc contractility, whereas il- (a th cytokine) decreased msc contractility. by immunofluorescence, we observed that il- , ifn-gamma and tnf increased the incorporation of alfa-sm actin into the stress fibers of msc, whereas il- decreased that incorporation. our results suggest that th and th cytokines regulate msc physiology by acting on their contractility. aims: thapsigargin (tg) is a sesquiterpene lactone (sl) of guaianolide type isolated from the mediterranean plant thapsia garganica l. it is widely recognized as an inhibitor of sarco-endoplasmic reticulum ca + -atpase leading to elevation of intracellular calcium. this activity is shared by trilobolide (tb), a sl from laser trilobum (l.) borkh. tg has been shown to possess prospective immunotherapeutic properties. it kills slowly proliferating and non-proliferating cells, and inhibits replication of viruses. the aim of our work was to investigate possible immunostimulatory potential of tg and tb. methods: the effects of the agents were analyzed under conditions in vitro using rat and mouse resident peritoneal cells (pecs), and human peripheral blood mononuclear cells (hpbmcs). they were cultured in density of × /ml in complete rpmi- medium. supernatant levels of ifn-g were determined by elisa. production of no by animal pecs was assayed using griess reagent. possible contamination of the samples with lps was excluded using the chromogenic limulus amoebocyte assay. results: we have found that both tg and tb at as low doses as nm and nm induce ifn-g secretion in rat pecs and hpbmcs, respectively. the concentration of ifn-g produced by the highest dose of the agents ( mm) at the -h culture interval reached the values of approximately ng/ml and ng/ml in rat pecs and hpbmcs, respectively. the increase was apparent within the interval of - h in rat pecs. the -h interval was optimal for accumulation of ifn-g in cultures of hpbmcs. only modestly enhanced secretion of ifn-g was observed in mouse pecs. production of ifn-g was found to depend on activation of nf-xb and map kinases p and erk / . it was not suppressed by the calcium chelating agents bapta-am and tmb- . the tg-and tb-induced ifn-g production was associated with activation of the high-output production of no. conclusions: sesquiterpene lactones tg and tb are potent inducers of ifn-g in animal and human cells. the effect is independent of their serca inhibitory potential. underlying mechanism(s) of the immunostimulatory effects remain to be elucidated. acknowledgements: the work was supported by the grant gacr / / . pin is a peptidil-prolil-cis-trans isomerase that specifically binds phosphorylated ser/thr-pro protein motifs and catalyzes the cis/trans isomerization of peptides. mitotic proteins, cytoskeleton, transcription factors and apoptotic proteins are pin substrates and targeting sites. recent data show that pin interacts with apo-bec g (a g). the pin /a g interaction results in a reduced a g expression and a diminished a g-mediated restriction of hiv. two single nucleotide polymorphisms (snps) in the promoter region of the pin gene (- g/c and - t/c) modulate pin expression; in particular, the - gc genotype or cc haplotype are associated with reduced protein levels (neurobiol. aging, ; - , ) . the - c/g and - t/c polymorphisms in the promoter of pin gene as well as pin protein levels were analyzed in exposed seronegative individuals (esn), heterosexual partners of hiv-infected patients; hiv-infected patients (hiv) and healthy controls (hc). the genotype and allele distributions of the - snp was skewed in esn (genotype: p= . ; allele: p= . ). in particular esn showed a significantly lower frequency of the - gg genotype compared to hiv and hc (p= . and p= . , respectively) and consequently a lower g allele frequency (p= . and p= . , respectively). no significant differences were found for the - snp. these snps are in linkage disequilibrium and combine to form haplotypes. conclusions: our findings support the role of hiv viral replication as the most important promoter of immune activation and prove the importance of art in reducing immune activation and viral replication even in a sub-saharan african environment, where patients are exposed to an abundance of other infectious agents. our data further indicates that hiv replication rather than host genetics is the key determinator of circulating cytokine levels among the studied hiv infected participants. aims: acyclic nucleoside phosphonates (anps) are synthetic analogues of natural nucleoside monophosphates. they have proved to be outstanding antivirals inhibiting replication of both dna-viruses and retroviruses. tenofovir, -(r)- [ -(phosphonomethoxy) propyl]adenine [(r)-pmpa] is broadly used for therapy of aids, adefovir, -[ -(phosphonomethoxy)ethyl]adenine (pmea) has been approved for the treatment of hepatitis b. the aim of our work was to investigate possible interactions of anps with production of cytokines and nitric oxide (no) implicated in antiviral defence mechanisms. the immunobiological effects of anps were screened in vitro using mouse resident peritoneal cells. they were cultured in density of × /ml in complete rpmi- medium. secretion of cytokines was determined after the -h culture by elisa. production of no was assayed at the interval of h using griess reagent. approximately compounds belonging to several distinct anp groups were included in the study: a) pmea derivatives, b) pmedap i. e. -[ -(phosphonomethoxy)ethlyl]- , -diaminopurine derivatives, c) -[ -hydroxy- -(phosphonomethoxy)propyl]-adenine (hpmpa), and hpmpdap derivatives, d) guanidino analogues of pmpdap, e) -heteroalkyl substituted -amino- -guanidinopurines, and f) -amino- -(purin- -yl)propanoic acid derivatives. possible presence of lps in the stock solutions of the samples was checked and excluded using the chromogenic limulus amoebocyte assay. results: approximately compounds were found to activate the secretion of anti-hiv effective chemokines rantes and mip- a and cytokines tnf-a and il- . although these anps did not stimulate biosynthesis of no on their own, they substantially augmented production of no triggered by ifn-g. no clear-cut relationship between the chemical structure and biological effects of anps was observed. however, the most effective proved to be the n -cycloalkyl derivatives of pme-dap. the effects were produced in a dose-dependent fashion, exhibiting the immunostimulatory effects at as low concentrations as to mm. the remarkably enhanced secretion of chemokines was reached within - h of the cell culture. the effects were found to depend on activation of nf-kb. conclusions: it may be suggested that anps represent a new generation of antivirotics with combined antimetabolic and therapeutically prospective immunostimulatory properties. acknowledgements. the work was supported by the grant m . host related immune factors in childhood chronic hepatitis b and change of initial profile with ifn-a treatment needs to be clarified. sixteen patients were included, and million units of ifn-a treatment three times a week for months was initiated. before and after treatment: percentages of the il- and ifn-g in cd + t cells were assessed to determine intracellular t helper cell (th ) type cytokine expression. similarly, percentages of intracellular il- and ifn-g were detected to verify cytotoxic t cell (tc ) type cytokine expression in cd + t cells. percentage of th and tc type cytokine expression, (il- and il- ) were determined in cd + and cd + t cells, respectively. six ( %) of these were evaluated as having no response and the other half with partial/complete response. all patients had higher percentages of th cells with respect to healthy controls before treatment. tc percentages, both tc and tc , were significantly different between these groups, being higher in the patient group. when values of no responder group were compared with healthy controls, il- expression was higher and the percentages of th type cells were significantly low. il- expression in th and tc cells decreased after treatment in the unresponsive group. intracellular cytokine profiles of treatment responders and normal controls were not different. this has been the first study in children comparing baseline and post treatment intracellular cytokine profiles with healthy controls. the frequency ( , %) of high concentration of igg anti-oxldl antibodies in patients with hcv infection were significantly elevated in comparison to healthy subjects ( , % according to bibliographic data). the immune response was significant but it was not assosiated with the viral load. it is probable that humoral immunity plays a critical role and contributes in an immunoinflammatory reaction of hcv-infection. abstract withdrawn by author t. schwandt , f. juengerkes , b. schumak , g. gielen , j. kalff , p. knolle , b. holzmann , a. limmer university hospital bonn, institute of molecular medicine and experimental immunology, bonn, germany, university hospital bonn, department of surgery, bonn, germany, department of surgery, tu munich, munich, germany bacterial translocation is a possible risk of abdominal surgery and could be the cause of life-threatening consequences such as organ failure and septic shock. patients surviving septic shock often suffer from opportunistic infections as well as defects in adaptive immunity. here we investigated the influence of bacteremia and bacterial translocation on systemic adaptive immune responses using murine models. to mimic abdominal surgery, mice were subjected to intestinal manipulation (im). to study septic conditions, mice underwent colon ascendens stent peritonitis (casp) or received e.coli intravenously. we monitored the distribution of gut-derived bacteria by in vivo imaging (xenogen) and additional microbiological assays and determined antigen-specific ctl responses towards subsequent infection with recombinant adenoviruses (av). we detected comparable amounts of bacteria in lung, liver and spleen of mice that underwent casp or were injected i. v. with e.coli. in addition, mice infected systemically with av lacked an antigen-specific ctl response, whereas the ctl responses of locally av infected mice were not affected. in contrast, bacteria were detected in lung and liver but not in spleen of mice that were subjected to im or received e.coli by injection into the hepatic portal vein. here, the ctl response was not impaired. depletion experiments imply that kupffer cells as well as soluble mediators such as tumor necrosis factor alpha play an important role in trapping and clearance of translocated bacteria in liver and lung. our experiments demonstrated that translocation of bacteria did not cause immune suppression as long as they did not reach the spleen in high numbers. we suggest that liver and lung fulfill a filter function to prevent systemic distribution of gut-derived bacteria. failure of or bypassing these barriers might enable bacteria to access the spleen and thus cause systemic suppression of adaptive immunity, whereas induction of local immunity is not affected. objectives: varicella-zoster virus (vzv) is one of the most frequent pathogens for which a vaccine is available. tropism of vzv for skin is the most obvious manifestation of vzv infection, producing vesicular cutaneous lesions that are associated with varicella and herpes zoster. the striking difference in the clinical outcome of infection by rush inducing circulating virulent vzv strains and asymptomatic infection by the vaccine leads to the assumption that the virus interferes with cutaneous immunity. therefore, we comparatively investigated the impact of vzv clinical isolates and the vaccine on the reciprocal crosstalk of immature dendritic cells (idcs) and epithelial gd t cells. methods: skin punch biopsies of herpes zoster patients were analyzed by dual immunofluorescence microscopy. phenotypic changes of cutaneous dcs and monocyte-derived dcs after vzv infection were investigated by flow cytometry. the cytokine profile of vzv-infected dcs and epithelial gd t cells was determined through elisa. results: we observed that innate lymphocytes recognize dcs, which infiltrate herpes zoster lesions, after infection with vzv. strikingly, only the vaccine but not vzv clinical isolates could license the bidirectional crosstalk between idcs and gd t cells resulting in release of ifn-g and il- , the signature cytokines of antiviral immune responses. this is the first demonstration that virulent virus strains disrupt dendritic cell instruction whereas the corresponding vaccine does the opposite. we describe a novel immune evasion strategy in the skin, which provides the opportunity for efficient and symptomatic virus replication. this result is also of practical importance: future vaccine design has to ensure that candidate vaccines do not impair dc instruction in order to allow stimulation of powerful antiviral immune responses. a. jafarzadeh rafsanjan university of medical sciences, rafsanjan, iran, islamic republic of objective: it has been reported that the caga + h. pylori strains induce more severe gastric mucosal inflammation. the aim of this study was to investigate the association of the h. pylori virulence factor caga with serum levels of il- and il- in h. pylori-infected duodenal (du) patients and h. pylori-infected asymptomatic (as) carriers. methods: totally, h. pylori-infected du patients ( patients were positive for anti-caga antibody and patients were negative for anti-caga antibody), h. pylori-infected as carriers ( subjects were positive for anti-caga antibody and subjects were negative for anti-caga antibody) and healthy uninfected subjects (as a control group) were enrolled to study. a blood sample was obtained from all participants and the serum levels of il- and il- was measured by elisa method. the mean serum levels of il- in total du patients ( . pg/ml ± . ) was significantly higher than those observed in total as subjects ( . pg/ml ± . , p x . ) and healthy control group ( . pg/ml ± . , p x . ). in du group, it was found that the mean serum levels of il- in subjects with positive test for anti-caga ( . pg/ml ± . ) was significantly higher than those observed in subjects with negative test for anti-caga ( . pg/ml ± . ; p x . ). the mean serum levels of il- in du ( . pg/ml ± . ) and as groups ( . pg/ml ± . ) was significantly higher than those found in uninfected control group ( . pg/ml ± . , p x . and p x . , respectively). however, no significant difference was observed for mean serum levels of il- between du and as groups. moreover, in both du and as groups the mean serum levels of il- was not significantly differ in subjects with positive test for anti-caga and those were negative for anti-caga antibody. the results of the present study showed higher serum concentrations of il- and il- in h. pylori-infected subjects as compared with control group. in du group the expression of il- influenced by the bacterial caga factor. a. aral , , a. atak gazi university faculty of medicine, department of immunology, ankara, turkey, gazi university institution of health sciences, department of immunology, ankara, turkey objective: ebv is a human herpesvirus, which infects human b lymphocytes latently and immortalizes the cells due to transformation. ebv infection is asymptomatic in childhood while it may cause a self-limiting lymphoproliferative disorder called infectious mononucleosis (im) in adolescence. in immunodefective patients, the virus may lead to malignancies like burkitt's lymphoma, nasopharyngeal carcinoma and immunoblastoma. the virus can also cause latent infections. cytokine production in response to ebv infection differs according to the phase of the infection. neopterin, ifn-g and il- levels are elevated in acute ebv infection in vitro; but in chronic phase, particularly, il- elevation could not be detected. tnf-a enhances the effects of ifn-g on neopterin synthesis while neopterin enhances the secretion of tnf-a via various stimuli. ifn-g levels are elevated particularly in the acute phase of im. since the elevation of cytokine levels changes according to the phases of disease, it's thought that the association between host defense and viral replication depends on different parameters. ifn-g is the major stimulus for neopterin synthesis, which stimulates monocytes and macrophages primarily. increased production of neopterin in body fluids can be used to monitor the activation of cell mediated immunity. method: in order to analyze the effects of neopterin release and other cytokines, mononuclear cells have been isolated from peripheral blood samples of healthy donors and transformed via ebv. neopterin, ifn-g, tnf-a and il- levels have been measured with eia kits in culture supernatants. results: neopterin levels increased dependent on time and independent of ebv transformation. in ebv-transformated cell culture tnf-a levels increased beginning from the th hour and reached to maximum levels at the st week and decreased again at the rd week; however there were no significant differences between the levels among three weeks. likely tnf, ifn-g levels also increased at the st week and started to decrease at the rd week. il- reached to its peak at the rd week. conclusion: according to these results, neopterin levels, which increased dependent on time but independent of ebv transformation, may be a helpful marker for evaluating the acute response to viral infection but not for transformation. v. jurisic , m. jurisic university of kragujevac, school of medicine, kragujevac, serbia, university of belgrade, school of dentistry, belgrade, serbia tnf-alpha is a pleiotropic cytokine that is considered as a primary modifier of inflammatory and immune reaction in response to various inflammatory diseases and tumour. we investigated tnf concentration in radicular inflamed cysts and odontogenic tumours obtained from patients undergoing surgery, under local anaesthesia, and after aspiration of cystic fluid from non-ruptured cysts. further, we estimated the role of cyst wall on production of tnf-alpha in respect of presence of inflammatory cells, degree of epithelial proliferation and degree of vascularization. the concentrations of tnf-alpha in the cystic fluids were measured by elisa, while proteins analyzed after separation by two-dimensional gel electrophoresis. the presence of pericystic inflammed cells were analyzed in thick section for routine histological examinations and by immunohistochemisty for cd , cd and cd . tnf-alpha is elevated in both cysts fluid, but higher values were found in radicular inflamed cysts in comparison to odontogenic tumours. higher concentration of tnf-a were associated with higher protein concentration, higher presence of inflammatory cells in peri cystic tissues, cysts wall thickness and higher degree of vascularisation (mann-whitney u-test, p x . ) in radicular cysts. no correlation was found, based on these parameters in odontogenic tumours, but all sumple have detectable concentrations of tnf-alpha. objectives: interactions between the neuroendocrine and immune system play an important role in maintaining and restoring homeostasis. in susceptible individuals a dysfunction of the neuroendocrine system may be one of the risk factors involved in the pathogenesis of septicemia and bacterial infection at all. we will study prolactin role in defensive reaction of immune system to bacterial infection. as a type of this bacterial infection we have chosen septic status, where we are expecting the most significant alterations of immune reaction, and specially septic statuses in blood malignancies, where we are expecting deficiency of immune system. our idea is that prolactin takes part in this defensive reaction by its cytokine effects, and it has contraregulative role against activation of adrenocortical system. the aims of this study are to extend our knowledge about the activation of peripheral prolactin expression and by this way to contribute elucidation of its role in periphery. ) drawings blood from patients and healthy donors. blood of patients and healthy persons were sampled together with past histories after getting their acquainted approval. status of patient had to meet these conditions: a) the presence of systematic inflammatory response syndrome (sirs) according to standard clinical and laboratory criteria. b) positive hemoculture or determination of septic focus with demonstration of microbiologic source. ) detection of the gene expression of prolactin and tlr- in cd + peripheral blood monocytes from patients with septic status and from healthy controls has been performed by rt-pcr at the level of mrna and flow cytometry at the level of intracellular protein results: we have found statistical significant differences (p p . ) in expression profile between patient and control groups. these differences were at both levels of expression, mrna and protein. conclusion: septic statuses change tlr- and peripheral prolactin expression in cd + monocytes. the function of interferon (ifn)-induced immunoproteasomes (i-proteasomes) is at present almost exclusively acknowledged in connection with improved processing of mhc-class i antigens. the experiments performed here challenge this existing paradigm of i-proteasome function. we demonstrate in vivo and in cellulo that the key role of i-proteasomes resides in the protection of cells against the formation of protein aggregates, which is ultimately crucial for preservation of cell viability under ifn-induced oxidative stress. ifns up-regulate the ubiquitylation machinery and enhance the formation of oxidant-damaged, nascent, poly-ubiquitylated proteins, which essentially require i-proteasomes for their efficient degradation. i-proteasome deficiency results in the formation of aggresome-like induced structures and increased sensitivity towards apoptosis. enhanced degradation of poly-ubiquitin conjugates designed to protect cells, will therefore also increase the peptide supply for antigen presentation. thus, only by executing their physiological function in the maintenance of protein homeostasis i-proteasome induction also provides a mechanism for cellular immune-adaptation. background: revived by the description of new functions, b cells are considered to be essential in the genesis of autoimmune diseases. in strong support of this interpretation, baff would play a key role in their physiology. however, the correlation between circulating baff levels and disease activity is not clearly established. conflicting results concerning levels of baff and b-cell repopulation after rituximab treatment have been reported. in fact, basal serum levels of baff reported in literature vary according to the antibodies (ab) used in the elisa and the mode of calculation. the aim of this study was to understand these variations. material and methods: different anti-baff abs were tested to verify whether they recognize glycosylated baff purified from u culture supernatant. sera from patients with autoimmune diseases were also tested. a western-blot analysis of sera was performed to evaluate anti-baff abs specificity and the best combination of anti-baff abs validated for elisa. then, different commercial baff elisa-quantification kits were compared to our "in-house" elisa. results: unexpectedly, the binding of some anti-baff ab was restricted to glycosylated baff. however, both glycosylated and non-glycosylated forms of baff were found in sera from patients with autoimmune diseases. most of the kits commercially designed to quantify baff suffer from some limitations. some sera are indeed positive with a kit and negative with another and vice versa. furthermore, there seems that rheumatoid factors (rf) interfere and correlate with the optical density of the anti-baff elisa. conflicting results concerning levels of baff and disease activity or auto-abs titers should be reconsidered in light of the glycosylation status of baff. (table- ). when tb household contacts and healthy controls were compared, cfp and esat seemed to be more useful than tst in tb contacts for displaying ltb (table- ) . although cfp spot numbers were much more than esat spots at the beginning and follow up period, statistically there was no difference in terms of median values(p: , )( table- ). both esat and cfp spots were prone to decline during the follow up period. [ is some evidence to suggest that aspects of innate immunity (e. g. triggering of cytokine production by dendritic cells) may be impaired by hcv. to gain insight into some features of the innate immune response activated in vivo in the context of acute hcv replication, we analysed cytokine and chemokine levels in serum samples from chronic hcv patients undergoing liver transplantation. luminex multiplex assays and elisas were used to quantitate serum levels of g analytes immediately prior to liver transplantation and at sequential time points up to days post-transplantation. the increase in serum hcv rna levels associated with acute viral infection of the transplanted liver was found to be associated in most patients with elevations in serum levels of cytokines and chemokines including ifn-gamma, tnf-alpha, ip- , il- and il- . notably, the pattern and magnitude of systemic analyte elevations were very similar to those accompanying the acute burst of viral replication in primary hcv infection. high-magnitude elevations in systemic type ifn levels were not observed in either setting, which may reflect an in vivo impairment of plasmacytoid dendritic cell functions by hcv similar to that observed in in vitro studies. we suggest that the liver transplant setting can be used as a model to study aspects of the innate immune response activated in acute hcv infection. to test the hypothesis that virus interactions with sialic acid receptors may play a role in innate antiviral immunity, we used recombinant viruses and differentiated cultures of human airway epithelial cells (hae). the hemagglutinin of the pandemic virus a/hong kong/ / (h n ) (hk) differs from its putative avian precursor by amino acid substitutions. we generated the complete recombinant virus rhk and its ha variants with amino acid reversions back to the ancestral avian sequence (rhk- aa-i r, n d, k n, s n, g a, human ( - ); rhk-r -l q, s g and rhk- aa-i r, n d, k n, s n, g a, l q, s g, avian ( - )). among these variants, the double mutant rhk-r and the seven mutant (rhk- aa) had a typical avian-virus-like receptor-binding specificity owing to substitutions l q and s g.we infected hae cultures with the viruses and collected samples from the apical and basolateral sides of the cultures at different times post infection. virus titers were determined in mdck cells, and concentrations of about pro-and anti-inflammatory mediators and chemokines were measured using a multiplex bead assay.concentrations of most cytokines progressively increased at the apical side of the cultures in the course of the infection. many cytokines, including t-cell-attracting chemokines such as ip- and rantes, were induced to similar levels by different viruses. however, some mediators were induced significantly stronger by avian-like viruses rhk-r and rhk- aa as compared to rhk and rhk- aa. in particular, avian-like viruses stimulated a higher release of potent chemo-attractants of innate immune cells, such as g-csf and il- , shedded adhesion molecules (cd , vcam- , icam- ), and pro-apoptotic factors (trail). remarkably, the patterns of secreted cytokines differed between the apical and basolateral sides of the cultures. whereas avian-like viruses typically induced similar or higher levels of cytokines at the apical side than did rhk and rhk- aa, the human-like viruses were stronger inducers of basolaterally secreted mediators. these data provide the first direct experimental evidence that receptor specificity of influenza viruses can significantly affect patterns of innate immune responses in human airway epithelium. further studies are warranted to determine the role of the observed effects in the host range and pathogenicity of influenza viruses in humans. a total of newborn infants were enrolled in the study. forty-nine newborn infants (group i), who met the criteria of sepsis, had a routine sepsis evaluation as well as measurement of pct, il- , and neutrophilic cd levels, procalcitonin and il- were measured by elisa technique while, neutrophilic cd by single colour flowcytometric technique. of these "infected" infants, had positive blood culture (subgroup ia: culture-positive sepsis), and infants were diagnosed to have clinical sepsis with negative blood cultures (subgroup ib: culture-negative sepsis). another newborn infants classified as control group (group ii) . results: sensitivity, specificity, positive predictive value, and negative predictive value for diagnosis of sepsis were analyzed for pct, il- , cd , and crp. il- had the highest sensitivity and specificity, % and %, respectively, using cutoff n . pg/ml. for pct, the highest sensitivity and specificity, % and %, respectively, were at a cutoff value of n . pg/ml. neutrophilic cd had maximal sensitivity and specificity of % and %, respectively, at cutoff value of . %. combinations of different markers may improve the sensitivity and specificity of biomarkers such as the tests used in this study. we found that the best combination was il- and neutrophilic cd , which together provided sensitivity and specificity of % and %, respectively, and npv %. the combination of il- and crp had high sensitivity and moderate specificity, % and %, respectively. conclusions: il- and neutrophilic cd levels determination can be used as good tests for early detection of neonatal sepsis. procalcitonin measurement might be used as an additive parameter to improve the diagnostic efficacy of the other markers in neonatal sepsis, but it is not helpful as a single test. objectives: the assembly and activation of inflammasomes are essential processes in the immune response to many inflammatory stimuli. inflammasomes are typically formed by at least one member of the cytosolic innate immune sensor family, the nod-like receptors (nlr). the nlr family members nalp , naip or ipaf and the adaptor asc are involved in caspase- activation in response to bacterial infection, triggering the processing and secretion of il- b and il- . recent studies have demonstrated that tlr-dependent inflammasome activation in macrophages is modulated by autophagy, a homeostatic mechanism for the catabolism of cytosolic constituents. autophagosome biogenesis and maturation requires activation of the class iii pi k, vps and can be inhibited with the pi k inhibitors wortmannin and methyladenine ( ma). in contrast, activation of akt, via class i pi k, results in inhibition of autophagy. the aim of this study was to determine the nature of this inflammasome and whether autophagy influences il- b secretion in dendritic cells. methods: murine bone marrow-derived dendritic cells (bmdm) were treated with tlr ligands in combination with ma, wortmannin or an akt inhibitor. supernatants were analysed for il- b by elisa. results: ma enhanced il- b secretion by bmdc treated with the tlr and tlr ligands poly(i:c) and lps, but not with any other tlr ligands tested. similar results were obtained using wortmannin. this increase in il- b secretion was greatly reduced in bmdc from nalp -/mice compared to wild type c /bl controls. treatment with the akt inhibitor had no effect on lps-induced il- b secretion by bmdc. tlr-dependent secretion of il- a was also enhanced by treatment with ma. conclusions: these data demonstrate that il- b secretion by bmdc in response to treatment with pi k inhibitors, in combination with lps or poly(i:c), is dependent on the nalp inflammasome. this response is limited to tlr and tlr agonists. inhibition of akt had no effect on lps-induced il- b production, suggesting that the effect of wortmannin and ma on inflammasome activation is not dependent on the inhibition of akt activation by class i pi k. objectives: in the last few years, several evidences have shown the modulation of toll-like receptors (tlrs) by g-protein coupled receptors, i. e. our group has recently demonstrated the attenuation of tlr signaling by the inflammatory lipid mediator sphingosine -phosphate (s p) through receptors and in human monocytes-macrophages, which could explain some of the s p anti-atherogenic effects. since adhesion of monocytes to endothelial cells is considered an important event in atherogenesis, our goal was to investigate the putative implication of tlr-s p receptors crosstalk on the expression of adhesion molecules in endothelial cells. methods: for the study, in vitro cultured endothelial cells from arterial and venous origin were challenged with a combination of tlr ligands and s p, and later analyzed by flow cytometry. a pharmacological analysis of the s p receptor subtype involved in the response was also performed. results: data from flow cytometry experiments revealed that icam- expression was increased following lps and s p concomitant stimulation in both venous and arterial cells, suggesting that tlr and s p receptors cooperate in the expression of icam- . conversely, no cooperation was observed when tlr ligands were used. in order to elucidate which s p receptor subtype was involved in the increase of icam- expression, we used a pharmacological approach with s p receptor inhibitors and pertussis toxin. results showed differences between arterial and venous cells. while in arterial cells elevated icam- after lps and s p challenge was significantly reduced by blocking s p receptor and the effect was pertussis toxin-insensitive, in venous cells the response was pertussis toxinsensitive and not blocked with inhibitors of s p receptors and , which suggest that s p receptor might be involved in the effect. conclusions: altogether these data demonstrate that tlr and s p receptors can interact to increase adhesion molecules such as icam- in human endothelial cells, and the s p receptor subtype involved in the effect differs between arterial and venous cells. with ssc without pah) and a pool of sera of healthy controls (hc) were tested. results: in dimension immunoblot, serum igg from ssc patients, patients with ipah and hc tested individually reacted with - , - and - protein bands in a human vsmc protein extract with qualitative and quantitative differences between groups, respectively. in dimension immunoblot, igg of pools of patients with ipah, igg of pools of patients with ssc with or without pah, and igg of a pool of hc recognized ± , ± , ± and protein spots respectively. twenty one protein spots were recognized by more than % of igg of pools of sera in each group of patients and not by igg of hc. twenty seven protein spots were recognized by the great majority of igg of pools of patients with a higher intensity than igg of pools of hc. identified proteins were constituents of cytoskeleton, proteins involved in oxidative stress as stress-induced phosphoprotein and peroxyredoxin and proteins involved in regulation of cell energy metabolism as triosephosphate isomerise. we have identified anti-vsmc abs in the serum of patients with idiopathic and ssc-associated pah. these abs bind to cytoskeleton, oxidative stress and cell cycle antigens. objectives: this study aimed to verify that subcutaneous lymph node transplantation inducing lymphatic regeneration is possible in healthy adult rats, as obtained in other species. methods: this rat model was used to determine the effects of lymph node fragmentation as well as sheep erythrocytes and platelet-rich plasma injection on the regeneration of the transplanted lymph nodes. results: this rat model is adequate to study the regeneration of transplanted lymph nodes. lymph node fragmentation seems to affect transplant regeneration negatively. an immune challenge by injection of sheep erythrocytes in the drainage area of the transplanted lymph nodes does not improve fragment regeneration. however, injection of syngeneic platelet-rich plasma containing several growth factors resulted in an improvement in regeneration. conclusion: lymph node fragment regeneration, although still experimental, could be relevant for lymphedema prevention. acquired lymphedema has a high prevalence in developed countries as a consequence of the removal and/or radiotherapy of tumor-draining lymph nodes in cancer patients. this disease causes lifelong disability due to chronic swelling and increased risk of infections. it currently lacks an effective treatment. methods: patients suffering from different diseases were enrolled in our study. patients were suffering from bone diseases (osteomyelitis, necrosis, tumour) whereas of them were suffering from inflammatoty diseases (soft tissue inflammation, diabetic ulceration). blood specimens were collected before hyperbaric oxygen treatment and the serum levels of icam- and vcam- were assesed by an enzyme immunoassay (elisa). results: out of the patients ( , %) had elevated levels of the intercellular adhesion molecule. out of the patients suffering from bone diseases ( , %) had raised values (mean value ng/ml) whereas patients out of the suffering from soft tissue diseases and diabetes ( , %) had raised values (mean value , ng/ml). reference value for icam- was - ng/ml. vascular cell adhesion molecule's assesment revealed no elevated levels in our patients. conclusions: our study revealed a high rate of patients ( %) having increasd levels of icam- . high icam- levels were more prevalent in patients suffering from soft tissue inflammatory diseases and diabetes ( , %) than in patients with bone diseases ( . %). mean values were found , ng/ml and ng/ml accordingly. those findings verify the positive correlation between icam- and inflammatory diseases and tissue damage but not for vcam- . colorectal cancer (crc) was the first solid tumour to be successfully targeted with anti-angiogenic therapy in the clinic. tumour angiogenesis is critical for cancer progression in that it permits expansion of the tumour mass and fosters malignant dissemination. angiogenesis is a multistep process involving endothelial cells as well as numerous stromal components within the tumour microenvironment that also represent potential therapeutic targets. inflammation dependent-angiogenesis is increasingly recognised as a central force in tumour growth and progression, while use of anti-inflammatory drugs has been found to reduce incidence of crc carcinoma potentially through repression of tumour angiogenesis. we investigated the link between inflammatory angiogenesis and colorectal cancer in archival tissues across a range of pathologies that represent diverse steps in the progression of crc: cases of ulcerative colitis (urc), adenocarcinomas developed from preexisting tubular or tubulo-villous adenomas, tubular or tubulo-villous adenomas with low grade dysplasia, and infiltrating adenocarcinomas. immunohisto- objectives: to determine the effect from the administration of preoperative pravastatina to therapeutic dose in the expression of cd in the leucocitaria adhesion to endotelio vascular in the isquémico-reperfundido miocardico weaveal endotelio vascular en el tejido miocardico isquémico-reperfundido by the circulation extracorpórea (cec). methods: they were included in way randomizada double blind patients with intervened controlled hiperlipidemia of surgery coronary low circulation extracorpórea (cec). mg of pravastatina oral hours they were administered before the procedure (group study, n= ) or placebo (group placebo, n= ), and control (group control, n= ). samples of outlying veined blood were extracted to the hours. the separation of leukocytes was made in peripheral blood, to determine the expression of cd in such. in all the samples one quantified the intensity of the expression pattern and the percentage of leucocitarias cells. results: types of patterns were distinguished: cytoplasmic, of membrane and compound. the intensity of the expression was classified in degrees: degree . without expression. degree . weak; degree . moderate; degree . intense. in the group control: most of the samples they presented/displayed a mixed pattern (cytoplasmic and of membrane) with an intensity degree - . the placebo group: mixed pattern, degree - . group study ( mg. oral pravastatina): most of the cells they presented/displayed a predominance of membrane pattern: degree - . the percentage of cells that expressed cd was greater in the group study ( mg. oral pravastatina). the preoperative oral pravastatina to therapeutic unique dose ours study produces a greater expression cd answer induced by the cec; it seems that these molecules located in the leukocytes participate in the adhesion to the activated endoteliales cells, necessary for the extrusion of the lymphocytes through endotelio towards the inflammatory center and in quimiotaxis of the leukocytes towards the inflammation sites. several surface molecules on endothelial and epithelial cells undergo regulated cleavage by the disintegrin and metalloproteinases adam and adam . we recently identified transmembrane chemokines, junctional adhesion molecule-a (jam-a), and members of the proteoglycan family as novel substrates for these proteases. here we demonstrate that cell lines and primary cells of human endothelial or epithelial origin release considerable amounts of soluble jam-a and proteoglycan ectodomains. this release is enhanced by treatment with the proinflammatory cytokines ifng and tnfa. the enhanced release was not caused by an increased gene induction but rather associated with a reduction of the surface expressed molecules. both, constitutive and induced release required the presence of adam as demonstrated by specific inhibitors, lentiviral silencing experiments as well as treatment with the recombinant catalytic domain of adam . these data suggest that the proinflammatory cytokines ifng and tnfa induce enhanced proteolytic shedding of cell surface molecules on endothelial and epithelial cells. to investigate the physiological relevance of this induced shedding, mice were treated systemically with ifng/tnfa leading to increased presence of soluble jam-a in the blood serum. both cytokines also stimulated jam-a release from excised murine aortas with was associated with enhanced adam activity in the tissue. in the presence of the adam inhibitor induction of jam-a release was suppressed. in cultured epithelial cell lines enhanced shedding of jam-a or proteoglycans was not associated with increased mrna or surface expression of adams but rather with increased activity of cellular adam as shown by means of a synthetic substrate assay. our study demonstrates that the proinflammatory cytokines ifng/ tnfa upregulate adam -mediated shedding activity rendering the protease an important modulator of endothelial and epithelial surface molecules in inflammatory settings. rium, and the haplotype vegf- / vegf+ is associated with rcc risk ( p= , ), metastases ( p= , ), nuclear grade ( p= , ), tumor stage ( p= , ), and tumor size (p= , ). on the other hand, the polymorphism vegf - a/c is not associated with rcc risk and clinical parameters. our results shed a new light to the knowledge on the association between vegf polymorphism and rcc risk and development. these data could help to improve our understanding of the rcc pathogenesis and disease progression. pten is a lipid phosphatase, whose substrate is phosphatidylinositol , , -trisphosphate. therefore, pten is one of the main antagonists of the pi -kinase, which plays a major role in many important cellular functions, such as proliferation, migration or response to inflammatory stimuli. here we investigated the role of pten in collagen induced arthritis. we show that conditional deletion of pten under the lysm promoter (lysmcrepten flox/-) leads to a significant reduction in clinical severity of collagen induced arthritis (cia). histological analysis of cia, lysmcrepten flox/mice displayed significantly reduced joint inflammation as well as erosive bone destruction. total anti-collagen antibodies, however, as well as anti-collagen iggs were identical in both groups. upon analysis of inflammatory cytokines in serum after immunisation we found a significant reduction of il- as well as il- levels. furthermore, pten deficient macrophages and dendritic cells showed reduced induction of il- as well as il- and il- mrna upon stimulation with various tlr-ligands. since these cytokines play an important role in the induction of pathogenic th- t cells, we measured th- cytokines in lymph nodes after immunisation with collagen. although dendritic cell and macrophage recruitment to the draining lymph node was comparable in both groups, there was a slight reduction of il- and a strong reduction of il- mrna in the draining lymph node of immunized lysmcrepten flox/compared to wild-type mice. one of the mechanisms through which il- exerts its anti-inflammatory effects consists in promoting the release of anti-inflammatory molecules. in this context, particularly important is the production of il- ra, whose expression is induced by lps in human neutrophils and monocytes and significantly potentiated by the presence of il- . based on our previous observation that support a direct role of il- -activated stat in the enhancement of il- ra transcription induced by lps, we plan to characterize the transcriptional activators recruited to the il- ra promoter in vivo and responsible of the increased rate of transcriptional initiation upon exposure of lps-treated cells to il- . quantitative chromatin immunoprecipitation (chip) studies were employed to examine the in vivo binding of transcriptional activators to the il- ra promoter. crosslinked nuclear lysates were immunoprecipitated and min after il- addition with different antibodies and immunoprecipitated dna was analyzed by quantitative real-time pcr for the presence of target sequence located in the il- ra promoter. chip assays showed that the pol ii recruitment to the il- ra promoter induced by lps is significantly increased by il- , further strengthening the concept that the rapid enhancement of lpsinduced il- ra gene expression by il- initially occurs by targeting transcriptional events. as expected, real-time pcr of anti-stat immunoprecipitated dna showed statistically significant levels of stat binding to the il- ra promoter only in cells stimulated with lps in the presence of il- . surprisingly, anti-p and anti-p chip assays revealed enrichment of both p and p recruitment to the il- ra promoter when il- was added to lps-stimulated cells, suggesting that il- enhances the recruitment of nf-kb to the il- ra promoter. interestingly, when nf-kb is recruited to this promoter in lps + il- -treated cells, the overall nf-kb nuclear translocation (analyzed by western blot) and dna binding activity (detected by emsa analysis) were not modified with respect to cells stimulated with lps alone. the enrichment of nf-kb at the il- ra promoter site is dependent on il- -activated stat , since it is greatly reduced when stat activation by il- is impaired. the molecular mechanism through which il- -activated stat promotes the recruitment of nf-kb to the il- ra promoter is currently under investigation. major components of mast cell secretory granules are proteases. we could recently report that intracellular stored mast cell-produced cytokines regulate mc protease activities and provided evidence that il- acts as a specific negative transcriptional regulator of mouse mast cell protease- (mmcp- ). we examined the mechanisms underlying the repression of mmcp- gene expression. our data show that the "repressor" effects of il- on mmcp- promoter activity are still operating on the mmcp- bp long minimal promoter. moreover, il- deficiency in mast cells causes a specific dysregulated expression of the transcription factors c/ebpb and yy . furthermore, chromatin immunoprecipitation revealed that il- promoted specific reciprocal recruitment of c/ebpb but not yy to the mmcp- promoter. finally, il- deficient mast cells display a predominantly non-cpg methylated pattern of the mmcp- . thus, we proposed that the expression of mmcp- and possibly other immunoregulatory genes may be regulated by il- through epigenetic modification and by balancing the content and binding of c/ ebpb and yy in mast cells. i. nagy , k. filkor , a. vörös , l. kemény , a. szász bzaka, baygen, szeged, hungary, university of szeged, department of dermatology and allergology, szeged, hungary micrornas (mirnas) are evolutionary conserved small non-coding rnas that act as key regulators of gene expression at post-transcriptional level by targeting mrnas for translational repression and/or degradation. mirnas have been shown to have unique tissue-, developmental stage-and diseases-specific expression patterns. during the last years several studies highlighted that mirnas play critical role in the differentiation and function of the adaptive as well as innate immunity. little is known however, about the differential regulation of mirnas following the activation of pattern recognition receptors. in order to tackle this issue, we treated hacat keratinocytes with staphylococcus aureus-derived peptidoglycan (pgn) once or repeatedly, the latter mimicking persistent infection. after appropriate treatments we first analyzed the expression profile of mirnas mir- , mir- a and mir- , which are known to participate in immune processes of the skin. repeated pgn-treatment significantly decreased mir- expression; in contrast, pgn re-stimulation had no further effect on mir- a and mir- expression. next, we investigated the correlation between the expression of mir- and its two known direct targets: regulatory protein p and suppressor of cytokine signalling- (socs- ). although the gene-expression profile of neither p nor socs- changed, we found that the expression of mir- reversibly correlates with both p and socs- protein expression, a phenotype that we verified by two independent protein analysis methods (western blotting and immunofluorescent labeling). importantly, transfection of hacat cells with anti-mir- prior to pgn-treatment completely abolished both p and socs- down-regulation, revealing the involvement of mir- in pgn-induced transcriptional regulation. finally, methylation-specific pcr experiments unravelled the role of dnamethylation in regulating mir- expression upon pgn-treatment. taken together, our results strongly suggest that sets of mirnas may be differentially regulated during persistent infection. results: tgfb +/-had a lower incidence and burden of benign papillomas when compared to tgfb +/+ animals. however, more scc developed in the tgfb +/-mice. after acute and chronic promotion, tgfb +/-skin showed a reduced proliferative response with no increase in epidermal tgfb or nuclear p-smad compared to tgfb +/+ mice. tpa-induced pkca activity as well as phosphorylation of specific pkc substrates in keratinocytes correlated with tgfb gene dosage. further, pharmacological inhibition of alk suppressed tpa-mediated pkca activation suggesting that physiological levels of tgfb are required for maximal activation of pkc-dependent mitogenic responses. even though the tpa-induced inflammatory response was greater in tgfb +/-skin, tgfb +/+ papillomas had more tumor infiltrating neutrophils. tpa-induced proinflammatory gene expression was sustained in tgfb +/-skin and primary keratinocytes but it was elevated in v-ras ha -transduced tgfb +/+ but not tgfb +/-keratinocytes, indicating that tgfb switches from an anti-inflammatory cytokine in the skin to a proinflammatory factor in tumors dependending on an activated ras. despite this differential proliferative and inflammatory response to tpa and enhanced papilloma formation in the tgfb +/+ mice, there was no increase in conversion to scc in this genotype. conclusions: tgfb acts to promote benign tumors enhancing cell proliferation and inflammation through its ability to regulate pkc activation in skin, yet retains a suppressive function for malignant conversion. background: proto-oncogene survivin has been recently shown as a prognostic marker distinguishing patients with destructive rheumatoid arthritis (ra). in the present study we studied the relationship between survivin and urokinase (upa), a fibrinolytic serine protease being over expressed in the inflamed joints and exhibiting arthritogenic properties. material and methods: levels of survivin and upa were measured in the paired blood and synovial fluid samples of patients with ra, using elisa and compared to controls with non-inflammatory joint diseases. the ability of upa to induce survivin and requirement of upa receptor (upar) was studied in primary synovial fibroblasts and pbmc of ra patients, human monocytic (thp- ) and fibroblast (mrc- ) cell lines employing antibodies against upar, sirna technique, and synthetic inhibitors of intracellular pathways. the ability to prevent urokinase-induced arthritis by interruption of survivin expression was evaluated in mouse model of arthritis. results: in the present material of ra patients and controls the levels of survivin correlated to urokinase (upa) (r= . ), a plasminogen activator over expressed in inflamed joints and known to exhibit potent arthritogenic properties. we found that / ra patients had high circulating levels of survivin. these patients had erosive arthritis and were characterized by high levels of upa. in vitro studies showed that upa induced survivin in leukocytes and this process was dependent on signaling through upa receptor. in turn, survivin was required for expression of upar. additionally, survivin was essential for upa production in mrc- and synovial fibroblasts. down-regulation of survivin with sirna was followed by significantly reducion of upa synthesis. finally, treatment with downregulation of survivin by sirna in vivo efficiently abrogated upa-induced arthritis in mice model. these findings indicate that survivin is an essential mediator of arthritogenic properties of upa regulating its synthesis in synovial fibroblasts and upar expression in leukocytes. close correlation between survivin and upa in patients with ra supports the improtance of these interaction for the pathogenesis of arthritis. upon cell activation, ubiquitously expressed inositol , , -trisphosphate -kinase type b (itpkb) phosphorylates inositol ( , , ) trisphosphate (ins( , , )p ), a calcium-mobilizing second messenger with pleiotropic effects. itpkb inactivation leads to severe t cell deficiency, altered thymo-independent b cell responses and neutrophil hyperactivation. we here report that itpkb-deficient (itpkb -/-) mice also display profound alterations in mast cell development and function. indeed, while mast cell number, c-kit and fcepsilonri expression were comparable in itpkb-deficient and proficient mice, itpkb -/mast cells were almost completely devoid of granules. this phenotype could be partially reversed upon treatment with sodium cromoglycate. nevertheless, fcepsilonri or c-kit activation on mast cells led to increased ca + responses and to stronger erk phosphorylations. however, itpkb -/mice displayed an attenuated sensitivity to ige-mediated passive systemic anaphylaxis, correlated to the absence of fcepsilonri-dependant histamine release and to downregulation of h and h receptor expression due to high basal histamine concentrations. production of neosynthesized mediators remained normal. finally, itpkb deficiency also severely impaired scf-induced mast cell differentiation in vitro. taken together these results demonstrate that itpkb is a key regulator of mast cell activation. itpkb antagonists might thus be of therapeutic interest for programmed and progressive depletion of histamine stores. the large percentage of immune relevant genes that are alternatively spliced and the connections between splicing and disease, strongly indicate that alternative splicing plays a central role in the regulation and fine-tuning of physiological immune responses. il- b is an important proinflammatory cytokine produced by activated macrophages and monocytes. il- b is produced as an inactive cytoplasmic precursor that is proteolytic processed by the inflammatory caspase- to generate the mature secreted active form. caspase- is also synthesized as an inactive form that requires processing by the inflammasome to become active. we have used a subset of the trc lentiviral human library to generate loss-of-function phenotypes for most of the splicing factors and splicing regulators. we were able to silence the expression of genes involved in splicing with an average -fold coverage. after the primary screen and several rounds of phenotypic validation, we have identified genes that significantly affect the production of il- b by thp- cells after a h challenge with pfa-fixed e. coli, as measured by elisa. knockdown levels were analyzed by qrt-pcr for the most significant candidates to validate the phenotypes observed. exon array analysis are being performed to identify possible targets of the most significant splicing factor candidates obtained by the shrna screening in order to dissect their mechanism of action in the regulation of the inflammasome and il- b secretion. tissue transglutaminase (tg ) has a critical role in the pathogenesis of chronic inflammatory diseases such as celiac or neurodegenerative diseases. we have previously described the key role of tg in cystic fibrosis (cf), a genetic disease characterised by chronic lung infections and inflammation. in cf, mutation on the cftr gene results in an increased tg expression and activity leading to functional sequestration of the anti-inflammatory pparg and increase of the classic parameters of inflammation. here we tested whether in vivo inhibition of tg can reverse inflammation in chronic inflammatory diseases. to assess the importance of tg not just in cf but in chronic inflammatory diseases in general, we injected cystamine, a potent tg inhibitor, in a transgenic mouse model cf and in the taz transgenic mice that spontaneously develop autoimmune thyroiditis. intraperitoneal administration of cystamine had a significant impact on the lung epithelium in the cf model, where it decreased tg expression and activity. the treatment was also able to dampen all the classic inflammatory parameters as well as restoring normal cellular levels of functional pparg. interestingly, cystamine injections could also block inflammation in the taz tcr transgenic mouse model with chronic thyroiditis, highlighting the pivotal role of tg in generating inflammation in two very different pathologies. this work underlines the critical role of tg in inflammation and provides new opportunities to develop therapeutic strategies for sufferers of chronic inflammatory diseases. angiogenesis, the growth of new blood vessels, is a process that is essential during tissue repair, foetal development, and female reproductive cycle. angiogenesis is also a relevant process associated to many pathologic conditions including autoimmune diseases and tumors. we have shown that dendritic cells activated in the simultaneous presence of pro-and anti-inflammatory signals (alternatively activated dc, a-dc) display potent angiogenic activity in vivo which is mediated by the release of biologically active vegf-a. here, we investigates the molecular mechanisms leading to vegf-a secretion in lps+pge stimulated a-dc. preliminary results indicate no accumulation of hif- alpha in a-dc, therefore suggesting that vegf-a is induced by a non-classical, hif- alpha independent pathway. in addition, we found that vegf-a secretion depends on the activation of mapk p but not erk / or jnk. inhibitor studies, nascent transcript analysis and polimerase ii recruitment on the promoter show that the induction of vegf-a is largely due to new transcription and not to changes in mrna stability. chromatin immunoprecipitation studies aimed at the characterization of the modifications of vegf-a regulatory regions in a-dc and at the identification of transcription factors bound to vegf-a promoters are being performed. this will possibly allow the description of novel transcription factors involved in vegf-a activation in a-dc. wnt proteins are secreted palmitoylated glycoproteins with multiple functions in cell proliferation, migration as well as tissue organization. they are best known for their role in embryonic development and tissue homeostasis. deregulation of wnt signaling has been shown to promote carcinogenesis. recently we identified wnt signaling to be involved in the regulation of inflammatory processes: wnt a is induced in human macrophages in response to mycobacteria and conserved bacterial structures and contributes to the regulation of the proinflammatory cytokines il- and ifn-gamma. to gain deeper insights into wnt mediated modulation of inflammatory processes we now used murine bone marrow derived macrophages and analyzed the effects of the addition of exogenous wnt homologs. we monitored wnt-mediated activation of primary macrophages by measuring the activation of signaling pathways and transcription factors, analyzed the expression of target genes by real-time pcr and measured the secretion of inflammatory cytokines by elisa. exogenous wnt a -but not wnt a -was able to induce cytokine expression in primary macrophages. in infection experiments wnt a promoted the mycobacteria-induced macrophage activation and enhanced the expression of inflammatory mediators in murine macrophages. in contrast, addition of wnt a reduced the expression of inflammatory mediators upon mycobacterial infection. these data corroborate our previous findings and further support the notion that tlr/nf-kappab and wnt signaling, both being evolutionary highly conserved pathways, are functionally interconnected infection of immunocompetent mammals with t. gondii induces a chronic infection of the brain. t. gondii cysts persist in neurons and escape elimination by the immune system. in immunodeficient individuals, the infection can be reactivated resulting in a lethal toxoplasma encephalitis (te). in te, the parasite is primarily controlled by infiltrating t and b cells. also brain resident cells may contribute to control of the disease and however, the mechanisms of brain resident cells leading to the protection of the vulnerable brain in chronic te are largely unknown. in a previous study, we could show that expression of gp on astrocytes in mice is critical for survival of te. in the present study, we analyzed the function of neuronal gp in te. after infection with t. gondii, mice lacking neuronal gp (synapsin-cre gp fl/fl ) died significantly earlier in the chronic phase of infection than control gp fl/fl mice. death of synapsin-cre cre gp fl/fl was due to a severe encephalitis with larger inflammatory lesions and higher numbers of inflammatory leukocytes. additionally, te of synapsin-cre gp fl/fl mice resulted in a substantial apoptosis of neurons both in the vicinity of inflammatory lesions and also in brain areas without inflammation. in vitro, apoptosis of gp -deficient neurons was also significantly increased upon infection with t. gondii or stimulation with tnf as compared to gp expressing neurons. interestingly, the intracerebral parasitic burden was not increased in synapsin-cre gp fl/fl mice indicating that the immunoregulatory role of neurons is more important than their anti-parasitic function. t. objectives: persistent production of tnfa in many autoimmune diseases, including intraocular inflammation (uveitis), can lead to significant tissue damage. targeting tnfa with neutralising antibodies or tnf receptor fusion proteins is often, but not always, an effective therapy. high serum concentrations of tnfa, il- b, il- and il- have been detected in a spectrum of autoimmune diseases; while in contrast, the levels of il- , il- and tgfb are reduced. this suggests, indirectly, that failure to regulate an appropriate balance of inhibitory factors contributes to the pathogenesis or propagation of tissue inflammation in autoimmunity. thus, understanding the homeostatic control of tnfa by tgfb further may generate more effective therapies. as tnfa mrna ' untranslated region (utr) contains an au-rich element (are), which targets mrnas for degradation, we wished to test whether tgfb suppresses tnfa protein production by upregulating the rnabinding protein fxr , which can bind to tnfa mrna and inhibit translation. methods: using raw . cells and mouse bone-marrow derived macrophages stimulated with lps and tgfb , we assessed mrna expression by q-pcr and tnfa protein expression by flow cytometry. the 'utr of tnfa mrna was isolated and inserted into a luciferase reporter vector on a constitutive promoter. transfected raw cells were treated with lps and tgfb and luciferase expression was quantified. cells treated with lps and tgfb were also examined for fxr expression using pcr and western blot. following fxr knockdown using sirna, the influence of tgfb and lps on tnfa protein production was examined by flow cytometry. results: we find that while tnfa mrna expression remains constant, lps induced tnfa protein expression is suppressed by tgfb . using the luciferase-tnf- 'utr vector we show that tgfb targets the 'utr of tnfa. furthermore, tgfb and il- both upregulate fxr mrna and protein; and treatment with tgfb and lps can synergistically upregulate mrna expression, more than tgfb alone. following sirna inhibition of fxr , tgfb can no longer inhibit lps-induced tnfa production. comtb up-regulated mmp- and mmp- secretion from saecs, nhbes and fibroblasts to a peak of . +/- . ng/ml, at hours. interleukin- augmented comtb-stimulated up-regulation of mmp- and mmp- secretion from saecs and fibroblasts in a synergistic manner. in contrast, interleukin- down-regulated mmp- secretion from saecs by %. interleukin- up-regulated mmp- and mmp- secretion from fibroblasts but not from saecs. timp secretion from saecs was enhanced by interleukin- but there was no effect of interleukin- . mmp up-regulation by interleukin- and comtb was inhibited by the pi kinase inhibitor ly and on western analysis akt (protein kinase b) was phosphorylated at minutes. chemical inhibition of the p d isoform of pi kinase with ic abrogated the il- and comtb driven secretion of mmp- from the small airway epithelial cells. chemical inhibition of the tumour suppressor phosphatase, pten (phosphatase and tensin analogue on chromosome ) accentuated mmp- secretion. these inhibitory effects were confirmed with sirna. mmp- up-regulation was secondary to increased gene expression with promoter activity peaking h after stimulation. in summary, interleukin- and interleukin- drive transcription dependent mmp- and mmp- secretion from airway epithelial cells and fibroblasts. interleukin- also increases timp but down-regulates mmp- gene expression and secretion. this may contribute to the matrix degrading phenotype in tuberculosis. the pi kinase pathway is central in interleukin- driven tissue destruction in the context of m. tuberculosis infection. v. delgado-maroto , l.s. moreira , e. gonzalez-rey , m. delgado institute of parasitology and biomedicine lopez-neyra , csic, granada, spain, university of seville, sevilla, spain objectives: atherosclerosis is an inflammatory chronic disease characterized by the formation in the arteries of lesions that involve inflammation, lipid accumulation, cell death and fibrosis. over time, the rupture of these atherosclerotic plaques releases prothrombotic material to the blood and causes thrombotic occlusion at the site of disruption. atherosclerosis will probably become the most common cause of death within years. one of the initial hallmarks of the disease is the uptake of oxldl particles by macrophages, which leads to intracellular cholesterol accumulation and the formation of foam cells. t cells undergo activation after interacting with foam cells, which process and present local antigens including oxldl, generating a t helper response. cholesterol metabolism is regulated by factors such as pparg (proliferator activated receptor g), srb (a class b scavenger receptor), cd or abca , that can induce cholesterol exit from the macrophage which may help to solve the lesions. expression of these factors depends on intracellular camp. adrenomedullin (am), urocortin (ucn) and vasoactive intestinal peptide (vip) are novel neuropeptides synthesized by immune cells that have various characteristics to be considered as possible therapeutic agents for atherosclerosis. they are potent anti-inflammatory agents, which downregulate a broad spectrum of pro-inflammatory mediators, and inhibit th immune response. their mechanism of action involves binding to gpcr and adenylate cyclase activation with subsequent increase of camp in the cell, which is recognized as an anti-inflammatory response. methods: we investigate am/ucn/vip effect on bone marrow-derived macrophages stimulated with oxldl. we determine the levels of pparg , srb , cd , and abca . we also analyze the cholesterol metabolism of oxldl-stimulated macrophages after neuropeptides incubation using oil red o staining of lipids drops and tritium labelled cholesterol. objective: endovascular aortic repair (evar) is considered a minimally invasive procedure, and the patients are expected to be discharged after a day or two. however up to % develop a systemic inflammatory response syndrome (sirs), resulting in prolonged convalescence. as yet there is no satisfactory explanation to this severe response. previous studies have shown a high level of il- in the mural thrombus lining the aneurysm. the thrombus is manipulated during the procedure, but whether or not it is the source of circulating il- and/or other cytokines during and after the operation is unknown. methods: quantitative analysis of the pro-inflammatory cytokines il- , tnf-a, il- b, il- and il- , and the anti-inflammatory cytokine il- , in plasma from five patients, as of yet, was carried out by means of cytometric bead array, while analysis of plasma il- was performed using the luminex platform. the cytokine levels were compared to the clinical response, in terms of sirs. results: evar induced the production of il- and il- , and in some cases, of il- . the maximal plasma levels of il- and il- were found at hours and of il- between - hours. modest plasma levels of il- were also observed, with maximal production at various time points ( - hours). by contrast, production of tnf-a, il- b and il- did not occur to a significant extent, while production of il- occurred sporadically. although our preliminary data indicate that sirs is associated with enhanced cytokine responses, the production of il- , il- , il- and il- also took place in patients who did not develop sirs. conclusion: evar is associated with the sequential production of il- , il- , il- and il- , i. e. a mixed pro-and anti-inflammatory response, even in the absence of sirs; but the production seems to be exaggerated in patients developing sirs. further studies involving patients are in progress, and will clarify this. we hypothesize that sirs is elicited by il- , activated by manipulation of the mural thrombus. to reveal whether this is the case, studies involving immunohistochemistry of the thrombus, will be performed. il- is a novel il- cytokine family member that is expressed as an intracellular precursor (pro-il- ) and is thought to be cleaved by caspase- to yield a mature bioactive form of the molecule (mat-il- ). to date however, evidence of cell-associated proteolytic processing and caspase- dependent secretion of mat-il- has not been reported. here we show that pro-il- but not mat-il- is released from uvb-irradiated keratinocytes. we demonstrate binding of pro-il- to the il- r and also il- r-dependent bioactivity of pro-il- on mast cells. we propose a previously unrecognized role for pro-il- as a pro-inflammatory mediator and suggest a direct link between uvb-mediated epithelial cell damage and cutaneous mast cell activation. we have previously shown that induction of er stress and tlr signalling synergistically enhance il- p mrna expression in myeloid cells, and markedly increase secretion of il- , but not il- , by dendritic cells. the aim of this study is to investigate the mechanism of this synergy. we examined the il- promoter for potential binding sites for er stress induced transcription factors and identified a putative site for chop . chromatin immunoprecipitation (chip) assays using anti-chop and isotype control mab were performed using nuclear lysates from u cells and il- promoter dna measured by qpcr. chop binding on the il- promoter was detected following stimulation of u cells with lps or tp alone, but this was significantly enhanced when er stress and tlr stimuli were combined. il- promoter dna was not detectable following chip with the isotype control antibody. to confirm the role of chop in il- gene transcription, u cells expressing shrna's specific for chop or non-specific gene target were tested for their ability to express il- following tlr and er stress stimulation. u expressing three independent shrna targets for chop exhibited significant reductions in il- p mrna (up to % reduction of the response to lps+tp) compared to u expressing a control shrna. chop shrna expression did not affect the expression of other lpsresponsive genes, including il- , il- , ccl and sod . to identify if er stress induction of il- mediated by chop expression plays a role in a more physiological setting, we examined the role of chop in the induction of il- p gene expression following chlamydia trachomatis (ct) infection. infection of u cells with live but not g-irradiated ct induced expression of er stress response genes, including chop . u infected with live ct exhibited increased il- p mrna expression compared to u infected with nonviable bacteria. chop silencing significantly reduced the ability of live ct to induce il- p mrna, confirming the important role of chop in this response. these data suggest that er stress induction of chop could contribute significantly to the pathogenesis of diseases in which il- plays an major role, through induction of il- and il- producing cells. the clonal deletion of thymocytes by negative selection is an important process to ensure immunologic tolerance, even though the underlying molecular mechanisms are poorly understood. here, we show that gadd b, a regulator of mitogen-activated protein kinases, is critically involved in selection processes in the thymus. gadd b expression was inducible in different in vitro and in vivo models of negative selection. strikingly, only tcr-ligating peptides resulting in negative selection induced gadd b expression, while positively selecting ligands or dexamethasone, a tcr-independent apoptosis agonist, failed to do so. expression of gadd b maintained a sustained activation of p kinase and thereby promoted tcr-mediated apoptosis. in contrast, thymocytes from gadd b-deficient mice showed only transient p activation and reduced caspase activation. interestingly, we observed a switch to positive selection in gadd b-deficient mice since a higher percentage of single positive thymocytes was found. moreover, markers of positive selection as cd and cd were elevated on gadd b-deficient thymocytes. thus, we provide evidence that gadd b and a resulting persistent activation of p constitute a novel apoptotic pathway involved in negative selection. these results also provide evidence for the novel concept that not only the on-off switch of a signaling module but also its spatiotemporal regulation may crucially determine cell fate decisions. di santo institut pasteur, paris, france, monash university, victoria, australia > the thymus represents the ''cradle'' for t cell development, with distinct thymic stroma components providing multiple soluble and cellular membrane cues that foster in a step-wise fashion developing thymocytes. although il- is recognized as an essential factor for thymopoiesis, the nature of the thymic il- niche remains poorly characterized in vivo. > using a novel bacterial artificial chromosome transgenic mice in which yellow fluorescent protein (yfp) is under control of il- promoter, we identify a subset of thymic epithelial cells (tecs) that co-express yfp and high levels of il transcripts (il- hi cells). il- hi tecs arise during early fetal thymic development, persist throughout life, and co-express homeostatic chemokines (ccl , ccl , cxcl ) and cytokines (il ) that are critical for normal thymopoiesis. in the adult thymus, il- hi cells are found in cortico-medullary regions and display traits of both cortical or medullary immature tecs. interestingly, the frequency of il- hi cells decreases with age, suggesting a mechanism for the age-related thymic involution that is associated with declining il- levels. conversely, the frequency of il- hi cells is markedly increased under severe lymphopenia imposed by genetic mutations that cause an early and profound block in t cell development. this augment indicates that thymocyte-tec crosstalk may condition il- -expression by tecs. > together, our temporal-spatial analysis of il- -expressing cells in the thymus suggests that thymic il- levels are dynamically regulated under distinct physiological conditions. this novel il- reporter mouse provides a valuable tool to further dissect the molecular and cellular mechanisms that govern thymic il- expression in vivo. two lines of evidence have recently demonstrated that the pre-b cell receptor (pre-bcr) is associated with autoimmunity, through its surrogate-light-chain (slc) components l and vpreb. it has been shown that pre-bcrs are polyreactive for several self-antigens. the polyreactivity of the pre-bcr induces pre-bcr signaling and activation. furthermore, in human a self-reactive b cell subset was identified that co-expresses immunoglobulin light chain (ig lc) and the slc components. these vpreb + lc + b cells, found in healthy individuals, are potentially harmful as they express autoreactive antibodies associated with autoimmune diseases, like sle and ra. to elucidate the contribution of pre-bcr components to the development and activation of autoreactive b cells, we have recently generated a slc transgenic (slc-tg) mouse model in which all b cells express slc proteins. slc-tg mice exhibit spontaneous igm + plasma cell development. moreover, aging slc-tg mice have elevated anti-nucleosome igm levels, accompanied by immune complex deposition in the kidney, but do not display auto-immune pathology. nevertheless, vpreb + lc + b cells may induce pathology when self-tolerance mechanisms fail. to test this hypothesis, slc-tg mice were crossed on two autoimmune-prone genetic backgrounds: (i) em-bcl -tg mice with b cell-specific overexpression of the anti-apoptotic protein bcl and (ii) fcgriib-deficient mice. both in young slc-tg;em-bcl -tg double tg mice and in slc-tg;fcgriib -/mice spontaneous germinal center (gc) formation -which is associated with autoimmunity -was significantly enhanced, when compared with control em-bcl -tg and fcgriib -/mice. in slc-tg;fcgriib -/mice, numbers of splenic igg plasma cells and serum igg levels were˚ - fold increased. importantly, serum from young slc-tg;em-bcl -tg and slc-tg;fcgriib -/mice contained high titers of igg auto-antibodies in / and / cases, respectively. these values were increased when compared with control groups: / in fcgriib -/and / in bcl -tg. finally, we found that the collagen induced arthritis (cia) was significantly enhanced in slc-tg;fcgriib -/mice, compared to fcgriib -/mice. taken together, these findings demonstrate the slc components have the capacity to induce auto-antibody formation in the mouse and and to enhance autoimmune pathology in ra. t cells are generated from progenitor cells that enter the thymus from bone marrow via blood. these progenitor cells once within the thymus have little selfrenewal capacity. differentiation from hematopoietic stem cells to early t lineage cells proceeds through a series of intermediate precursor populations. however, it is largely unknown to what extent these cell populations contribute to t cell development in the presence of other precursor populations and how the earliest intrathymic t cell progenitors are generated from extrathymic precursors. to assess the relative contribution of potential precursors to t lineage differentiation we developed a strategy based on the depletion of well-characterized precursor populations rather than their enrichment and subsequent adoptive transfer together with an equal amount of congenic non-depleted bone marrow. thus, the physiological ratio of extrathymic precursors remained largely intact and we were able to address the question whether there is only one physiological t cell precursor or many. we showed that, under such competitive conditions, t lineage progenitors are confined to the cd + cd + fraction of bone marrow cells. notably, t lineage reconstitution was not restricted to either cd hi cells, representing multipotent progenitors, or cd + cells, representing common lymphoid progenitors, both of which contributed to t lineage differentiation with different kinetics. in conclusion, our data suggest that multiple physiological extrathymic t cell precursors exist, which are able to compensate for the loss of depleted populations. thus, our findings may have implications for devising strategies for improved t lineage reconstitution after hematopoietic stem cell transplantation. background: previous results from our group have demonstrated ephb and ephb expression on both thymocytes and thymic epithelial cells (tecs). we used chimeric models to determine that those molecules govern in an autonomous and non autonomous manner thymocyte and tec development, and how they regulate interactions between both cell types. objectives: in order to better define the importance in thymus of eph-ephrin b interactions we have analyzed the effects of the lack of ephrin b and/or ephrin b , the ligands of ephb and ephb receptors. this approach is specially interesting taking into account that eph-ephrin signaling is transmitted to both the two cells participating in the interaction and that the cell responses depend on the type of signals (reverse, forward or both), their direction and intensity. methods: for this purpose we have used cre-loxp recombination systems for deleting ephrinb or ephrinb genes specifically on either thymocytes or tecs. results: animals with ephrin b deficient thymocytes showed thymic hypocellularity and alterations on t-cell development whose severity depended on the background of the analyzed mice. in these mice only a few changes occurred in the cortical tec network. on the contrary, mice with conditioned deletions in tec, especially ephrinb /b double mutants, showed a more severe phenotype that began early in the ontogeny and resulted in very small thymi exhibiting an extremely compact cortical and medullary network, decreased numbers of cd + cells in the cortex, increased proportion of k +k + cells and high presence of cysts. in addition, t-cell development was partially blocked at the dn cell stage. conclusion: these data reveal an autonomous and non-autonomous role for ephrinb and ephrinb in the development of both t cells and tecs, confirming the importance of these molecules in the establishment of a crosstalk between the main two cell types of thymus. we discuss how eph-ephrin contacts modulate cellular homotypic and heterotypic interactions that take place during thymus organogenesis and in t cell differentiation. a. rolink , d. vanhecke university of basel, developmental and molecular immunology, basel, switzerland the importance of normal t lymphocyte development in the immune system is exemplified by the occurrence of inherited and acquired human immunodeficiencies where the development or functional maturation of t cells is defective. in order to identify molecules/genes and elucidate developmental processes that are essential for human t cell development we use a novel in vitro tool, the op -dl cell culture system ( ) . using this in vitro assay we obtain large numbers of human cycd + and cd + cd + double positive thymocytes starting from umbilical cord blood (ucb) derived cd + hsc. signals and molecules that are involved in t cell development are being addressed by using blocking antibodies and/or chemical inhibitors. similar as in mice we found an essential role for il- and notch mediated signaling in the development and survival of particular developmental stages of human thymocytes. among the molecules that are rapidly induced in cd + cells upon notch signaling is cd followed by cd . t cell specification is accompanied by the induction of cd a and loss of cd on cd + cd + cells. these cd -cd a + cd + cells become dependent on continuous il and notch signaling for sustained survival and further differentiation into cd + cd ab + dp thymocytes. we found that flt l is not essential for the differentiation of cd + cd + human thymocytes but that addition of exogenous flt l in the co-cultures increases the number of cd + precursors and consequently result in higher yields of developing cd + cd ab + dp thymocytes. finally few mature tcrab + t lymphocytes develop from the cycd + cd + cd ab + dp subset in this in vitro assay suggesting that op stromal cells lack the required selecting mhc-antigen complexes and/or costimulating molecules to induce and sustain positive selection of human thymocytes. this in vitro assay will allow us now, by using rna interference, to test additional genes for their role during human lymphoid development. from a clinical standpoint, a better understanding of the mechanisms controlling human t-cell development is a fundamental step towards the development of specific therapies for the treatment of primary and acquired immunodeficiencies as well as for the treatment of malignant t-cell disorders. brain derived neurotrophic factor (bdnf) promotes various neuronal functions such as survival, regeneration and synaptic plasticity. emerging evidence also indicates an essential role for bdnf in the immune system, e.g. in the b and t cell lineages. we therefore investigated the impact of bdnf on thymocyte development using bdnf knockout (ko) mice and conditional ko mice lacking bdnf specifically in t cells. in both models, we found reduced thymocyte numbers and a significant increase in double negative thymocytes. in contrast, the percentage of naturally occurring regulatory t cells and the expression of activation markers were unaltered. moreover, the lack of bdnf did not result in enhanced thymocyte apoptosis. the increase in double negative thymocytes was due to a partial block in the transition from the dn to dn stage, where bdnf and its receptor p are expressed as revealed by real-time pcr. the observed partial block in thymic maturation results in mild peripheral lymphopenia without affecting the activation status of peripheral t cells, their homeostatic proliferation and without compromising peripheral immune responses in general. in summary, our findings point to a critical role of t cell lineage derived bdnf in thymocyte development acting in an autocrine and/or paracrine manner. r. berga , c. lópez-rodríguez pompeu fabra university, barcelona, spain nfat is a transcription factor that belongs to the rel family (nf-kb and nfatc proteins). its expression in primary cells and organs is restricted to certain proliferative tissues, like activated t lymphocytes and thymocytes, where levels of nfat are relatively high and its subcellular distribution is predominantly nuclear. recent mouse models suggest that nfat participates in thymocyte development and also indicate its involvement in t cell proliferation and survival. nfat deficient mice present a t cell immunodeficiency consistent on lymphopenia, which is more accused for cd + lymphocytes. these observations are of substantial relevance as we and others have described that, in vivo, nfat -null mice are unable to mount cd +-and cd + -immune responses. data from our laboratory indicate that nfat -null mice suffer from hyperosmolarity in plasma (hypernatremia) as a result of the incapacity to induce an osmoprotective gene expression program at a systemic level. to selectively analyze the t-cell autonomous effects derived from the lack of nfat during the development of t lymphocytes, we developed mouse models that delete nfat at early (lck-cre + /nfat flox/flox ) or late (cd -cre + /nfat flox/flox ) stages of thymocyte maturation and that present isotonic plasma. our work indicates that nfat is expressed at all stages of t cell development. in addition, analysis of mouse models that lack nfat at different points of t cell development indicate that it participates at early stages of the ontogeny of t cells. objectives: apoptosis mediated by the tumor suppressor molecule p , is regarded as a major player in tumor prevention but this may not be its only role. we have investigated this by creating a mouse (m ¿ pro) lacking residues - of the proline-rich domain of p . methods: we compared the ability of various hemaptopoietic tissues from m ¿ pro mice and wild type mice to undergo apoptosis following irradiation or treatment with pro-apoptotic drugs. apoptosis was measured by staining with annexin v in vitro and by detection of caspase activation in vitro and in vivo. we also compared their ability to undergo cell cycle arrest using brdu staining. tumor development was monitored in cohorts of m ¿ pro, p null (p -/-) and wild type (p +/+) mice, with or without prior irradiation. results: apoptotic function was lacking in m ¿ pro mice, but they were able to arrest cell-cycle progression in hematopoietic tissues. m ¿ pro developed late-onset b-cell lymphoma, but not the thymic t-cell tumors found in p -/-mice. interestingly, m ¿ pro lymphomas were comprised of incorrectly differentiated b-cells. bcell irregularities were also detected in m ¿ pro prior to tumor onset, in which aged mice showed an increased population of inappropriately differentiated b-cells in the bone marrow and spleen. we propose that the apoptotic function of p has an important role in b-cell homeostasis, which, in turn, is important for prevention of b-cell lymphomas moreover, our data suggest that the apoptotic function of p is not important for preventing thymic t-cell tumors. s. myrczek , r. pardi , a. gessner microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany, vita-salute san raffaele university school of medicine, milano, italy jab is the catalytic subunit of the highly conserved cop signalosome. this complex plays a central role in various cellular processes as proliferation and cell cycle control. jab regulates the neddylation of ubiquitin ligases and thus contributes to degradation of many proteins. furthermore jab regulates the activity of ap transciption factors. to date jab is thought to be essential for every cell type as jab knock out mice are embryonic lethal and t cell development is blocked by t cell selective absence of jab . to investigate the function of jab in b cells we established a mouse strain deficient for jab selectively in b cells. mice with floxed alleles of jab kindly provided by r. pardi were crossed with a mouse strain expressing the cre recombinase under control of the mb -locus (m. reth, freiburg). ablation of jab expression resulted in an almost complete block of b cell development at the pro b cell stage. the absence of peripheral mature b and b cells and serum immunoglobulins resulted in chronic arthritis with high pathogen burden after experimental infection with borrelia burgdorferi. the observed block in b cell development is rescued by over expression of the anti apoptotic protein bcl under the control of the m enhancer. facs analyses revealed that all b cell subtypes analyzed in the jab -deficient, bcl -transgenic mice are present albeit at reduced numbers compared to wild type animals. serum immunoglobulin titers are detectable and after borrelia infection specific antibodies are produced. we confirmed the absence of jab in sorted spleen b cells by immunoblot analysis. in summary, we show for the first time that cells are viable and functional without jab when apoptosis is prevented. t. nitta , s. murata , k. tanaka , y. takahama university of tokushima, tokushima, japan, university of tokyo, tokyo, japan, rinshoken, tokyo, japan how self-peptides are generated and displayed in the thymus to select a useful and self-protective repertoire of t cells is largely unknown, whereas the role of thymic self-peptides in eliminating self-reactive t cells and thereby preventing autoimmunity is well established. a recently identified form of proteasome, termed thymoproteasome, is specifically expressed by thymic cortical epithelial cells (ctec) and is required for the optimum generation of cd t cells. here we show that ctec display a thymoproteasome-specific spectrum of class i mhc-associated self-peptides, which is essential for positive selection of major and diverse repertoires of class i mhc-restricted t cells. indeed, cd t cells generated in the absence of thymoproteasomes display a markedly altered tcr repertoire that is defective in both allogeneic and antiviral responses. these results demonstrate that thymoproteasome-dependent self-peptides are required for positive selection of a diverse repertoire of immunocompetent cd t cells. defects in helper t cell number or function causes susceptibility to infections and in some cases autoimmunity or allergy. our understanding of the genetic control of helper t cell differentiation into specific functional subsets is still far from complete. here we present the results to date from a genome-wide enu mutation screen for mice with inherited deficits in specific helper cell subsets. these deficits were detected by multi-colour facs analysis of peripheral blood samples, and by antibody production following immunization with heat-killed b.pertussis and cgg coupled with the hapten arsonate (aba) in alum, which induce internally polarized th and th antibody responses, respectively. using this screen, a number of new mutant strains have been isolated with complete or partial loss of cd + t cells or functional deficits that selectively interfere with th or germinal centre responses. in this talk i will present data from some of the first strains that have been identified including the first strains where we have been able to identify the causative mutation. systematic genetic analysis of helper t cell differentiation in the resulting strains will illuminate how t cell help is correctly polarized for immunity and to avoid immunopathology. intrathymic t-cell development provides a unique model system to study cell fate determination because of the well-defined cellular stages and the confined microenvironment of this process. in order to highlight the differences and similarities between fetal and adult t-cell development at the molecular level we performed a microarray study. labelled rna from facs purified fetal and adult dn c-kit high (etp), dn and dn thymocyte populations was hybridised to affymetrix mouse a- . genechips. the resulting data were grouped into four distinct gene clusters: cluster i contained genes over-expressed throughout adult development and included a large proportion of transcription factors ( out of genes), illustrating a significantly different transcriptional program acting during adult differentiation. conversely, cluster ii consisted of genes that were over-expressed in fetal progenitors and included signal transducers (out of genes) such as acvr , bmpr , fzd , chemokine receptors cx cr , cxcr and integrins a , a , a , ae and av, pointing to a difference in microenvironments. genes that showed uniform down-regulation during consecutive stages of fetal and adult development were restricted to cluster iii. amongst these were transcripts governing alternative developmental choices, therefore emphasising a common mechanism of lineage restriction during thymopoiesis. on the other hand, cluster iv was limited to genes that were homogeneously up-regulated during development. these included gata- , tcf- , notch- , rag- , rag- and pre-ta, which are indispensable for t cell development. interestingly, levels of expression of these genes were elevated in fetal progenitors, especially at the etp and dn stages, suggesting that the molecular program of t-cell development is more advanced in the early stages of fetal differentiation. discriminant analysis with the use of the support vector machine arrived at the same conclusion that demonstrated a nearby clustering of all fetal stages with the adult dn population, therefore implying a more committed state of fetal progenitors. finally, transcriptional signatures of each developmental stage were defined by "recursive feature elimination" with support vector machines. this approach can now be used to classify characterised and aberrant hematopoietic progenitors and thus construct an ontological scheme of hematopoietic development based upon transcriptional signatures of populations under normal and pathological conditions. tcrgd+ cells and tcrab+cd aa+ intraepithelial lymphocytes (iels) of the gut are unconventional t cells that reside in tissues and provide innate-like immune responses to "stressed-self". as these cells share common functional properties in the periphery, we have hypothesised a common mechanism of development in the thymus; their progenitors diverging from the conventional t cell developmental pathway based on tcr signal strength at the dn stage. the pre-t-alpha chain (pta) that pairs with tcrb to generate the pre-tcr, has two isoforms; pta a and pta b . both can form a functional pre-tcr with tcrb. ligand-independent signalling by the pre-tcr is a result of spontaneous oligomerisation (followed by internalisation), that is mediated through charged residues on the pta chain. pta b lacks out of of these essential residues and therefore, we speculate results in higher surface expression and different signalling capabilities. we have hypothesised that pta a and pta b permit differential signal strength through the pre-tcr at the dn stage, facilitating the divergence of the conventional and unconventional lineages of tcrab+ t cells. preliminary semi-quantitative pcr data suggest that pta a and pta b are differentially expressed in wt thymocytes at different stages of ontogeny. retroviral transduction of pta -/-e thymocytes with either pta a or pta b alone, followed by fetal thymic organ culture, confirmed the rescue of abt cell development by both isoforms. however the two isoforms appear to differentially regulate the kinetics of thymocyte development by - days of culture; pta b expression generates a greater percentage of tcrab+ cells while pta a expression results in the accumulation of isps. these results suggest different roles for the two isoforms of pta in the thymic development of abt cells. in order to determine the mechanism by which pta a and pta b may generate qualitatively different signals, site directed mutagenesis was used to produce mutant chains of pta a and pta b that lack the "dimerisation residues" necessary for internalisation of the receptors. in addition, bac transgenic mice that express singly either pta a or pta b under the pta promoter are being generated to fully characterise their role in conventional vs. unconventional lineage commitment. erythroid, myeloid and lymphoid cells are initiated in parallel in appropriate cytokine environments so that specific number of erythrocytes, myeloid cells, natural killer cells, thymocytes and t cells, and precursor of b cells can be detected and counted at day of culture. if needed for further functional analyses, long-term proliferating lines and clones of progenitor t and b cells can be established at this point of "in vitro" development. hence the "in vitro" differentiation of es cells to different hematopoietic cell lineages and their progenitors can be quantified. it allows for testing the efficiencies for hematopoietic development of genetically or epigenetically different es or ips cells. aim: increasing evidence includes wnt proteins inside the group of master-signalling pathways which govern immune and non immune differentiation systems. although their precise functions in bone marrow and thymus are still controversial, numerous studies show that wnt signalling is able to control the proliferation of hsc and thymic progenitors and might also affect both their cell-fate decisions and subsequent maturation. in the present work we analyse the effect of transient stimulation of canonical wnt pathway in the differentiation potential of lin -cd + cd ahuman thymic progenitors, a multipotent and heterogeneous cell population which has the capacity to develop into t cells, nk cells, monocytes, conventional dendritic cells (cdc) and plasmacytoid dcs (pdcs). methods: human thymus samples from patients aged month to years undergoing corrective cardiac surgery were obtained and used according to the declaration of helsinki. transient b-catenin stabilization was triggered culturing purified thymic lin -cd + cd precursors with recombinant wnt a ( ng/ml) or with licl ( mm) for hr. active b-catenin, was detected by flow cytometry using anti-human active b-catenin mab ( e ) under conditions of phosphatase activity inhibition. wnt a or licl pre-treated precursor were assayed in chimeric human-mouse ftoc, in il- and scf-supported cultures for generation of nk cells and in co-cultures with murine bone marrow stromal st cells suplemented with il- and flt l. phenotype of recovered cells, apoptosis and cytokine receptors were analysed by flow cytometry. expression profile of transcription factors was analysed by real-time quantitative rt-pcr . our results demonstrate that giving a boost to canonical wnt signalling triggered by transient exposure of thymic progenitors to wnt a or licl, change their differentiation capacity enhancing nk cell production. on the contrary, wnt a or licl pre-treated thymic progenitors generate a significant lower number of myeloid lineage cells, monocytes and cdc, as well as reduce their capacity to differentiate into pdc lineage. as a possible mechanism for this effect we show that wnt pre-treated progenitors change their expresssion of receptors for cytokines pivotal for their expansion and differentiation, such are il- and flt l and modify the transcription factor profiles of cd + cd thymocytes mainly increasing hes- and id expression levels. human th clones and circulating th cells showed lower susceptibility to the anti-proliferative effect of tgf-beta than th and th clones or circulating th oriented t cells, respectively. accordingly, human th cells exhibited lower expression of clusterin, and higher bcl- expression and reduced apoptosis in the presence of tgf-beta, in comparison with th cells. umbilical cord blood naï ve cd (+)cd (+) t cells, which contain the precursors of human th cells, differentiated into il- a-producing cells only in response to il- beta plus il- , even in serum-free cultures. tgf-beta had no effect on constitutive rorgamma t expression by umbilical cord blood cd (+) t cells but it increased the relative proportions of cd (+) t cells differentiating into th cells in response to il- beta plus il- , whereas under the same conditions it inhibited both t-bet expression and th development. these data suggest that tgf-beta is not critical for the differentiation of human th cells, but indirectly favors their expansion because th cells are poorly susceptible to its suppressive effects. m. irla , w. reith university of medecine, pathology and immunology, geneva, switzerland objectives: medullary thymic epithelial cells (mtecs) are specialized for inducing central immunological tolerance to self-antigens. to accomplish this, mtecs must adopt a mature phenotype characterized by expression of the autoimmune regulator aire, which activates the transcription of numerous genes encoding tissue-restricted self-antigens. the mechanisms that control mature aire(+) mtec development in the postnatal thymus remain poorly understood. however, the generation of mutant mice exhibiting blocks in thymocyte differentiation at different stages, together with studies on embryonic development of the thymus, have demonstrated that reciprocal interactions between developing thymocytes and tec control not only t cell development but also the differentiation and organization of tec, a phenomenon designated as 'crosstalk'. the aim of the project outlined here is to elucidate the cellular and molecular mechanisms by which thymocytes control the numbers of mature mtec, key mediators of central tolerance. we have demonstrated by generating different transgenic mouse models, that although either cd (+) or cd (+) thymocytes are sufficient to sustain formation of a well-defined medulla, expansion of the mature mtec population requires autoantigen-specific interactions between positively selected cd (+) thymocytes bearing autoreactive t cell receptor (tcr) and mtecs displaying cognate self-peptide-mhc class ii complexes. these interactions also involve the engagement of cd on mtecs by cd l induced on the positively selected cd (+) thymocytes. conclusion: this antigen-specific tcr-mhc class ii-mediated crosstalk between cd (+) thymocytes and mtecs defines a unique checkpoint in thymic stromal development that is pivotal for generating a mature mtec population competent for ensuring central t cell tolerance. q. qiu , i. ravens , g. bernhardt hannover medical school, institute of immunology, hannover, germany cd is originally identified as human poliovirus receptor (pvr) and as rodent tage , which is overexpressed in rodent colon carcinoma. cd is also known as necl- , a particular notable nectin-like molecule belonging to immunoglobulin superfamily, owning its unique expressing frofiles. cd expression is very low in most adult organs, but is abundant in the developing or regenerating liver. in addition, cd is overexpressed in transformed cells and promotes the cell cycle. thus, cd seems to be an oncofetal protein that functions in embryonic development and cancer progression. t-cell development is characterized by the progression through several phenotypically distinct stages, defined as double negative (dn), double positive (dp) and single positive (sp) based on expression of the co-receptors cd and cd ; the dn subset is further subdivided into four stages (dn - ) by differential expression of cd and cd . thymocytes at different stages of development occupy distinct spatially restricted domains in the adult thymus, indicating that differentiation occurs concomitantly with a highly ordered migration. during their final maturation in the medulla, semi-mature sp thymocytes down-regulate activation markers and subsequently exit into periphery. while semimature cd + sp are sensitive to negative selection, it remains elusive when negative selection occurs in the cd lineage. here we show that the frequency of terminally matured cd + sp cells but not that of cd + sp present in thymus varies depending on age. in mice lacking expression of the adhesion receptor cd , a selective deficiency of mature cd + sp thymocytes was observed emerging first in adolescent animals at the age these cells start to accumulate in wild type thymus. evidence is provided that the mature cells emigrate prematurely when cd is absent thus cutting short their retention time in the medulla. moreover, in unmanipulated wild type mice semi-mature cd + sp thymocytes are subjected to negative selection as reflected by the diverging t cell receptor repertoires present on semi-mature and mature cd + t cells. in cd deficient animals, a shift in the tcr repertoire displayed by the pool of cd + sp cells was found demonstrating that cd is involved in negative selection. in the adult, steady-state, homeostatic conditions, lymphohematopoietic cell lineages display high rates of cell turnover. yet, the frequencies of simultaneously cycling cells are small, except in intermediate cellular stages of transit-amplifying precursor cell stages. the analysis of the molecular targets controlling these proliferation rates may provide relevant information to understand differentiation pathways along the ontogeny as well as mechanisms of leukemic transformation (passegué et al. j. exp. med. , , . during development, hematopoietic stem cells and their derived cell lineages need to expand to cope with continuously-increasing somatic demands. by using complementary, quantitative analyses (brdu labelings, hoescht , propidium iodide), we are dissecting the proliferation rates of hematopoietic cell lineages and their differentiation stages along the whole mouse gestation from e (e, gestational day) on, in yolk sac, splanchnopleura/agm, blood, liver, spleen and bone marrow. we have observed that around half of cd + ter + erythroid and cd + cd b + myeloid cells are simultaneously cycling (s/g /m) in the post-gastrulation mouse embryo (e - ). the peak of lymphohematopoietic cell proliferation occurs at e in a sort of wave-like pattern. these high-proliferation frequencies are present not only in immature, but also in mature cells, the latter thus displaying a different behaviour from the one present in the adult. later on, the proliferating cell subsets are restricted to fetal liver, whereas the equivalent cells become arrested in the periphery. interestingly, nucleated erythroid cells suddenly go into quiescence - hours before they enucleate, suggesting that this cell arrest is required for the enucleation process. we are also analysing the proliferation state of the first b and t lymphoid progenitors emerging at e - that give rise to perinatal lymphocytes and, in some cases, to innate-like lymphocytes displaying self-renewal in the adult. we attempt to dissect the mechanisms regulating proliferation and death in the embryo versus those of adult lymphohematopoietic precursors, which may influence the functional activities of the mature cells. objectives: the role of cd -cd interactions in t cell activation of antigen presenting cells and b cells is known, but a role for this receptor-ligand pair in hematopoiesis control has not been described. following an initial discovery that b lineage cells in the bone marrow (bm) as early as pro-b cells express cd , we hypothesised a role for cd -cd interactions in the control of b cell haematopoiesis. the objectives of this study were to investigate this hypothesis further. methods: flow cytometry was used to investigate cd expression by precursor b cells using b cell specific markers. reverse transcription of bm stromal cell rna and pcr were used to assess the presence of cd message and cell lineage specific mrna. irradiation and bone marrow transplantation (bmt) in both directions between cd -/-and wt mice was used to assess potential functional contributions of stromal or haematopoietic cd on reconstitution of b cell numbers following depletion. we show that cd is expressed by pro-b cells, and these cells proliferate in response to cd signalling in vitro. pcr identified a source of cd , negative for cd eta, in the bm of wt mice showing this cd is not provided by activated re-circulating t cells. we have shown that when cd -/-mice are recipients, but not donors of bmt, b cell recovery after irradiation is significantly delayed regardless of the donor cell source. in the in vitro experiments we found that the pta gene is expressed from the dn (cd -/cd -/cd + /cd -) to the dp (cd + /cd + ) stage, whereas no yfp expression could be observed in the b lineage. the in vivo analysis of thymocytes confirms the appearance of yfp positive cells during t cell development from the dn stage on. in the bone marrow we found yfp + /b + and yfp + /b populations. thus these pta expression analyses show closely similar pattern to those observed with hucd preta-reporter transgenic mice (gounari f. et al. , martin et al. . the bac pta reporter system can be used together with specific markers of other hematopoietic lineages and their progenitors to trace lymphopoiesis. gounari f et al., nat. immunol. , - ( ) martin c. h. et al., nat. immunol. , - ( . the individual functions and the reason for the tightly regulated expression of igm and igd during b cell development are poorly understood. our data show, that igd requires stronger stimuli than igm to induce b cell activation and that this silences autoreactive vdj recombination products when expressed as igd. in agreement with this, mhc and dhc, the respective heavy chains of igm and igd, differ dramatically in pre-bcr signaling, which represents the prototype of an autoreactive receptor. together with published data, our results reveal a novel role for igd and suggest that the differential expression of igm and igd is important to raise the activation threshold of mature b cells, thereby avoiding hypersensitivity and ensuring tolerance towards self-antigens. p. d. rymkiewicz , g. klein zmf (center for medical research), section for transplantation immunology and immunohematology, tübingen, germany thymic conduits which are exclusively found in the medullary region of the thymic lobules have been recently identified. the core of the conduits consist of fibrillar collagen bundles and is surrounded by a basement-membrane-like structure which contains the typical basement membrane components such as laminins, collagen type iv, nidogens and perlecan. a marker molecule for the conduits in the human thymus is the laminin isoform lm- which is synthesized by the medullary thymic epithelial cells (tecs) which tightly surround the conduits. functionally the conduits are too small to transport cells but they are able to transport small molecules x kda.mmp- , a secreted member of the matrix metalloproteinase superfamily, is a protease capable of digesting lm- . in the human thymus medullary, but not cortical thymic epithelial cells strongly express mmp- . by western blotting the zymogen and the activated form of mmp- can be detected in whole thymus lysates, whereas in lysates from isolated tecs mainly the activated form is present. an in situ zymographic analysis revealed an increased proteolytic activity in the medullary region of the thymus. using confocal laser scanning microscopy double immunofluorescence staining showed that lm- and mmp- can be found in close neighbourhood, but they do not exactly co-localize. why activated mmp- which can be secreted by medullary tecs does obviously not destroy the surrounding basement membrane of the conduits has not been solved so far. two natural inhibitors of mmp- , timp- and timp- , are found in the thymic medulla, but they are not expressed and secreted by tecs. whether mmp- plays a role in processing medullary chemokines which are produced by the thymic epithelial cells is presently under investigation. to study the process of t cells differentiation in more detail, we intend to establish an inducible gene expression system (tet-on system) in primary t cells. the tet-on system comprises two retroviral vectors. the response vector contains an inducible modified minimal cmv promoter which per se is unable to induce expression of the gene of interest (goi). the second vector encodes a transactivator which is constitutively expressed and undergoes conformational changes upon binding of doxycycline. in this state, the transactivator enables the minimal cmv promoter to transcribe the gene of interest. therefore, co-transduction of both vectors is required to achieve transcription of the gene of interest. to date we have tested two tet-on systems (revtet system and retro-x tet-on advanced inducible expression system) that differ in the sequence of their inducible promoters. to monitor successful transfection in retrovirus-generating phoenix cells and transduction in t cells, respectively, we have cloned the reporter gene gfp under the control of a constitutive cmv promoter, into the response vector of the revtet system. this allowed identification of transduced gfp-positive cells via facs. however, when we used a red fluorescent protein, tomato, as a surrogate goi, we detected considerable leakiness of the promoter irrespective of the presence of the transactivator or doxycycline. in contrast, we found comparably low leakiness when using the retro-x tet-on advanced inducible expression system. here, co-transfection of phoenix cells with the transactivator and supplementation of doxycycline yielded an induction of - % compared to only % basal rate. therefore, the retro-x tet-on advanced inducible expression system appears suitable for our studies. future experiments will aim at establishing this system in primary t cells. although a number of different experimental approaches has been used to elucidate impact of basal levels of adrenal gland-derived glucocorticoids (gcs) on t-cell development, and thereby t-cell-mediated immune response, their relevance for these processes is still far from being understood. the study was undertaken to explore relevance of basal levels of gcs for t-cell differentiation/maturation. eight days post-adrenalectomy in adult male rats thymocyte yield, apoptotic and proliferative rate and relationship among major thymocyte subsets defined by tcrab/cd /cd expression were examined using flow cytometry analysis. it was found that adrenal gc deprivation affects: i) thymocyte apoptosis, producing thymic hypercellularity and ii) kinetics of t-cell differentiation/maturation leading to an overrepresentation of the cd +cd + double positive (dp) tcrab low cells entering selection, and their cd +cd + dp tcrab-immediate precedents followed by underrepresentation of the selected cd +cd + dp tcrab high and the most mature cd -cd + and, particularly, cd +cd -single positive (sp) tcrab high cells. the study suggests that withdrawal of adrenal gcs produces alteration in thymocyte selection processes that may affect diversity of functional t-cell repertoire and generation of potentially self-reactive cells as indicated by the reduced proportion and number of cd -cd -double negative tcrab high cells. in addition, it indicates that gcs influencing post-selection maturation of thymocytes play a regulatory role in controlling mature cd +cd -/cd -cd + sp tcrab high cell ratio. in the thymus a specific subset of thymic stromal cells -medullary thymic epithelial cells (mtecs) -express a highly diverse set of tissue-restricted antigens (tras) representing essentially all tissues of the body, which is known as promiscuous gene expression (pge). this allows self-antigens, which otherwise are expressed in a spatially or temporally restricted manner to become continuously accessible to developing t cells. the scope of central tolerance is to a large extent dictated by the pool of promiscuously expressed genes. thus, even lack of a single tra can result in spontaneous organ-specific autoimmunity. promiscuously expressed gene which have no structural or functional commonality display two prominent features, they are highly clustered in the genome and show a preference for tras. for better understanding these features, we set out to precisely define the genomic organization of this gene pool. in particular, we probed to what extent and according to which rules predefined genomic clusters of tras are transcribed in mtecs. our analysis proceeded from the bioinformatic definition of tra clusters via gene expression analysis in mtecs using whole genome arrays to the in depth analysis of selected tra clusters by rt-pcr at the population and single cell level. patterns emerging from these studies will hopefully yield insight into evolutionary mechanisms responsible for selecting this gene pool. conceivably, positional cues in the genome and/or particular properties of self-antigens (e. g. immunogenicity) could have been driving forces during the co-evolution of pge and adaptive immunity. although catecholamines have been shown to influence thymocyte proliferation and differentiation, long-lasting b-adrenoceptor (ar) blockade failed to show any significant effects on thymic cellularity. bearing that in mind, the present study was undertaken to explore: i) a -ar expression on thymic lymphoid and nonlymphoid cells and ii) putative role of a -ar-mediated mechanisms in modulation of thymic cellularity and t-cell development. for this purpose a -ar expression on thymic cells was assessed using both immunocytochemistry and flow cytometric analyses, while their putative modulatory role in thymopoiesis was estimated by analyses of thymocyte proliferation and apoptosis, as well as expression of major thymocyte differentiation antigens (cd / cd /tcrab), in adult wistar rats subjected to -day-long treatment with a -ar blocker urapidil ( . mg/kg body weight/day s. c.). the a -ar immunoreactivity was found in both thymocytes (mainly less mature cd and cd low cells) and thymic nonlymphoid cells (thymic epithelial cells located mainly at cortico-medullary junction and cortical ed -postive cells, which comprise macrophages and dendritic cells). chronic treatment with urapidil increased thymic weight and caused the organ hypercellularity. the thymic hypercellularity reflected, at least partly, increased frequency of proliferating thymocytes, which was followed by diminished thymocyte apoptosis. in addition, in these rats changes in distribution of major thymocyte subsets delineated by cd / cd /tcrab expression were observed. these changes comprised of an increase in the percentage of cd + + tcrabthymocytes, which was accompanied by the reduction in that of cd + + tcrab low cells in urapidil-administered rats, and divergent changes in the percentage of the most mature single positive tcrab high thymocytes. compared with saline-administered controls, the percentage of cd + -tcrab high thymocytes in urapidil-administered rats was increased, while that of the cd - + tcrab high was reduced. in addition, the percentage of cd + t regulatory and cd +tcrab+ nkt cells was increased. collectively, this study clearly showed the expression of a -ar on both lymphoid and nonlymphoid thymic cells, and indicated that a -ar-mediated mechanisms may be implicated in modulation of multiple steps of t-cell development. we have recently shown that the thymus is a common target for mycobacterial infections. of notice, while bacterial growth is arrested in the spleen around weeks post infection with mycobacterium avium, it takes several weeks longer within the thymus to reach a bacterial plateau. this observation suggests that a specific immune response occurs in the thymus, although this seems to be distinct from that occurring in the spleen. since t cell differentiation occurs, to a large extent, in the thymus, and depends, among other factors, on the antigens encountered within the thymus and on the cytokine milieu of the organ, we decided to characterize the pattern of the thymic immune response against mycobacteria and investigate possible consequences on the normal function of this organ. methods: c bl/ mice infected with m. avium ( cfus, iv) were sacrificed at different time points after infection ( , and weeks). non-infected animals were used as controls. bacterial load was assessed in the spleen and thymus and cytokines (such as ifn-g, tnf, il- ) were quantified by rt-pcr in tissues (normalized for hprt expression) or by elisa in the supernatants of cultured thymocytes and splenocytes. statistical significances were determined by anova. we observe increased levels of ifn-g in infected thymi at weeks post infection. this increased expression of ifn-g is concordant with the late bacterial growth arrest within the thymus. at weeks post infection this difference is still present. throughout the course of infection no significant differences are found in the expression of tnf and il- in this organ. in the spleen, ifn-g reaches a peak of expression earlier ( weeks post infection) and this is accompanied by increased tnf expression. conclusion: our cytokine analysis of the thymus and spleen confirm that an immune response against mycobacteria is mounted within the thymus, although different in timing and pattern from the one in the spleen. since the cytokine milieu influences t cell differentiation within the thymus, our observations raise the question on the consequences of such response on the normal function of this organ. such implications should next be investigated. precise regulation of eukaryotic gene expression requires interactions between distal cis-acting regulatory sequences with the looping out of the intervening dna, but how trans-acting regulatory proteins work to establish and maintain dna loops during gene activation remains largely unexplored. lps-induced transcription of the mouse igx gene in b lymphocytes utilizes three distal enhancers and requires the transcription factor nf-xb, whose family members include rela and c-rel. using chromosome conformation capture technology in combination with chromatin immunoprecipitation, here we demonstrate that lps-induced igx gene activation creates chromosomal loops by bridging together all three pair-wise interactions between the distal enhancers and rna polymerase ii, the apparent molecular tie for the bases of these loops. rela and actin polymerization are essential for triggering these processes, which do not require new transcription, protein synthesis or c-rel. we have thus identified both essential and non-essential events that establish higher-order chromatin reorganization during igx gene activation. this investigation was supported by grants gm and ai from nih and grant i- from the robert a. welch foundation to wtg, and by grants hl and hl from nih to lst. allelic exclusion of immunoglobulin (ig) genes supports burnet's clonal selection theory. the recognition that m-chain expression is sufficient for the maintenance of the silenced allele status by a process of feedback inhibition is yet not enough to explain the earlier monoallelic activation by the rag complex. attempts to prove the probabilistic or epigenetic nature of monoallelic v(d)j recombination were insightful and favor the epigenetic hypothesis, mainly by the observation that, like autosomal imprinted or x-chromosome inactive genes, ig genes are differentially marked at the chromatin level, and replicate asynchronously in virtually any cell of the mouse organism ever since the embryonic life (mostoslavsky, singh et al. ) . we are testing this hypothesis, i. e., that an epigenetic event has previously marked, on each b cell progenitor, which of the ig alleles is going to be activated for rearrangement and expression. for this, we are generating b cell clones from b cell progenitors of (c bl/ x balb/c)f mice, because the original strains have ig heavy chain (igh) alotypes distinguishable by monoclonal antibodies. we are analyzing if individual heterozygous clones show biased expression of a particular igh allele, and we expect to map the cell stage at which the (epigenetic) allelic marks are fixed. we have already shown, for the igh gene, a clear segregation of monoallelic expressors among b cell clonal lines that were generated from the common lymphoid precursor, but no allele bias was observed among multi-potent progenitor or hematopoietic stem cell b cell clones. this result, although suggesting epigenetic silencing starting at the common lymphoid precursor stage, does not favor the prevailing epigenetic hypothesis in its original formulation. we are currently exploring this result by the analysis of the igh silenced allele rearrangement status in sorted fractions of igm+ b cell clones. we are also testing the same epigenetic hypothesis for the ig kappa light chain gene (igk), using f mice in which the igk constant region from both alleles can be distinguished by antibodies. a. giniewski , s. lang , m. stein , t. winkler friedrich-alexander university, erlangen, germany vdj recombination is considered to be regulated by lineage and stage specific changes in accessibility of the loci to rag recombinase. accessibility is expected to correlate with certain histone modifications such as acetylation (e. g. h ac) or methylation of h on lysine (h k me / ). previous studies in our lab revealed three regions in the intergenic part of the distal v h cluster (ivars), which are associated with high levels of active chromatin marks (h ac and h k me / ) only in pro b cells but not in pro t cells. it is also known that vdj h recombination is accompanied by sense and antisense transcription, however, little is known about the function and origin of the antisense transcripts. since one ivar (ivar # ) shows promoter activity in antisense orientation, it was analysed in more detail. we found three transcription start sites by 'rlm race at the ivar # element. background: t-all is a malignancy of the lymphoblast committed to the t-cell lineage with translocations between tcr genes and oncogenes as a genetic hallmark. these translocations are thought to be driven by v(d)j-recombination mechanisms. we believe that these mechanisms only partly facilitate the occurrence of tcr translocations and that the accessibility of involved genes plays an intrinsic role in promoting these events. the lmo locus, is thought to be accessible only during the double-negative (dn) and thymocyte stages based on mrna expression, implying that translocations between lmo and tcr genes can only occur within these stages. gene expression as a readout for accessibility can not elucidate the involvement of other oncogenes such as tlx (hox ), which are not expressed in thymocytes, as being accessible for translocations to occur. objectives: we aimed ) to evaluate lmo and tlx breakpoint-site accessibility during thymocyte development; ) to determine in which stage of development there is an increased chance for lmo or tlx translocation to occur based on this accessibility. methods: dna of immunologically "healthy" sorted thymocytes was isolated using faire (formaldehyde-assisted isolation of regulatory-elements). this dna was used to quantitatively assess lmo accessibility during thymocyte development at both the transcription start site (tss) and negative regulatory element (nre), and within different in t-all documented breakpoint-sites of both the lmo and tlx loci. results: quantitative analysis on the tss showed a correlation with mrna expression, with the dn and dn development stages showing the highest accessibility. the nre, showed an inverse pattern of accessibility to the tss region. analysis of breakpoint-sites revealed the highest accessibility levels within the earliest stages of development, dn , dn , dn and immature single positive (isp) stages for both lmo and tlx . conclusion: our findings show that both the lmo and tlx loci are accessible during thymic development irrespective of gene expression and that this accessibility is not restricted to dn and dn stages, suggesting that these loci are much more active than assumed, thus increasing the opportunity for translocations to occur. we addressed this issue, by building a model able to account for of v-ja gene rearrangements observed experimentally during thymus development of mice. we developed, based on experimental data, a numerical model on the whole tra/trd locus to estimate va and ja genes accessibility to rearrangements. the progressive opening of locus to v-j gene recombinations is modeled through windows of accessibility of different sizes and with different speeds of progression. furthermore, the possibility of successive secondary v-j rearrangements was introduced in the modeling. the model points out some unbalanced v-j associations resulting from a preferential access to gene rearrangements and from a non-uniform partition of the accessibility of the j genes, depending on their location in the locus. the model shows that to successive rearrangements are sufficient to explain the use of all the v and the j genes of the locus. finally, the model provides information on the kinetics of rearrangements and on the frequency of each v-j association. the model accounts for the essential features of the observed rearrangements on the tra/trd locus and may provide a reference for the repertoire of the v-j combinatorial diversity. the genetic programs of b-cell differentiation and the first dj h gene rearrangements appear in the post-gastrulation mouse embryo (e - ), shortly after the first multipotential hematopoietic progenitors do emerge. these dj h joints represent the unselected baseline of the ig repertoires. we have undergone a systematic sequencing of embryo dj h joints obtained from normal balb/c embryos and heterozygous embryos obtained from rag -/mothers mated to balb/c males (to discard any mother-derived contribution), as well as newborn and adult control groups. the embryo dj h s displayed unexpected mechanisms of diversity, including short stretches of non-templated n nucleotides in one-third of the studied sequences (in the absence of tdt expression) and frequent dj h s with large nucleotide deletions, as a consequence of ligation to joint-distal microhomology sites. because the dna polymerase m (polm), a highly-homologous tdt member of the x dna polymerase family, showed an increased expression in the embryo, we analysed the dj h s of polm -/mouse embryos. we observed that polm was mainly responsible for introducing n nucleotides at the mouse embryo dj h joints. also, and based on its dna-dependent polymerization ability, polm filled-in small sequence gaps at the coding ends, and ligated highly-processed ends by pairing to internal microhomology sites, although at the cost of germline sequence losses and the generation of "useless" gene products. we think that, more than attempting to increase diversity, polm acts as a "connector" in the embryo, subsequently participating in the repair of rag-induced double-strand breaks, to preserve genomic stability and cellular homeostasis in cells with high proliferation rates. along the end of gestation, further selective pressures acting over these first v-dj h products will contribute to establish the differential neonatal ig repertoires. although mortality from infectious diseases peaks during infancy, many vaccines are ineffective in early life. most children with infantile bronchiolitis are under months of age, and most cases are due to respiratory syncytial virus (rsv) infection, for which vaccines continue to be elusive. we now show that, compared to adults, the antibody response to rsv infection is very poor in neonatal mice and is unaffected by cd cell depletion. however, cd depletion in infancy led to a remarkable boosting of antibody responses during adult re-challenge. to test the possibility that poor antibody boosting is caused by rsv-specific cd t cells killing rsv-infected b cells, we sorted cells from the lungs of infected neonates. viral copy number was high in neonatal b cells, but viral load in surviving b cells was unaffected by cd cell depletion. in addition, fas ligand (fasl) deficient gld mice responded to rsv infection in the same way as normal mice, indicating that fasl is not required for the inhibition of antibody responses. this new mechanism of regulation of b cell responses by cd t cells has important implications for vaccine development against neonatal infections. ( ) showed that irf knockout mice had significantly reduced numbers of pre-pro-b cells in marrow and a phenotype similar to agammaglobulinemia. these results prompted us to consider icsbp/irf as a candidate gene in the pathogenesis of defective early b cell development. therefore we decide to undertake direct sequencing of the gene encoding icsbp/irf in a small cohort of patients with autosomal recessive agammaglobulinemia. methods: eight patients affected by agammaglobulinemia were included in this study. all patients were under regular ig replacement therapy. informed consent was obtained from all patients. genomic dna was extracted from whole blood and amplified with specific primers designed on the flanking regions of every exon. direct gene sequencing of the eight exons of icsbp/irf were obtained using abi prism sequencer. results: seven of the eight patients result wild type while only one patients present a synonymous snp in exon v, yet documented as rs . conclusions: although recent findings indicated that irf function is essential for early b cell development, our data in a small cohort of patients affected with autosomal recessive agammaglobulinemia did not evidence any mutations in icsbp/irf that may be responsible for this disorder. the hh/ptch signaling system is known to control the development and neoplastic transformation of several cell types. however, the role of hh/ptch for the differentiation of b and t lymphocytes from hematopoietic stem cells (hsc) has not been assessed so far. to analyze the function of hh/ptch for lymphopoiesis in vivo, we have employed a genetically engineered mouse mutant in which the ptch gene can be conditionally inactivated by virtue of the cre/loxp recombination system. we show that targeted disruption of ptch in the adult organism results in a dramatic specification and differentiation defect of the lymphoid lineage leading to rapid disappearance of newly generated b and t lymphocytes from peripheral lymphoid organs. the developmental block occurs at the level of the common lymphoid progenitor cell (clp), which defines an early branching point of hsc differentiation and lineage commitment. in contrast to the lymphoid lineage, development of cell types of the myeloid lineage from common myeloid progenitors (cmp) appears normal. our data identify hh/ptch-induced signaling as a key regulator for proper development of immunocompetent lymphocytes. hence, the progression of tumors, which are initiated upon oncogenic hh/ptch mutations, may be further promoted due to impaired tumor surveillance by a compromised immune system. l. calderon dominguez , t. boehm max-planck institute for immunbiology, developmental immunology, freiburg, germany in many organ systems of animals and plants, specialized niche microenvironments maintain and specify stem and progenitor cells. the ability to modify or artificially create such niches in vivo and in vitro has many implications for stem cell research and therapy. by analysis of several mutant mouse strains and subsequent transgenesis in the mouse, we disentangle and individually modulate niche functions responsible for collection, maintenance and specification of multipotent thymocyte progenitors. we demonstrate how an epithelial niche, rendered functionally inactive by disruption of the foxn transcription factor, can be specifically rebuilt in a modular and combinatorial fashion to only attract, or attract and maintain, or attract, maintain and specify progenitor cells into the b and t cell lineages, respectively. the strategy of engineering niche functions in a modular fashion might be applicable to other progenitor cell systems. silencing of dc-sign using lentiviral rna interference revealed its critical function for pd-l expression on dcs after m. tuberculosis infection. as a counterpart to expression of its ligand, we showed that cd and cd t cells from tuberculosis patients highly express pd- when compared to healthy uninfected individuals. in addition, analysis of pd- expression in lung biopsies from tuberculosis patients revealed that pd- is expressed on cd and cd t cells confined to lung granulomatous lesions. finally, blocking of the pd- /pd-l axis using monoclonal antibodies abrogated the down-modulation of t cell proliferation and ifn-g production induced by manlam, a mycobacterial cell wall glycolipid and ligand for dc-sign. taken together, our results suggest that the pd- /pd-l pathway is involved in the exhaustion of t cell responses to m. tuberculosis. the inflammatory canonical nfkb pathway is critically involved in virtually all aspects of inflammation in general. yet, the role of the alternative, non-canonical nfkb pathway in inflammation and adaptive immunity remains largely elusive. the alternative pathway is primarily mediated through the nfkappa-b inducing kinase (nik) which in turn leads to the phosphorylation and the cleavage of p to p . among the receptors engaging nik is the ltbr, which is also required to form the anlage for secondary lympoid tissues (slts). due to a point mutation within nik, alymphoplasia (aly) mice do not develop slts and are highly immunodeficient. however, while the immunodeficiency of aly mice is widely held to stem from their developmental malformation, it has been overlooked, that the mutation of nik itself could potentially lesion the development of immune responses. to verify this notion, we generated a series of bone marrow chimeric mice (bmc) in which the absence of slts was disconnected from the hematopoietic loss of nik function. we generated mice, which lack all slts, but are equipped with a normal systemic immune system (wt aly), and conversely, mice with normal slts, but lacking nik in all leukocytes (aly wt). surprisingly, we discovered that nik is vital for the development of autoimmune disease, while slts (ie. lns, spleen etc.) are essentially dispensable for cell-mediated immunity. we found that nik is required for the polarization of effector t cells and that th and th cells cannot be generated in the absence of nik. preliminary data implicate the involvement of nik in a discrete and novel pathway required for the formation of cell-mediated immune responses. the family of nfat (nuclear factor of activated t-cells) transcription factors is indispensable for t cells, for example playing an important role in cytokine gene regulation. in peripheral cd + t cells, nfatc and c are predominantly expressed. nfatc is synthesized in six isoforms which have partly opposing functions regarding activation and apoptosis. here we address the functional difference of the short isoform nfatc /a, which is highly induced upon t cell activation, and the long constitutively expressed isoform nfatc /c. as demonstrated by y h screen and co-ips, nfatc /c-specific c-terminus can be highly sumoylated. confocal microscopy studies revealed that upon sumoylation nfatc /c -but not the unsumoylated nfatc /a -translocates to promyelocytic leukemia-nuclear bodies (pml-nbs). this leads to interaction with hdacs followed by deacetylation of histones (co-ips), which in turn induces transcriptionally inactive chromatin (chip and confocal microscopy). as a consequence, multiple expression studies revealed sumoylation dependent suppression of the nfatc target gene interleukin- . other lymphokines like ifng and il are reversely regulated. interestingly, ntreg cells which do not express il exerted only nfatc /c, but no nfatc /a expression (qrt-pcr). these findings demonstrate that the modification by sumo converts nfatc from an activator to a site-specific transcriptional repressor, revealing a novel regulatory mechanism for nfatc function. therefore, especially ntreg cells and anergized cd + t cells might be regulated by the long sumoylatable isoform nfatc /c. lnk/sh b and aps/sh b , two members of the lnk/sh b family of adaptor proteins, play an important role as negative regulators in b cell lymphopoiesis. they possess several protein-protein interaction domains and motifs that allow their interaction with different signalling effectors. mice deficient for these proteins demonstrated that lnk inhibits expansion of pro/pre-b cells while aps controls mature b- cell population, suggesting specific roles for these adaptors during b cell development. however, the molecular mechanisms underlying their regulatory function in these cells, have not been identified. to address this question, we used primary and b cell lines at different stages of differentiation as our cellular system. analysis of lnk/aps expression pattern showed that lnk is expressed at all developmental stages, while aps is only detected in immature and mature cells. we then first examined the role of lnk in il- signalling in pre-b cells overexpression of lnk dramatically inhibits il- -dependent growth demostrating that lnk negatively regulates il- pathways. furthermore, we showed that il- stimulation induces lnk phosphorylation and its subsequent association with important signalling effectors, notably the e ubiquitin ligase cbl. we next analyzed the role of aps in mature b cells by imaging and biochemical techniques. our results showed that aps colocalizes with the bcr complex after bcr triggering. interestingly, lnk is not recruted to the bcr signalosome in these cells, suggesting that interaction of the adaptors with the receptor complex regulates their function at different development stages. moreover, we showed, for the first time, that aps can associate, upon bcr stimulation, with the signalling molecules cbl and vav . to address the functional implications of these interactions, we examined specific b cell responses, notably bcr trafficking and cytoskeleton remodelling. we demonstrated that overexpression of aps enhances ligand-induced endocytosis of bcr, possibly through interaction with cbl and affects the kinetics of bcr-induced cell spreading. our results therefore suggest a regulatory function of aps in bcr internalization and cytoskeleton dynamics. altogether, our findings demonstrate that lnk and aps display sequential specific regulatory roles during b cell development that are important for maintaining b cell homeostasis. signaling through the t-cell receptor (tcr)-cd complex is a critical event in adaptive immunity. it is still not clear how ligand binding to the tcr is communicated across the plasma membrane and leads to phosphorylation of the cytoplasmic domains of the cd complex. it is widely accepted that dimerization or multimerization of tcr is required for tcr triggering. in our model t-cell activation is initiated by recognition of monomeric mhc/peptide complexes on the surface of antigen presenting cells (apc). critical to tcr triggering is the movement of the t-cell across the apc. engagement of a mhc/peptide complex on the surface of the apc will change the mobility of the tcr leading to partitioning with lipids of lower mobility that are enriched in signaling molecules critical for t-cell activation. furthermore, the change in mobility will lead to dislocation of the itams from the plasma membrane so that they become accessible to tyrosine kinases. to test the hypothesis we established a new approach where we created a soluble bifunctional complex composed of a pmhc and a fab that recognizes an epitope tag that we express on the t-cell surface. binding of the fab to the expressed epitope tag will constrain the lateral mobility of the tcr that is engaged by the pmhc arm of the same complex. the bifunctional complexes induced activation and proliferation as well as ca influx and cytokine production in human cd + t-cell clones that displayed the epitope, but not in t-cells that did not display the epitope. activation required interaction of the fab with its epitope on the t-cell surface because no activation was observed when soluble epitope peptide, which acts as a competitor for the fab binding site, was added. these results demonstrate that a monomeric copy of a pmhc is sufficient to trigger tcr and that formation of a tcr dimer is not an obligatory step in t-cell activation. the bifunctional complex we generated may also have a great immunotherapeutic impact. exchanging the fab with a fab or cytokine directed to a surface molecule may allow an antigen specific stimulatory or inhibitory modulation of t-cell responses. adaptor proteins are crucial in signal transduction, cell cycle regulation, apoptosis and stress response. adaptor proteins containing characteristic sh or sh domains known to mediate protein-protein interactions are key players in these processes. sly (sh domain protein expressed in lymphocytes ) was identified as a putative adaptor protein containing a sh and a sam domain as well as a bipartite nls. sly belongs to a family of three molecules: sly , sly and sash .in humans, the sly gene is located on chromosome , in mice on chromosome . sly is widely expressed for example in immune tissue as well as in hematopoietic cells, brain, lung and pancreas. subcellular fractionation showed that the sly protein is located in the cytoplasm and the nucleus and to a lesser extend in the plasma membrane.to elucidate the function of sly we searched for possible interaction partners by yeast two hybrid screening with a mouse t cell lymphoma library. this approach identified sin -associated polypeptide p (sap ) as a putative interaction partner of sly . sap is a conserved member of the sin a-hdac corepressor complex that contains histone deacetylase (hdac ) and histone deacetylase (hdac ) and acts as a transcriptional repressor for a variety of genes. we confirmed this interaction by implementing coimmunoprecipitations with lysates from transiently transfected t cells. in addition, we could show a direct interaction between sly and hdac . to investigate the functional impact of this molecular interaction, we performed hdac enzymatic activity assays. we were able to show that sly increases the activity of hdac in whole cell lysates and, more precisely, in nuclear extracts of t cells. the interaction of sly with sap and hdac indicates a transcriptional function of this protein. within the sin a-hdac corepressor complex sly might act as a switch for the activity of hdac . cd -cyt and cyt are co-expressed in human t cells and undistinguishable from the cell surface. in order to determine their specific role in t cell activation, we have expressed chimeric proteins consisting of the extracellular domains of cd (b cell marker) fused to the transmembrane and intracellular domain of cd -cyt or cyt in primary t cells. we show that these two isoforms differently control human t cell function. specific cyt coengagement controlled il- secretion, while cyt coligation inhibited ifng production. moreover, our preliminary data suggest that cd -cyt inhibits the phosphorylation of several molecules known to be activated by cd stimulation. these data suggest that these two isoforms act as molecular switches for t cell activation, either promoting or turning off t cells. they demonstrate for the first time the distinct roles of cd cytoplasmic isoforms in primary human t cell activation. this also suggests that the modulation of their expression and/or activation might provide new therapeutic avenues. nck is a ubiquitously expressed adapter protein that is almost exclusively built of one sh domain and three sh domains. nck connects receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. in t cells, nck participates in different and interdependent signalling pathways linking t cell activation and effector function with actin remodelling proteins that in turn initiate changes in cell polarity and morphology. we previously showed that nck directs the death factor fasl to the cytotoxic immunological synapse when t cells encounter putative target cells. we now performed a systematic screening for interaction partners of the four individual interaction modules of nck in primary and leukemic t cells. we precipitated putative binding partners from untreated or pervanadate-treated pha blasts, jurkat and hut cells with gst fusion proteins containing full length nck, the three sh domains or the individual sh and sh domains. binding proteins were excised from gels after staining with coomassie, silver or flamingo pink and processed by tryptic in gel digestion for mass spectrometrical analysis. as expected, we observed major differences in nck binding proteins precipitated from resting versus activated t cells. we not only verified established interactions (e. g. with the tcr signalling components slp and cd epsilon, the actin-regulatory proteins wasp and wip and the nuclear protein sam ) but also identified novel nck-interacting proteins. the interaction with the actin-binding protein hip once more underscores the fundamental role of nck in tcr-mediated actinreorganization. the identification of the nuclear proteins sfpq/nono points to novel, yet unknown functions of nck that might be associated with the recently reported nuclear translocation/localization of nck. accordingly, employing laser scanning microscopy, we clearly detected nck within the nucleus also in human t cells. the present data highlight that nck serves versatile functions in t cells, which include the different interdependent pathways of tcr-induced actin reorganization but also novel, yet poorly defined protein networks that are associated with a nuclear translocation of nck. cytotoxic t lymphocytes (ctl) mediated killing is tightly regulated according to the strength of t cell receptor signal. killing is regulated by the delivery of perforin-containing lytic granules moving along microtubules towards the centrosome, which polarizes and docks at the central supramolecular activation complex (csmac) within the immunological synapse. although much has been learnt about the mechanisms controlling the strength of tcr signal and the mechanisms required for release of the lytic granules, little is known about how the strength of the tcr is able to control the degree of ctl-mediated killing so finely. here we examine how the strength of tcr signal controls polarization of the secretory apparatus leading to ctl-mediated killing using tcr transgenic ot-i ctl. decreasing the tcr signal by reducing the concentration of ova peptide or using the weak agonist peptide, g , results in a slight reduction in the number of ctl target cell conjugates formed, and the number of conjugates in which a csmac (visualized by a patch of lck-staining at the immunological synapse) was formed. tcr signals result in reduced or absent (in the case of g ) staining with psrc and perk antibodies in the immunological synapse and reduced or absent (g ) degranulation as measured by cd a assays. the centrosome docks at the csmac of the immunological synapse even with relatively weak tcr signals, but the lytic granules require a certain threshold of signaling to successfully polarize to the immunological synapse. inhibitors support a role for pi k in granule polarization. together these data demonstrate that the strength of tcr signal controls the level of ctl mediated killing at the single cell level by controlling, the number of conjugates formed, the formation of the csmac and the accumulation of psrc and perk at the synapse. the centrosome polarizes to the csmac even with relatively weak tcr signals, but granule recruitment requires a higher threshold of signaling. these findings reveal how ctl can fine tune the degree of killing in response to tcr signals at the single cell level. cytotoxic t cells play an essential role in the immune system, particularly in the elimination of tumor and virus-infected cells. cytolytic t-cell activity is mediated through the pore-forming molecule perforin allowing granzymes to enter the target cell and to initiate apoptosis. perforin and granzymes are stored in specialized secretory granules, called secretory lysosomes. they are capable of undergoing regulated secretion in response to a t cell receptor engagement which involves binding to a cognate mhc class i-peptide complex. the intracellular transport of lysosomal proteins from the golgi to the lysosomes is mediated by the cationindependent mannose- -phosphate receptor which exhibits structural and functional similarity to the vps p-receptor sortilin. sortilin was characterized predominantly in neuronal cells where its function in protein sorting was identified. in the secretory pathway, sortilin is putatively involved in trafficking of proteins in the constitutive and regulated pathway. to explore whether sortilin has a broader functional relevance, we asked if sortilin might act as an alternative receptor for the cation-independent mannose- -phosphate receptor in cytotoxic t cells. first, we demonstrate that sortilin is expressed in t cells. to examine its function during an adaptive immune response, we analysed sortilin-deficient cytotoxic t cells derived from a knockout mouse strain. in strong contrast to the results reported from neuroendocrine cells, we obtained a reverse phenotype in sortilin-deficient cytotoxic t cells. whereas the regulated release of secretory lysosomes was enhanced, the constitutive release of interferon-g was found to be decreased. the enhanced release of cytotoxic molecules from sortilin-deficient cytotoxic t cells translated into an increased cytotoxicity in vitro. thus, the deletion of sortilin imposed a specific phenotype in cytotoxic t cells which could not be compensated for by other sorting receptors. our localisation studies of sortilin in t cells were consistent with the results previously described in neuronal cells which indicated that sortilin acts as a sorting receptor during the anterograde transport of lysosomal hydrolases from the trans-golgi-network to endosomes and lysosomes. taken together, we suggest that sortilin might play a modulatory role in the regulation of the adaptive immune response through the control of the constitutive and regulated secretory pathway. there is growing interest in the soluble splice variant of ctla- (sctla- ) as an immune inhibitor secreted by t cells, because genetically determined variation in its production is associated with susceptibility to autoimmune disease. however, little is known of the biology of sctla- in immune responses. using a specific anti-human soluble ctla- monoclonal antibody, jmw- b that selectively binds the soluble isoform but not membrane bound ctla- , or cleaved fragments of it, we demonstrate that sctla- plays a vital role in regulating antigen-specific immune responses. we used antibody blockade to show that antigen-specific t cell responses are strongly enhanced upon blockade of sctla- , secreting increased amounts of cytokines including interferon-g, il- and tnf-a, but lower amounts of il- . soluble ctla- was also prepared from sera for use in experiments by antibody based affinity purification techniques. addition of sctla- induced secretion of the immunoregulatory cytokine il- by human pbmcs both in an antigen-selective and dose-dependent manner, while antibody blockade abrogated that effect. the immunosuppressive indoleamine , dioxygenase enzyme cascade was also initiated by sctla- . it is clear that the importance of this natural soluble molecule has been overlooked and like membrane-bound ctla- it is crucial to t cell inhibition. membrane-bound ctla- exists as a homo-dimer on t cells but sctla- is usually considered to be monomeric in form, implying its functional capacity is diminished because of an inability to cross-link b ligands on antigen presenting cells. a third important observation from this study is that sctla- exists both in serum and culture supernatants as a natural kda homo-dimer, and not as a monomer. this goes some way to explaining why this molecule has such potent immunoregulatory effects on antigen-specific immune responses. together, these results lead us to reappraise sctla- , concluding it to be a mediator of negative feedback, secreted as a recall regulatory t cell response to antigenic stimulus, rather than a product of resting t cells. this work also raises the possibility that where il- dependent regulation is most critical, boosting sctla- secretion by regulatory t cells could be a novel therapy for immune mediated diseases. recently, we identified a new adaptor protein, swiprosin- /efhd- , in lipid rafts of b cell lines that undergo apoptosis after b cell receptor (bcr) stimulation. swiprosin- /efhd is expressed in immature b cells of the bone marrow, in resting and activated splenic b cells, in t cells, macrophages, mast cells and some nonlymphoid tissues. ectopic expression of swiprosin- /efhd- in the immature murine b cell line wehi enhanced spontaneous and bcr-induced apoptosis. in contrast, shrna-mediated down-regulation of swiprosin- /efhd- impaired spontaneous and bcr-elicited apoptosis, but not bcr-induced g cell cycle arrest. to understand how swiprosin- /efhd enhances pro-apoptotic bcr signals, we analyzed whether swiprosin- /efhd is involved in proximal bcr signalling. in fact, ectopic expression of swiprosin- /efhd enhanced bcr-induced calcium flux in wehi cells, whereas shrna-mediated down-regulation of swiprosin- / efhd impaired bcr-elicited calcium signals. concomitantly, gst-pulldown experiments revealed that swiprosin- /efhd interacts with phospholipase cg (plcg ) and with the tyrosine kinase syk (splenic tyrosine kinase), both of which are important for bcr-induced calcium flux. the interaction of plcg and swiprosin- /efhd was further established by co-immunoprecipitation. reconstitution of bcr-elicited calcium signals through complementation of swiprosin- /efhd silenced wehi cells with swiprosin- /efhd- was inhibited by the syk inhibitor bay - . in analogy, swiprosin- /efhd regulated syk activity positively. moreover, swiprosin- /efhd re-expression accelerated tyrosine phosphorylation of several proteins, specifically tyrosine phosphorylation of plcg and of syk tyrosine residue , which is involved in syk activation. finally, reconstitution of swiprosin- /efhd knock-down cells with swiprosin- /efhd mutants revealed that the n-terminal putative sh -binding site, the first ef-hand, and to a lesser extent, the second ef-hand and the c-terminal coiled-coil domain, are important for bcr-induced calcium flux in wehi cells. interestingly, swiprosin- /efhd re-expression in swiprosin- /efhd -silenced cells induced already in unstimulated cells raft partitioning of syk, plcg and the bcr, which was reversed after min of bcr stimulation. in summary, swiprosin- /efhd is an accelerator of proximal bcr signalling and acts through syk and plcg by assembling a syk-dependent calcium initiation complex in lipid rafts. this might be relevant for memory b cell signalling or central b cell tolerance. to test the biological relevance of cbl-b e ligase activity, these mice were analyzed for t cell proliferation, susceptibility to autoimmunity, in vivo t cell tolerance responses, and tc tumor rejection. results: when stimulated, t cells from rf mutant mice hyperproliferate compared to wild type t cells, even in the absence of cd co-stimulation. preliminary data also suggest that rf mutant mice are more susceptible to autoimmunity. in addition, rf/p mice die within hours after a second challenge with p peptide, indicating a severe defect in t cell tolerance induction. more importantly, cbl-b e ligase dead mice can spontaneously reject tc tumors. conclusion: cbl-b e ligase dead mutant mice phenocopy total body cbl-b knock out mice, thus indicating that cbl-b e ligase activity is indispensable for its regulatory in vivo functions. intriguingly, our data suggest that its inactivation could be sufficient to confer anti-tumor activity. to further elucidate the cellular mechanism of cbl-b mediated tumor rejection we have now generated the conditional cbl-b e ligase dead mutant mice to for the first time study the cbl-b ubiquitination function in a tissue specific and temporal fashion. our research is also currently focused on identifying the relevant in vivo cbl-b ubiquitination substrates. interferon alpha (ifn-a) has been broadly used in the treatment of specific malignancies and chronic viral diseases. for a long time it was thought that the direct inhibitory effects on malignant or virus infected cells were the major mechanisms involved in the response to ifn-a therapy. however, recent studies in mice have revealed that ifn-a/b also exerts effects on several host immune cells. ifn-a has been shown to enhance cd t cells (ctls) responses against soluble antigens in mice. this immunostimulatory activity of ifn-a results at least partly from its direct ability to induce maturation of dendritic cells. several studies have recently demonstrated that ifn-a/b also acts directly on murine ctls, inducing clonal expansion and differentiation into effector and memory cells. to date, little is known about the effects of ifn-a on human ctls. to approach this issue, magnetically sorted untouched human cd + cd ro -t cells (mainly naï ve cells) were unstimulated or stimulated with human ifn-a and gene expression profiles were compared using an affymetrix human array. interestingly, ifn-a stimulation of highly purified human ctls without any other concomitant signals remarkably enhanced the expression of several molecules involved in death receptor signalling (trail) and chemotaxis (ip and itac). in a second genome-wide array analysis, we analyzed the effects of ifn-a on human ctls responding to antigen (signal ) and co-stimulatory signals (signal ), provided by beads coated with anti-cd /cd antibodies. gene expression patterns were compared for cells stimulated with anti-cd /cd beads alone or along with ifn-a. ifn-a regulates the expression of a number of genes that promote proliferation, activation and survival of ctls, tcr stabilization, chromatin remodelation, and, importantly, enhances the expression of genes involved in ctls effector functions (granzyme-b, ifn-g, trail, fasl) and chemotaxis (ip , itac). the enhanced expression of granzyme-b, ifn-g, trail and ip were further confirmed at the protein levels by flow cytometry analysis and/or elisa. enhancement of granzyme-b-and trail-mediated cytolitic functions was also found by functional assays using anti-cd -coated p cells and trail-sensitive caki-i cells as targets. our results show that ifn-a provides a strong signal- to human ctls leading to their differentiation into effector ctls. t cell activation is an important process of the adaptive immune system, which requires recognition of mhc-associated antigens by antigen presenting cells (apcs) via the t cell receptor (tcr). to induce a productive t cell response the interaction of t cells with apcs needs to be stabilized by adhesion molecules. junction adhesion molecules (jams) are a recently discovered group of immunoglobulin (ig) superfamily proteins, which are involved in the regulation of various inflammatory and vascular events. the third member of the jam protein family, jam-c, is highly expressed in platelets and endothelial cells, whereas expression in t cells is largely unknown. to investigate the regulation of jam-c in t lymphocytes, we determined jam-c gene expression in quiescent and activated human t cells. treatment with the polyclonal t cell activator phytohemagglutinin (pha) increased surface and total jam-c expression in t cells time-and dose-dependently, as determined by flow cytometry and immunoblot analysis. by contrast, no up-regulation of jam-a in activated t cells was detectable. the highest level of jam-c up-regulation by pha was observed in cd + foxp + and cd + cd high t cells. moreover, t cell receptor activation with combined anti-cd and anti-cd stimulation induced jam-c expression in t cells. jam-c induction occurred at the mrna level suggesting a transcriptional regulatory mechanism of jam-c expression. accordingly, we studied the regulation of the human jam-c gene promoter in transiently transfected t cells. luciferase activity of a jam-c promoter gene construct with three potential consensus sites for the transcription factor nfat was markedly induced in activated t cells. finally, pretreatment with two pharmacological inhibitors of calcineurin, cyclosporin a and fk- , but not with mapk inhibitors, blocked jam-c induction in activated t cells. in summary, the present data indicate that jam-c is induced in activated human t lymphocytes via a transcriptional mechanism and suggests a major regulatory function of jam-c for the t cell response. hiv- infection leads to immune dysfunction owing to a successive loss of the cd + t cell compartment. the molecular mechanisms underlying this depletion are not well-understood but may involve the viral nef protein. nef is a multifunctional accessory protein that is required for full hiv- virulence and the maintenance of high viral loads. nef enhances viral infectivity and replication by downregulating cell surface receptors, e. g. cd and mhc class i, and modulating signal transduction pathways. the latter is thought to raise the cellular activation level and in this way may increase the infected cell's susceptibility to apoptosis. in this study we identify a signaling complex assembling at the n-terminus of nef, which contains the kinases lck and pkcv. formation of this complex, termed nakc for nef-associated kinase complex, led to activation of lck, as assessed by in-vitro kinase assay, and recruitment of pkcv to membrane rafts, as detected by discontinuous sucrose density gradient ultracentrifugation. recruitment of pkcv to membrane rafts is a hallmark of t cell activation and has been associated with activation of the nfxb transcription factor. however, contrary to our expectations, nef-mediated nakc formation did not activate nfxb. instead, it led to a strong induction of erk / . this correlated with a nakc-mediated increase in hiv transcription that was demonstrated by luciferase reporter assays suggesting that erk / directly targets hiv transcription, possibly via induction of transcription factors. to our surprise, however, the effect of nakc on hiv transcription was found to be independent of ap- , nfat and nfxb suggesting an alternative mechanism of nakc-mediated enhancement of hiv transcription. on the basis of our previous results we propose that nef enhances hiv transcription via removal of inhibitory factors and thus derepression of the hiv promoter. how erk / is involved in this mechanism and whether nakc targets other cellular promoters, which may enhance the cellular activation level and thus sensitize the cell to apoptosis, remains to be determined. p. otahal , t. brdicka , v. horejsi institute of molecular genetics as cr, praha, czech republic aims: c-terminal src kinase (csk) and cd are key regulators of src-family kinases in leukocytes. while cd is a transmembrane phosphatase, csk is localized mostly in cytosol. however, a fraction of csk is found at the cell membrane and in lipid rafts where it inhibits signaling by phosphorylating inhibitory tyrosine of src-family kinases. currently, it is accepted that sh domain of csk binds phosphotyrosine of transmembrane adaptor protein pag and via this interaction is recruited to the cell membrane and lipid rafts. however, pag knock-out mice still have cell membrane-associated csk and do not show any apparent dysregulation of signaling which would be expected due to the low levels of membrane csk. thus, the mechanisms of membrane targeting of csk remain unclear. to analyze the role of membrane and lipid raft targeting of csk on lymphocyte signaling we targeted csk to different membrane compartments by fusing csk with transmembrane domains of lat, lax, cd and n-terminal part of src kinase. methods: csk chimeras containing n-terminal membrane targeting motif and c-terminal orange fluorescent protein were cloned into retroviral vector pmxs. jurkat t cells expressing individual constructs were subsequently prepared and analyzed for the inhibitory effect of these csk chimeras on t-cell receptor (tcr) signaling by measuring calcium flux and cd upregulation. the efficiency of inhibition depended on the membrane targeting motif, while lat-csk chimera completely inhibited tcr signaling and src-csk chimera inhibited the signaling only partially; lax-csk and cd -csk chimeras showed almost no inhibition of tcr signaling despite efficient presence at the plasma membrane. conclusions: our data demonstrate that the function of csk strongly depends on its targeting to the specific areas of plasma membrane. it also strongly supports the idea that membrane compartmentalization is critical for regulation of t-cell signaling. peripheral cd t cell tolerance can be generated outside lymphatic tissue in the liver. however, the course of events leading to tolerogenic interaction of hepatic antigen presenting cells with circulating t cells is unclear. here, we demonstrate that systemically circulating antigen was preferentially taken-up by liver sinusoidal endothelial cells (lsec) and not by other antigen presenting cells in the liver or spleen. uptake and cross-presentation of circulating antigen was followed by rapid antigen-specific naï ve cd t cell-retention in the liver but again not in other organs. using bone-marrow chimeras and tie- kb mice, we could show that antigen cross-presentation by lsec was both essential and sufficient to cause antigen-specific t cell-retention under non-inflammatory conditions, which was followed by cd t cell proliferation and expansion, but ultimately led to the development of t cell tolerance. our results show that cd t cell tolerance towards circulating systemic antigens is predominantly generated in the liver by lsec, which preferentially take-up and cross-present circulating proteins to cd t cells, leading to their rapid local antigen-specific retention and subsequent tolerisation. these insights broaden our understanding not only of physiological immune regulation towards circulating antigens but also of therapeutic manipulation of cd t cell responses. alphapix is a rho gtpase guanine nucleotide exchange factor domain-containing signaling protein that associates with other proteins involved in cytoskeletalmembrane complexes. it has been shown that pix proteins play roles in some immune cells, including neutrophils and t cells. in this study, we report the immune system phenotype of alphapix knockout mice. we extended alphapix expression experiments and found that whereas alphapix was specific to immune cells, its homolog betapix was expressed in a wider range of cells. mice lacking alphapix had reduced numbers of mature lymphocytes and defective immune responses. antigen receptor-directed proliferation of alphapix deficient t and b cells was also reduced, but basal migration was enhanced. accompanying these defects, formation of t-cell-b-cell conjugates and recruitment of pak and lfa- integrin to the immune synapse were impaired in the absence of alphapix. proximal antigen receptor signaling was largely unaffected, with the exception of reduced phosphorylation of pak and expression of git in both t cells and b cells. these results reveal specific roles for alphapix in the immune system and suggest that redundancy with betapix precludes a more severe immune phenotype. s. merluzzi , s. parusso , b. frossi , g. gri , c. pucillo university of udine, dstb, udine, italy in this study, we investigated whether primary mcs could modulate the activation and proliferation of primary b cells. we performed co-culture assays using mouse splenic b cells and bone marrow-derived mcs. naï ve and activated b cells proliferation could be induced by nonsensitized mcs while an increase in b cell proliferation was observed when mcs are activated. moreover, b cell proliferation was partially abolished when mcs and b cells were separated by the transwell membranes suggesting that cell-cell contact is important in this event. using both il- -/-mcs and anti-il- receptor antibody, we demonstrated that in co-culture of primary b cells and mcs, il- derived from activated mcs is a key cytokine implicated in the b cell proliferation. moreover, we showed that activated mcs can influence the surface expression of costimulatory molecules as cd on naï ve b cells and the interaction of cd on b cell surface and cd l on mcs is important for the further differentiation of b cells to plasmacells. indeed, we presented for the first time evidence that cytokines produced by activated mcs and interaction between cd l e cd on mc and b cells respectively can contribute to differentiate mature b cells to iga secreting cells. in conclusion, in the present report, we showed a novel role of mcs as promoter of both the survival and activation of naive b cells and of the proliferation and further differentiation of activated b cells through soluble factors production and cell-cell contact, suggesting that mcs can contribute to the regulation of specific immune response. e. fourmentraux-neves , n. bercovici , a. caignard inserm u , paris, france inhibitory killer ig-like receptors (kir dl - / ) which bind to hla-c molecules are expressed by human natural killer cells and effector memory cd + t cell subsets. these receptors suppress cd + t cell activation through recruitment of the src homology domain-containing protein tyrosine phosphatase (shp- ). to further analyse the yet largely unclear role of inhibitory kir receptors on cd +t cells, kir dl transfectants were obtained from a cd + t cell line and primary cells. the transfection of cd + t cells with kir dl dramatically increased the t cell receptor (tcr)-induced production of il- independently of ligand binding, but inhibited tcr-induced activation after ligation. kir-mediated tcr activation requires intact itim motifs, involves kir dl -itim phosphorylation, shp- recruitment, zap- and pkc-v phosphorylation. synapses leading to activation were characterized by an increase in the recruitment of p-tyr, shp- and p-pkc-v but not of shp- . in contrast, the kir dl /hla-cw interaction led to a strong synaptic accumulation of kir dl and the recruitment of shp- / , inhibiting tcr-induced il- production. kir dl may induce two opposite signaling outputs in cd + t cells, depending on whether the kir receptor is bound to its ligand. these data highlight unexpected aspects of the regulation of t cells by kir dl receptors. b cell receptor (bcr) binding by antigen initiates activating signaling cascades and facilitates the exposure of specific b cells to powerful co-stimulatory signals, such as t cell help or toll-like receptor ligands. the role of bcr binding in modulating the access to these second signals is complex and varies between stimulatory conditions. by quantitative tracking of b cell responses in vitro we can measure which signals affect b cell proliferation or differentiation, or both, and thereby establish a novel understanding of how b cells respond appropriately to different combinations of stimuli. we utilised hel-specific bcr transgenic sw hel mice to assess the effect of a specific antigen signal on b cell responses to the t-independent mitogen lipopolysaccharide (lps). the presence of antigen renders a greater proportion of cells responsive to lps stimulation and profoundly influences effector cell differentiation. antibody secreting cell formation is dramatically inhibited by hel, but we found that isotype switching to igg is strongly upregulated. both of these alterations to differentiation outcomes occur independently to the proliferative effects induced by antigen. when b cells are exposed to antigen for a limited period of time, switching to igg still occurs but some capacity to differentiate to antibody secreting cells is recovered, leading to effective secretion of igg antibody during these conditions. the observed igg switching behaviour mimics that of b cells responding to lps and il- , but is mediated by a different, stat -independent pathway. these data are indicative of the important role specific antigen signals play in regulating b cell responses in stimulatory environments. a. quintana , c. schwindling , m. pasche , c. junker , c. kummerow , u. becherer , e.c. schwarz , j. rettig , m. hoth saarland university, biophysics, homburg, germany, saarland university, physiology, homburg, germany the adaptive immune response requires the interaction between antigen-presenting cells and t cells. this cell-cell interaction, called the immunological synapse (is), facilitates the activation of t cell receptor-mediated signalling cascades including a rise of cytosolic calcium through the activation of crac/orai channels. to allow sustained activity of crac/orai channels, the calcium-dependent inactivation of the channels through local calcium microdomains has to be prevented. objectives: the purpose of the study was to analyze local and global calcium signals in t cells and to test the hypothesis that the is controls these signals through mitochondrial positioning. methods: we used different microscopy techniques including very fast wide-field microscopy with subsequent deconvolution, total-internal reflection microscopy, and confocal microscopy in combination with electrophysiological techniques in primary human t helper cells and cell lines. to test the statistical significance of our data, we used two-sided student t-tests or non-parameterized tests. results: following is formation, we found that mitochondria translocated to the is in a calcium-dependent way. the distance between mitochondria and the plasma membrane at the is was lower than nm. following accumulation at the is, mitochondria limited calcium entry to the orai channels localized right at the is by preventing their calcium-dependent inactivation. in contrast, no calcium influx was observed at sites where no mitochondria were accumulated as orai channels were inactivated at these sites. mitochondrial positioning at the is thus induced local calcium influx at the is without the necessity to enrich orai channels at the is. mitochondria took up calcium at the is distributing it further into the cytosol by releasing it at different sites, which kept the local domain at the is low enough to prevent calcium dependent orai inactivation and to prevent excessive calcium clearance by the calcium aptases in the plasma membrane, which could inhibit an efficient t cell activation. conclusion: mitochondria positioning at the is controls local calcium entry through orai channels. mitochondria prevent orai inactivation and excessive calcium clearance at the is to facilitate calcium-dependent t lymphocyte calcium signalling. we aimed to determine the functional correlates of cd + t cell tolerance and immunity in vivo. ovalbumin (ova)-specific transgenic cd + t cells were adoptively transferred into syngeneic mice immunized with soluble ova protein ± lipopolysaccharide (lps) by the i. v. route, and analyzed for a variety of immunological parameters over a period of days. under tolerogenic conditions (ova alone), cd + t cells showed substantial early activation, but their activation profile differed markedly, both in magnitude and quality (icos, - bb), from t cells activated by ova+lps. this difference in activation also translated into differing cd + t cell expansion and contraction kinetics in the early phase of the t cell response (days - ). in the late phase of the primary response (days - ), under immunizing conditions, the large majority of transgenic cd + t cells in the spleen developed into mature effectors with a prominent capacity to secrete il- , ifn-g, and il- a, and only few ova-specific foxp + regulatory t cells ( x %) were observed. germinal centers were prominent and ova-specific ig of all isotypes were generated. in contrast, under tolerizing conditions, antigen-specific cd + t cells failed to migrate into the b cell follicles, but production of ova-specific igm was nevertheless observed. in these animals, the proportion of splenic ova-specific regulatory t cells ( %) was substantially increased. on day , both groups of mice were re-challenged via the airways with ova+lps to functionally assess their immune status. in tolerized animals, the transgenic t cell population in the lung infiltrate was composed of ova-specific regulatory t cells ( %) or t cells with a reduced capacity to secrete effector cytokines. in contrast, in immunized animals, this population almost exclusively consisted of cd + effector t cells with a pronounced inflammatory cytokine profile (ifn-g, il- a). with this model we provide a comprehensive analysis of the many functional correlates of "immunity" versus "tolerance" to soluble protein antigen in vivo. we identify and characterize a number of the key players (cell surface molecules, cytokines, cell subsets) representing the decision between immunity and tolerance in the immune system. mast cells (mcs) are well-recognized as key effector cells in immunoglobulin e (ige) -associated immune responses and as prototypic regulators of innate immunity. the characteristics, importance, and molecular requirements for interactions between mast cells (mc) and cd t cells (tc) remain to be elucidated. using myelin/oligodendrocyte glycoprotein (mog), we demonstrated that mcs induce antigen-specific cd tc activation and proliferation. the antigen crosspresentation by mcs induces the secretion of interleukin- , interferon-g and macrophage inflammatory protein- by cd tc. in vivo evidence that mcs modulate t cell responses has been obtained so far in the murine experimental autoimmune encephalomyelitis (eae), the standard animal model for multiple sclerosis, in which both cd tc and mcs are now recognized as key players. one of the main central nervous system (cns) antigens recognized by autoreactive tc in eae is the myelin oligodendrocyte glycoprotein (mog). to investigate the in vivo-relevance of the identified mc-cd tc interactions, we have employed the eae as a model of organ specific autoimmune disease in wild type mice and mc-deficient w/w sh mice. wt and w/w sh mice were immunized with the mog - protein. our results provide direct evidence that mc contribute to cd -specific priming in eae and show that the tc proliferation failure is specific for cd tc from mog - -immunized w/w sh mice. the role of mc-cd tc interaction in induction of autoimmunity will be further investigated in eae. in summary, we provide the first evidence that mcs regulate antigen-specific responses of primary cd tc in vitro and in vivo. our study further supports the emerging concept that mcs, protagonists of innate immunity are also important regulators of adaptive immune responses and corresponding cd tc responses. this newly uncovered mc function might be of great biological relevance in situations where effector cd tc are critically involved, e. g. viral infections or infections with intracellular pathogens and/or autoimmune diseases such as multiple sclerosis. activation of resting t cells in vitro is triggered by combined t cell receptor (tcr) and cd engagement and can be modulated by simultaneous ligation of various other surface receptors. although the fasl is best known for its capacity to initiate cell death in fas-bearing cells, it has recently been implicated in the regulation of t cell activation. thus, a crosstalk between the tcr and fasl is likely, but far from being biochemically elucidated. we report that fasl engagement by immobilized but not soluble fasfc fusion protein and anti-fasl antibodies blocks the activation of primary human peripheral t cells even in the presence of cd costimulation at the level of an early signal initiation. inhibition is thus associated with a reduction of tyrosine phosphorylation of a number of key elements in tcr signal transduction and also with a lowered calcium response. the data presented stress the importance of the fas/fasl-system for signal initiation via the tcr/cd complex and provide further arguments for a retrograde signaling capacity of fasl or a crucial role of fas as a costimulatory molecule. golgi network (tgn). moreover, trim specifically associates with the cytoplasmic tail of ctla- , but not via any conventional motifs in this region. overexpression of trim augments ctla- surface expression, whereas down-regulation of trim expression by shrna results in disturbed ctla- localisation, mainly restricted to the tgn. ctla- vesicles and surface expression were significantly reduced but not abolished, suggesting that other factors are involved in ctla- trafficking. here, we identify additional transmembrane adapter protein (trap) family members as novel binding partners and regulators of ctla- expression. although there is some redundancy amongst traps, our results highlight the importance of this family of proteins in ctla- transport to the cell surface. it is imperative to reveal the mechanisms by which ctla- is transported to the cell surface, given that minor changes in expression can have major effects on t-cell function and in the development of autoimmunity. natural killer t (nkt) cells are found within the liver and are known to exhibit immune regulatory function. upon recognition of glycolipids presented on cd molecules, nkt cells are activated and release cytokines, including ifn-g, il- and il- . nkt cells are efficiently recruited to the liver via cxcr -dependent chemotaxis toward cxcl and constitute a large proportion of the liver-resident lymphocytes. we have previously shown, that liver sinusoidal endothelial cells (lsec) can scavenge circulating soluble antigens, and can cross-present these antigens to naive cd t cells. cross-presentation leads to initial t cell activation and expansion, but ultimately these cd t cells are rendered tolerant. as both naive t cells and nkt cells come into close contact with lsec in the hepatic sinusoids, we investigated whether nkt cells can modulate cd t cell tolerisation via interaction with lsec. to this end we analysed cd d expression on lsec and their ability to activate nkt cells by presentation of the cd d-binding glycolipid a-galactosylceramide (agalcer). we found that lsec express functional cd d, as agalcerpresenting-lsec were capable to induce tnf-a, il- , il- and ifn-g production in nkt cells in vitro. the interaction of agalcer-presenting-lsec with nkt cells led to the upregulation of cd and b -h on lsec. as naï ve cd t cell tolerisation by lsec critically depends on b -h , we hypothesise that hepatic nkt cell activity may contribute to the immunological capabilities of the liver by regulating the tolerogenic function of lsec. improved antibody responses by class-switched memory b cells require enhanced signaling from their antigen receptor (bcr). however all bcr classes on naïve and antigen-experienced b cells utilize the canonical iga/igb subunit for signaling. we identified the signal amplification mechanism of the igg-and ige-bcr. for these isotypes tyrosine-based signaling is not confined to iga/igb but extends to a conserved tyrosine residue in the cytoplasmic segments of immunoglobulin heavy chains. the phosphorylated immunoglobulin tail tyrosine recruits the adaptor grb in order to sustain protein kinase activation and generation of second messengers causing robust cellular proliferation. hence membrane-bound igg and ige not only recognize antigen but also exert bcr-intrinsic costimulation to render memory b cells less dependent on t cell help for activation. objectives: the majority of circulating human gd t cells harbor tcr containing vg , jg . , and vd gene products. they recognize nonpeptide antigens like (e)- hydroxy- -methylbut- -enyl pyrophosphate derived from pathogenic microbes and isopentenyl pyrophosphate (ipp) in malignant cells. recently, we and others found out that gd t cells express a variety of costimulatory molecules including icos, and pd- . one of the inhibitory receptors, pd- , is a member of cd /ctla- family and contains a single ig v-like domain in its extracellular region. pd- can bind to two b homologue molecules, pd-l and pd-l . it has been reported that interaction of pd- with its ligands resulted in peripheral immune regulation and tolerance in ab t cells. in this study, we show that pd- is expressed on activated human gd t cells and regulates the effctor functions of gd t cells. methods: peripheral blood mononuclear cells were resuspended in yssel's medium and stimulated with -methyl- -butenyl- -pyrophosphate plus il- to obtain gd t cells. pd-l + and pd-l -human tumor cell lines were established from cancer patients. in order to prepare anti-pd-l mabs, the pd-l extracellular domain was expressed in e.coli as inclusion bodies and refolded in the standard arginine-based buffer. mice were immunized with the refolded protein and mabs were established. to determine the function of pd- in gd t cells, we determined cytokine production and cell mediated tumor lysis by activated gd t cells in the presence of inhibitors of pd- /pd-l interaction. results: gd t cells expressed pd- upon simulation with nonpeptide antigens and many tumor cell lines expressed pd-l . we first examined whether or not the engagement of pd- receptor could modulate the cytotoxic activity of gd t cells. pd-l -expressing tumor cells tempered cytotoxic activity of pd- + gd t cells, and cytokine production such as tnf-a was down-regulated by pd- engagement. in addition, inclusion of anti-pd-l mab reversed cytotoxic activity and cytokine production when pd-l -expressing tumor cells were challenged by pd- -expressing gd t cells. conclusion: pd- delivers inhibitory signals in gd t cells upon engagement with pd-l . peripheral tolerance plays an important role in preventing t lymphocyte responses to self or harmless antigens. one of the mechanisms that contribute to this form of tolerance is anergy, which is characterized by a lack of proliferation and il- production by t cells in response to antigenic challenge. the acquisition of the anergic phenotype is an active process, with negative regulators of t cell signalling being induced. among these are the e ubiquitin-protein ligases which recognize target proteins for ubiquitination and catalyse the transfer of ubiquitin to them, directing them to the proteasome or to the endosome-lysosomal pathway, and hence downregulating their activity. the e ubiquitin-protein ligases cbl-b, itch and grail have been shown to be upregulated in anergy and to ubiquitinate and downregulate tcr signalling elements. our objectives were to determine the expression of cbl-b, itch and grail in antigen-specific cd + t cells in both the induction and maintenance phases of anergy, in vitro and in vivo, and to investigate their functional signalling role(s) in the maintenance of the tolerance phenotype. in order to accomplish these objectives we induced priming or tolerance of ovalbumin (ova - peptide)-specific t cells from do . tcr transgenic mice in vitro or, following adoptive transfer of near physiologically relevant numbers of such cells into recipients, in vivo and correlated functional outcome (via proliferation and cytokine readout assays or antibody production) with e ubiquitin-protein ligases expression and the ubiquitination status of the tcr signalling machinery. cbl-b, itch, grail and target ubiquitination status, in terms of tissue, cellular and subcellular protein expression, modification and localisation, were assessed by a combination of immunoprecipitation and western blotting studies. moreover, we have performed quantitative analysis at the single cell level by tracking such antigen-specific cells in vitro and in vivo by using laser scanning cytometry. our current work focuses on the functional consequences of adenoviral transfection of such antigen-specific t cells by mutant e ligase-, signal target-and ubiquitin-constructs. collectively, these approaches have facilitated the dissection of the potential differential roles of ubiquitin signalling in priming and tolerance of antigen-specific t cells. s. j. keppler , p. aichele immh, university freiburg, immunology, freiburg, germany interleukin (il- ) is produced by cells of the innate immune system during infection and plays an important role in controlling various pathogens. it was postulated recently that il- has a direct influence on cd + t cells in vitro, enhancing expansion and the development of effector functions as a third signal, additionally to tcr engagement (signal ) and costimulation (signal ). we analysed direct il- signaling to cd t - signaling exhibited normal degranulation activity, cytolytic functions, ifn-g and tnf-a production. however, cd t cells lacking il- signaling failed to up-regulate klrg and to down-regulate cd in the context of listeria but not viral infections. thus direct il- signaling to cd t cells determines the cell fate decision between short-lived effector cells (slecs) and memory precursor effector cells (mpecs), dependent on the pathogen-determined local cytokine milieu. cd + t lymphocytes are required for effective host defense against pathogens but also for mediating effector responses against uncontrolled proliferating self tissues. we could now reveal that individual cd + t cells are tightly controlled in their effector functions by cd (ctla- ). we demonstrate that signals induced by cd reduce the frequency of interferon-gamma (ifn-g) and granzymeb expressing cd + t cells. for this novel function cd specifically represses the transcription factor eomes, but not t-bet. a cd mediated induction of the inhibitory transcription factor ckrox has been ruled out. ectopic expression of eomes reversed cd -mediated inhibition of effector molecule production. additionally, enhanced cytotoxicity of individual cd + t cells differentiated in the absence of cd signaling could be demonstrated in vivo. the novel insights that cd -mediated signal transduction in vivo indeed alters cd + t cell cytotoxicity qualitatively at the single cell level and not only quantitatively by enhancing expansion extend the understanding how to selectively modulate immune responses of cd + t cells. objectives: atp constitutes a damage associated molecular pattern (damp) and contributes together with pathogen associated molecular patterns (pamp) to the efficient priming of the innate immune system. atp is a ubiquitous extracellular messenger, which activates plasma membrane receptors for extracellular nucleotides termed p receptors. p x - receptors open to non-selective ion channels, whereas p y , , , , - are g-protein coupled receptors, which bind preferentially adp, udp, utp or udp-glucose. as the role of p receptors in the control of b cell activation has been poorly investigated, aim of the present study is to understand better the mechanisms of intracellular atp production and release by human b cell subsets. methods: intracellular atp measurement has been performed using a bioluminescence assay while extracellular atp has been measured by hplc. storage and release of atp by b cells have been elucidated using confocal and tirf microscopy, to study vesicles distribution and dynamics near the plasma membrane. results: in both human naive and memory b cell we observed a prominent increase of atp synthesis upon tlr but not bcr stimulation. glycolytic pathway rather than oxidative phosphorilation was involved in atp synthesis. p x antagonists inhibited both proliferation and differentiation to plasma cells of human b cells thus suggesting that atp is released in the pericellular space. labelling of resting and activated human memory b cells with quinacrine, a nucleotide binding component, revealed a typical vesicular pattern of atp, confirmed with subcellular fractionation on sucrose equilibrium gradients. tirf imaging showed a fluorescently labelled vescicle underwent fusion with the plasma membrane after stimulation with anti-ig and this event was ca( +)-dependent. conclusion: these data provide evidence that atp is produced by b cell preferentially by glycolytic pathway and vesicular exocytosis is a key mediator of atp release in human b cells. atp released in the pericellular space might act as an autocrine and paracrine signalling molecule that regulates the functions of b cells. o. ballek , a. brouckova , d. filipp institute of molecular genetics as cr, laboratory of immunobiology, prague, czech republic two src family tyrosine kinases lck and fyn are critical for the proximal t-cell signaling. we have previously demonstrated that induced lck activation outside lipid rafts (lr) results in lck translocation to lr. central in this sequence of events is the rapid translocation of kinase active lck to lr, yet the mechanism underpinning this process is unknown. the main aim of this study is the characterization of molecular mechanisms and its functional elements regulating the early recruitment of signaling molecules to lr and forming immunological synapse. we have recently characterized the c-terminal yqpqp sequence as a novel cis-acting component essential for partitioning of lck to lr. here we report that the expression of the c-terminal truncate of constitutively active lck ( ¿ fqpqp) in nih t cells failed to phosphorylate several proteins detected in the presence of untruncated kinase active y flck. comparative -d gel analyses followed by ms/maldi identified rack as a candidate protein for interaction with the c-terminal tail of lck. co-expression in nih t cells of ha-tagged rack with either a wild type lck or constitutively active y flck revealed a significantly enhanced complex formation between y flck and rack compared to that of wtlck. ectopic expression of y flck with its domain-inactivating mutations showed that lck-rack interaction depends on functional sh , sh and the c-terminal tail sequence of lck. lck-rack interaction is readily detectable also in primary cd + lymph node t cells. upon their activation, only the pool of lck molecules associated with high molecular weight complexes can translocate to lipid rafts. co-purification of rack with these fractions further suggests that it plays a role in the translocation of lck to lr. in addition, lck and rack co-redistribute to both forming immunological synapse and to antibody-mediated capping clusters. moreover, the importance of interaction between activated lck and rack in the context of lck translocation to lr is further strengthen by the observation that rack is associated with elements of cytoskeleton. these results are the first to characterize rack as a candidate molecule involved in the regulation of lck translocation to lr through linking the c-terminal sequence of lck to cytoskeletal network. human b cells are currently not known to produce the pro-apoptotic protease granzyme b (grb) in physiological settings. we have discovered that b cell receptor stimulation with either viral antigens or activating antibodies in the context of the acute phase cytokine interleukin (il- ) can induce secretion of substantial amounts of grb by human b cells. grb response to viral antigens was significantly stronger in b cells from subjects recently vaccinated against the corresponding virus as compared to unvaccinated subjects. both, naï ve and memory b cells differentiated into grb-secreting cells, which featured a homogeneous cd +cd +cd -cd -igd-phenotype, improved survival and enhanced expression of co-stimulatory, antigen-presenting and cell-adhesion molecules. b cellderived grb was enzymatically active and its induction required activation of similar signaling pathways as in cytotoxic t cells. our findings suggest grb-secreting b cells play a role in early anti-viral immune responses, thereby contributing to the elevated serum grb levels found in various viral diseases. further studies will elucidate whether b cell-derived grb induces cytotoxicity towards virus-infected cells or exhibits other functions. results: transfer of otii cells augments the wild type th response to alumova inducing large early germinal centres and massive plasma cell formation with more than % of these switching to igg . the plasma cells up-regulate cxcr , but not cxcr , a chemokine receptor that attracts plasma cells to inflammatory sites. oti cells respond to alumova by producing ifng, a th -associated cytokine. when both oti and otii cells are transferred switching is diversified with plasma cells being igm (˚ %), igg a (˚ %), igg b (˚ %) or igg (˚ %). in addition to cxcr , some % of these plasma cells strongly express cxcr . the induction of cxcr in these plasma cells correlates with their increased expression of the transcription factor t-bet, which has been linked with igg a switching during th responses. this is functionally significant for oti-dependent cxcr expression, as well as induction of switching to igg a, are dependent on t-bet expression by the responding b cells, although t-bet-deficient b cells still switch to igg b. t-bet is known to be induced in b cells exposed to ifng or tlr stimulation. these two hypothetical mechanisms are currently being tested in mice injected with blocking anti-ifng antibodies or mice deficient in myd . objective: a successful immune response against malaria has to be tightly controlled. the early production of pro-inflammatory cytokines is required to control the growth of intraerythrocytic parasites but the same cytokines are also involved in the induction of severe malaria in both humans and mice. activation of t lymphocytes through tcr signalling takes place within the context of numerous other cell surface protein interactions. to prevent unnecessary activation of t cells the immune system has developed an intricate balance between positive and negative costimulatory signals. costimulatory signals determine whether antigen recognition by t lymphocytes leads to full activation or to anergy. in contrast negative costimulators expressed by t cells seem to mediate the regulation of immune responses and thus play a pivotal role in the maintenance of peripheral tolerance. recently, btla (b and t lymphocyte attenuator, cd ) was described as a novel negative costimulatory receptor. btla is predominantly expressed on t and b cells and dampens t cell activation. in this study, we analyzed the function of btla during experimental malaria infection. to study the function of btla we employed a mouse model of blood-stage malaria using p. yoelii nl infection of btla-deficient and hvem-deficient mice. p. yoelii provokes a high parasitemia in infected mice that is cleared within three weeks from time of infection. immunity in this model depends on cd + t cells and hence the role of negative costimulators that modulate t cell function can be studied using this model. results: peak parasitemia of p.yoelii-infected btla-deficient and hvem-deficient mice was much lower compared to wild type mice. the increased immunity of btla-deficient mice depends largely on cd + t cells. we found that btla::hvem interaction regulates the size and the cytokine-production of the responding t cell pool. however, in contrast to the ctla- pathway, the manipulation of btla::hvem interaction triggers no pathology during infection. the hvem::btla interaction dampens the protective immune response during experimental malaria and thus manipulation of this pathway is an attractive target for therapeutic interventions. . so far, the contribution of actin regulatory proteins to this process remained largely unknown. here we demonstrate that the actin-bundling protein l-plastin is indispensable for segregation of lfa- and cd in the psmac of untransformed human t-cells. in marked contrast, tcr/cd accumulation in the csmac is not dependent on l-plastin. the relocalization of l-plastin in the immune synapse occurs within seconds of t-cell/apc contact formation and relies on actin polymerization. importantly, binding of calmodulin to l-plastin is required for the maintenance of l-plastin in the immune synapse and inhibition of calmodulin prevents psmac formation. thus, receptor segregation in the immune synapse is a consequence of the combined activities of the actin-bundling protein l-plastin and calmodulin. protective t cell responses are based on expansion and persistence of clones with optimal affinity for antigen. presently, it is unknown which mechanisms guard the selection and expansion of the highest affinity clones from the very diverse naï ve pool. rapid cell division creates shifts in selective pressure, which is a basic biological prerequisite for elimination. therefore we hypothesized that apoptosis might play an important role during this phase of t-cell biology. here we show that the balance between the pro-apoptotic protein noxa and its antagonist mcl- regulates interclonal t cell competition during acute and chronic immune activation. we found p -independent noxa gene induction and mcl- downregulation upon t cell activation. concomitant we observed the release of death-inducing factor bim from mcl- , which was delayed in noxa -/cells. using ot- cells and altered peptide ligands we observed that the level of mcl- downregulation in activated t-cells depended on the antigen affinity of the t-cell receptor. since mcl- -/mice are embryonic lethal, noxa -/mice were used to study the functional implications of this mechanism in vivo. at a young age noxa -/mice have a normal lymphoid compartment, but accumulate effector t cells over time. upon acute influenza infection, normal levels of effector cells were generated. however, the quality of the antiviral (np ) response was impaired in these mice as many subdominant clones persisted in the effector t-cell population of noxa -/mice at the peak of infection. this increased diversity correlated with exacerbated pathology and a reduced rate of viral clearance. in a model of chronic immune activation, effector t cells rapidly accumulated in the noxa -/mice and infiltrated the peripheral organs, culminating in severe multi-organ pathology and premature death. these results establish a novel role for the noxa/mcl- axis during immune responses and suggest that the formation of a high-affinity effector population of restricted clonal diversity depends on a darwinian selection of t-cells during the expansion phase based on antigen affinity, with survival of only the fittest clones. cytotoxic t lymphocytes (ctls) are essential for immunosurveillance, a process that requires the presentation of virus-or tumor derived antigenic peptides in context of antigen presenting cells. insight into intracellular mechanisms facilitating lytic granule release and formation of the immunological synapse as a prerequisite for target cell destruction was primarily obtained from loss-of-function mutations in hereditary human diseases and gene-mutated mice. here, we refer to estrogen receptor-binding fragment-associated antigen (ebag ) as a negative regulator of secretory lysosome release. in gene deleted mice we show that loss of ebag confers ctls with enhanced cytolytic capacity, in vitro and in vivo. here, we show that ebag , which was previously identified as a snapin-interacting protein in neuronal cells, interacted with the adaptor molecule g -adaptin in t cells. both interactions suggested an involvement of ebag in endosome-lysosome related organelle biogenesis and membrane fusion. efficiency of granzyme b sorting towards secretory lysosomes was improved, which was consistent with the enhanced kinetics of cathepsin d proteolytic processing. while the formation of the immunological synapse remained unaffected in ebag -/-ctl, relative size distribution of lytic granules revealed a shift towards smaller granule diameters and volumes in ebag -deficient ctls. these data imply a role for ebag in regulating the formation of mature ctl granules and identify ebag as a tunable inhibitor of ctl-mediated adaptive immune response functions. finally, ebag defines a novel negative regulator of secretory lysosome release in ctls. thus, the elucidation of the ebag -related pathway might provide a fresh impuls on therapeutic approaches in the treatment of autoimmune disorders. the liver is known to induce tolerance, rather than immunity, through tolerogenic antigen presentation or elimination of effector t cells. recently, we could show that liver sinusoidal endothelial cells (lsec) inhibit activation of naive cd t cells by antigen-presenting dc. this regulatory effect of lsec on dc function was mhc-independent and not limited to soluble mediators, but required physical contact. interestingly, interaction with lsec led to reduced dc expression levels of cd / and il- . in addition to indirect inhibition of t cell activation by de-licensing of dc, we now detected another influence of lsec in form of direct inhibition of t cell priming. in the presence of lsec, stimulation with acd /acd or pma/ionomycin could not significantly activate cd and cd t cells. thus, lsec did not only inhibit t cell priming triggered by tcr activation but also after elicitation of ca + influx into the cytoplasm. furthermore, we found that ifn-g secreted by t cells in the early phase of activation is crucial for licensing the inhibitory function of lsec. taken together, these data indicate that the inhibitory effect of lsec is mediated by a machinery induced at the early phase of t cell activation, however, interferes with late events in the t cell activation cascade. we propose a model of "inducible inhibition", where on the one hand naive t cell priming is directly inhibited by lsec, and on the other hand tolerogenic priming by antigen-presenting lsec is still allowed. taken together, these results reveal a novel principle, operative in hepatic tolerance induction, in which lsec not only tolerize t cells themselves, but also inhibit the responsiveness to local activation stimuli. m. almena , s. carrasco , i. merida centro nacional de biotecnología csic, inmunología y oncología, madrid, spain self-tolerance acquisition is essential for the immune system to control its own response. t cells achieve self-tolerance trough thymic selection and anergy, two processes where rasgrp -ras-erk signal intensity is critical to determine the final cell outcome. rasgrp is a gef for ras that is activated in a diacylglycerol (dag)dependent manner. dag is generated by plcg after tcr stimulation and is consumed by diacylglycerol kinases (dgk). dag generation, as a result of the concerted regulation of these two enzymes, activates ras, providing a mechanism to translate the strength of the stimulus into a quantitative cell response. . analyze the impact that dag metabolism plays in t cell tolerance in vivo, using transgenic mice where dag generation is impaired. . develop a method to sense dag production and localization, in both thymocytes and peripheral t cells, and its correlation with the strength of the stimulus used. methods: we generated transgenic mice expressing a constitutively active dgk in t cell lineage. this protein was anchored to the plasma membrane, thus diminishing the lipid levels in this specific location after tcr stimulation. ot-i cd cells expressing gfp-c domains were used with peptide-pulsed apcs to study dag generation and dynamics by confocal microscopy. results: transgene expression was obtained in thymic and peripheral t cells. no major defects were observed in t cell subsets but analysis of peripheral t cells demonstrated important defects in t cell activation. we are currently studying thymic selection breeding our transgenic with h-y mice, in order to check if t cell populations are being properly selected. using t cell-apc conjugates with peptides with different tcr binding affinities we found a clear correlation between the strength of the stimulus, dag production and ras-mapk activation. conclusion: our data demonstrate that dag generation not only activates c domain containing proteins but regulates a mechanism by which t cells sense the magnitude of the stimulus received, translating it into the intensity of the generated response, a process essential in t tolerance. future experiments will help to define the exact contribution of the lipid to tcr signaling pathways and to t cell homeostasis. the inducible costimulator (icos, h , cd ), a cd -like costimulatory molecule, has an important role in the development of efficient t cell responses. early data showed that icos costimulation produced th biased responses and high production of the anti-inflammatory cytokine il- , and was essential to the development of germinal centres. however, icos can also help in the il- -dependent differentiation of inflammatory th cells. these different functions could be due to differences in the apcs bound by icos-expressing t cells and/or because of the intervention of distinct molecules binding the cytoplasmic domain of icos. icos shares with cd a yxxm cytoplasmic motif that can bind, upon tyr phosphorylation, the regulatory p subunit of class ia pi- kinases. these can complex with one of the three kda catalytic subunits (p a, p b, and p d) expressed by leukocytes that generate pip affecting cell growth, cell cycle progression, survival, intracellular traffic, cytoskeletal changes and migration. there is also evidence that the regulatory and catalytic class ia pi k isoforms fulfill specific functions in macrophages and lymphocytes. we have used proteomic and immunochemical approaches to identify molecules binding the phosphorylated or unphosphorylated cytoplasmic domain of icos, and particularly the presence of distinct pi kinase isoforms. then, the functional importance of these molecules has been analyzed by using pharmacological inhibitors specific for downstream mediators of icos activation. pull down of t cell lysates using phosphorylated or unphosphorylated synthetic peptides covering the cytoplasmic domain of icos was carried out. proteomic and immunoblot analysis of bound proteins showed that phosphorylated icos bound the different pi -k regulatory (p a, p b, p a) and catalytic (p a, p b, and p d) pi -kinase subunits expressed by leukocytes. these data were confirmed in icos immunoprecipitates from pervanadate-activated cells. icos bound regulatory and catalytic subunits in the order p a g p a g g p b and p a g p d g g p b, in agreement with quantitative rt-pcr and immunochemical estimation of subunit abundance in the t cells and t cell lines used. the use of specific pi -kinase inhibitors has confirmed the relative importance of the catalytic isoforms in icos function, including reorganization of actin cytoskeleton induced by icos ligands, or costimulation of tcr/cd -induced secretion of il- and il- . during the process of antigen recognition between t-cell and antigen-presenting cell (apc), structural and spatial changes take place at the cell-cell contact, where the molecules involved in the formation of the immune synapse (is) reorganize, displaying a segregated localization. in this context, the translocation of the microtubule-organizing center (mtoc) is an early event that occurs during the formation of the is, bringing with it the golgi apparatus, thus providing the basis for a polarized secretion. however, the molecular mechanisms involve in the localization of the mtoc at the contact area between the t cell and the apc are not completely understood yet. we have studied the possible role of scaffolding protein akap , a member of the a-kinase anchoring protein (akap) family that localizes at the centrosome and interacts with pka regulatory subunit and other signalling molecules, in mtoc polarization and immune synapse formation. either the overexpression of gfptagged c-terminal cg-nap/akap construct that acts as a dominant negative, or sirna knockdown of endogenous akap expression in t cells prevents the correct organization of cd z and pkcv to the is and mtoc reorientation towards t cell-apc contact area in antigen and superantigen-dependent human models, resulting in a disorganized is; lfa- localization was also analyzed to assess p-smac architecture and, interestingly, confocal d reconstruction revealed that lfa- ring was not clear in the akap -disrupted cells. moreover, akap was required for tcr signalling since the knock down with specific sirna and overexpression of c-terminal of akap decrease the phosphorylation of molecules such as lat, plcg and pkcv. these defective activation events as reflected in a reduction of il- production. together, our results underscore a key role for akap in the organization of the immune synapse and in the antigen-specific reorientation of the mtoc. the tcrbeta/ptalpha pre-tcr complex signals the expansion and differentiation of developing thymocytes. functional properties of the pre-tcr rely on its unique ptalpha chain, which suggests the participation of specific intracellular adaptors. in fact, we have recently identified cms, a member of the cin /cms family of adaptors, as a ptalpha-binding protein that specifically interacted with the human ptalpha cytoplasmic domain via its sh domains, and to the actin cytoskeleton via its c-terminal region. we found that cms co-localized with polymerized actin in pre-tcr clusters at the pre-tcr activation site, and also in the ptalpha endocytic compartment. since actin polymerization plays a critical role in regulating signalling through the alpha/beta tcr in mature t cells, we decided to investigate the potential function of cms as a regulator of actin polymerization and pre-tcr signalling in pre-t cells. using pre-t cells expressing a mutant pre-tcr lacking the cms-binding motif in the ptalpha tail and short hairpin irna-based gene silencing, we demonstrate that binding of cms to ptalpha contributes to cytoskeleton dynamics and pre-tcr-mediated signalling in human pre-t cells. cms-deficient cells specifically showed defects in pre-tcr-induced ca + mobilization and cell activation involving the pi k, nfat, plcg and erk signalling pathways, together with defects in actin polymerization and cell motility. cms therefore links cytoskeleton dynamics with the function of discrete pre-tcr signalling components, suggesting the functional implication of cms in human t-cell development in vivo. abstract withdrawn by author j objectives: most signaling pathways engaged after bcr activation have been described. however, several negative regulators of these pathways are unknown. the characterization of these regulators is important to understand the control of transduction pathways in adaptative immunity. carabin (tbc d c) has been recently described as a negative regulator of tcr signaling. it interacts with calcineurin and inhibits the formation of calcineurin/calmodulin complex, blocking nfat nuclear transport. moreover, carabin maintains ras protein under an inactive form, thus inhibiting ras-mapk cascade. expression of carabin is finely regulated following tcr signaling, and its knockdown (kd) enhances t cell activation. considering the important molecular similarities of antigen receptor signaling pathways in t and b cells, we studied the role of carabin in b cell. could carabin play a role of negative regulator of b cell function? methods: we studied by quantitative rt-pcr ) the expression of carabin in different purified subsets of bone marrow and splenic mouse b cells, as well as ) the kinetic of expression of carabin in bcr stimulated murine splenic mature b cells. ) we then studied the phenotype of carabin kd (shrna expressing) a b cells after bcr stimulation. ) the expression of carabin is significant in murine b cells, with an increase during b cell development, from bone marrow pro/preb to immature, to splenic t tot b cells and to follicular mature b cells. ) the kinetic of expression of carabin in bcr stimulated murine mature b cells suggests a fine regulation of carabin expression. ) bcr simulation, but not lps stimulation, of carabin kd a b cells shows an acceleration of ras target erk / phosphorylation, without any for the phosphorylation of mapk jnk, which is not targeted by ras. conclusion: carabin is expressed in murine b cells in a developmental regulated manner, with the highest expression in mature compartment. bcr stimulation leads to a fine regulation of carabin expression in wild-type mature b cells, and to a faster activation of erk / pathway in carabin kd b cells. altogether, these results strongly suggest a role of carabin as a negative regulator of b cell function toll like receptors are pattern recognition receptors, which recognize invariant pathogen associated molecular patterns. toll like receptor (tlr ) binds doublestranded rna, a nucleic acid frequently associated with viral replication. we observed that freshly isolated human cd + t cells express tlr and respond to the well characterized synthetic tlr ligand polyinosinic-polycytidylic acid [poly(i:c)]. the expression of activation markers and cytokine production by cd + t cells upon t cell receptor (tcr) stimulation is enhanced in response to co-stimulation via tlr . tlr stimulation on its own had no effect on expression of activation markers and cytokine production. to elicit the molecular basis of a potential cross-talk between tcr and poly(i:c) induced signaling, we used jurkat cells to perform luciferase assays. we observed that costimulation with poly(i:c) in comparison to tcr stimulation alone enhanced nf-kb but not nfat activation in jurkat cells. similarly to jurkat cells, tcr stimulation activated nf-kb in primary cd + t cells. this effect was further enhanced by additional poly(i:c) stimulation as shown by real-time-pcr and western blot analysis. on the other hand, we observed that poly(i:c) stimulation on its own activated the transcription factor interferon regulatory factor (irf ) as revealed by realtime-rcr analysis of ifn b and irf , whose transcription depends on the activity of irf . combined tcr and poly(i:c) stimulation further enhanced the transcription of these two genes. these results indicate that tlr signaling modulates tcr-driven responses and vice versa both in jurkat cells and in freshly isolated cd + t cells. this study was supported by dfg spp "innate immunity" (ka / - ). the initiation of protective t cell responses requires the recognition of mhc-bound peptides from pathogen or tumor antigens by the t cell receptor (tcr). how this signal is transmitted across the t cell membrane to the cytoplasmic signaling motifs is still unknown, and is the focus of this project. in textbooks, the cytoplasmic domains are depicted as flexible chains in the cytoplasm, but biochemical studies show that the cd e cytoplasmic domain (cd e cd ) binds to synthetic lipid vesicles that contain acidic phospholipids. this binding is predominantly due to electrostatic interactions between basic residues of cd e cd and acidic phospholipids. in the cell, acidic phospholipids are enriched in the inner leaflet of the plasma membrane. phosphatidylserine in particular is concentrated on the inner leaflet of the plasma membrane due to active transport mechanisms, explaining how such charge-charge interactions are generated. to study the interaction of the cd e cd with the membrane in live cells, we have developed a fluorescence resonance transfer (fret) assay which measures the proximity between a fluorescent protein (tfp) attached to the c-terminus of cd e cd and a fluorescent membrane dye (r ). with this assay, we show that the cd e cd is membrane-bound in resting cells and that binding is abrogated by introduction of mutations that disrupt lipid binding in the biochemical assay. additionally, in vitro analysis confirm functional domains for cd e cd lipid binding and conformational change. finally, nmr spectroscopy analysis reveals key features in membrane binding dynamics of cd e cd to lipid bicells. membrane binding by the cd e cd could thus be subject to dynamic regulation during the engagement of the tcr and further activation of the t cell. m. xydia , y. ge , u. quitsch , p. beckhove german cancer research center (dkfz), heidelberg, germany in peripheral tissues and the factors affecting their proliferation. cd + t cell help is believed to contribute to optimal cd + memory expansion via cd l on cd + t cells binding cd on dendritic cells. however, a few reports suggest that cd l-cd engagement may mediate direct cell-cell contacts between cd + and cd + t cells. in this study, we investigated the importance of cd -cd co-operation and cd l-cd interactions for t em proliferation. methods: we isolated human cd + and cd + t em cells from peripheral blood of healthy donors by facs or macs sorting. separated or mixed cd + and cd + populations were activated in vitro using anti-cd /cd beads. proliferation was measured by [ h]-thymidine incorporation, in some experiments after irradiation of one t em subset and/or incubation with blocking mabs against cd or cd l. furthermore, facs staining was used to assess cell-surface markers. statistical comparison was performed by student's t test. results: upon activation mixed t em populations showed a highly better proliferative response than separated cd + or cd + t em cells, demonstrating that optimal t em expansion requires direct cd -cd interactions. surprisingly, not only cd + but also cd + t em cells proliferated much more in mixed populations compared to the separated ones, indicating that optimal cd + t em proliferation depends on signals from cd + t em cells. activation induced the expression of cd on both populations and cd l on subsets of cd + and cd + t cells. blocking of cd l on cd + t em cells impaired significantly cd + t em proliferation, which confirms that the improved expansive potential of cd + t em cells in mixed populations depends on cd l co-stimulation by the cd t em subset. conclusions: our data demonstrate for the first time that activated cd + t em cells deliver help to the cd + t em subset via cd l-cd signalling and may play an important role for cd + t em expansion upon stimulation. the t cell surface glycoprotein cd , a member of the scavenger receptor cysteine-rich (srcr) family of proteins, targets to the immunological synapse upon t cell binding to antigen presenting cells (apc). however, it has not been established whether this translocation is due to the binding of a ligand expressed in the apc, or to intracellular interactions with signaling molecules or components of the cytoskeleton, that may control cd localization upon t cell:apc conjugation. we have questioned which domains of cd mediate the localization within the is, and for this we have expressed cd mutants as gfp fusion proteins in human t lymphocytes. we have also used jurkat cell lines expressing different cd mutants. t cells were incubated with superantigen-loaded raji b cells, and following the establishment of stable interactions between the cells, we analyzed the localization of cd by immunofluorescence and confocal microscopy. interestingly, our results show that the translocation of cd depends on sequences within the cytoplasmic domain, as a cd deletion mutant lacking most of the cytoplasmic tail, cd .k stop , is randomly distributed through the whole cellular surface, even in sustained t-apc interactions. the cytoplasmic domain relevant to cd translocation was mapped within amino acids glu and his since the cd .h stop mutant, just short of aa is still able to translocate to the is, whereas cd .e stop , that lacks important tyrosine residues, is no longer transported to the is upon t cell: apc interactions. although these studies do not exclude a role for the extracellular domain binding to an elusive apc-expressed ligand, they suggest that a major mechanism of regulation of cd translocation is dependent on molecular association of a short stretch of its cytoplasmic region (glu -his ) to intracellular signaling effectors. however, in hodgkin's lymphoma (hl) ebna- is missing, but lmp- is still expressed. using a hl derived cell line, we have shown that the cytokine il- can induce lmp- expression in vitro and can replace ebna- . we have investigated the molecular events for this mechanism. stat proteins bind to the palindromic ttc(n) x gaa sequence, where × is , or . a high affinity stat binding site is spaced by nucleotides. we found three potential stat binding sites in the lmp- promoter, which we named lrs, tr and edl . they were spaced by , and nucleotides, respectively. electrophoretic mobility shift (emsa) experiments were performed with nuclear extracts prepared from il- -treated or non-treated kmh -ebv cells. dna binding activity was analyzed using a double stranded oligonucleotide corresponding to the germline (gl) epsilon promoter, which is known to contain a high affinity stat binding site, or lrs-stat . a stat complex binding to the gl-epsilon promoter and lrs-stat was induced by il- . the specificity of the stat complex was shown by supershift experiments with anti-stat , but not anti-stat antibodies. when gl-epsilon or lrs-stat was used as cold competitors in a -fold excess, both unlabelled probes could compete out the labeled probe, providing evidence that the lrs-stat contains a functional stat binding site. oligonucleotides, corresponding to lrs in which the stat site had been mutated, could not compete for stat binding. interestingly, the unlabeled lrs-tr with nucleotides as spacer could also function as competitor. however, when ttc/gaa palindrom was spaced by nucleotides (lrs-edl ), it could not compete. thus, expression the transforming protein lmp- can be induced directly by the t cell derived cytokine il- in a stat dependent manner. it is likely that this mechanism operates in vivo as well and determines expression of the ebv encoded protein lmp- and thus the pathogenesis of ebv carrying hls. established knockout/ knockin mice with a fasl deletion mutant that lacks the intracellular portion (fasl ¿ intra). co-culture experiments confirmed that the truncated fasl protein is still capable of inducing apoptosis in fas-sensitive cells. preliminary immune histochemistry data suggest that, in contrast to published data, the absence of the intracellular fasl domain does not alter the intracellular fasl localization in activated t cells. we are currently investigating signalling and proliferative capacity of b-and t-cells derived from homozygous fasl ¿ intra mice. our data point to a rather inhibitory role of fasl reverse signaling during immune responses. during an immune response numerous receptor-mediated signals delivered to t cells direct their proliferation, survival and differentiation. we are using a quantitative model and in vitro methods to assess the "calculus", or decision-making algorithms t cells use to process these multiple signals. previous experiments with ot-i cd t cells revealed that tcr affinity regulated both the frequency of cells responding and the average time taken for cells to reach their first division (ji (ji . : . furthermore, affinity was the sole regulator of the rate of cell death in subsequent divisions. here we examine the same question for cd t cells. again we find that lower affinity peptides stimulate t cells to divide rapidly, however, a high proportion of cells die within each division round, revealing an important potential mechanism for affinity maturation and selection of dominant clones over time. in contrast varying the number of dendritic cells used to stimulate cd + t cells primarily affect the proportion of cd + t cells going into division rather than affecting division time or cell death in subsequent divisions. currently we are using these quantitative methods to measure the effect of cytokines and co-stimulatory molecules cd , cd and cd on parameters of cd + t cell proliferation to inform quantitative models of the immune response under different conditions. our goal is to develop quantitative models of t cell behaviour that can accommodate information at the molecular, cellular and population level. interaction between cd , a member of the tumor necrosis factor receptor superfamily constitutively expressed on antigen-presenting cell as b cells, and cd l, a member of the tumor necrosis factor family transiently expressed on activated t cells, are essential for the development of humoral adaptative immune response. various studies have shown that dual stimulation of b cell through antigen binding on bcr and cd leads to an enhancement of ig and cytokine production. the current dogma postulates that these signals are necessary and sufficient to drive naive b cell proliferation and differentiation to ig secreting plasma cells. however, recent evidence suggests that the innate immune responses could regulate humoral adaptive immune response. indeed, b cells can be activated through engagement of a variety of innate immune receptors, including toll-like receptors (tlrs). soluble cd l is unable to induce murine b cell proliferation. however, we and others have shown that recombinant mouse cd l (rmcd l) can increase proliferation induced by tlr (poly ic) and tlr (lps) agonists. by contrast, we never observed any synergy between rmcd l and tlr / (pam csk ) or tlr / (pam csk ) agonists. to go further in the study of cd l/tlr agonist synergetic effect, we have developed trimeric synthetic molecule to mimick cd l, named mini-cd ls, based on a c -symmetry core holding cd -binding motif lys-gly-tyr-tyr. in surface plasmon resonance experiments, mini-cd ls bind to immobilized human cd and compete with the binding of cd l homotrimers and diplayed effector functions that matched those of the much larger recombinant cd l homotrimers as maturation of mouse dendritic cells and activation of in vivo immune response in a mouse model of trypanosoma cruzi infection. as soluble cd l, mini-cd ls synergize tlr (lps), tlr (poly ic) and tlr / (r ) agonist-induced murine b cell proliferation but no synergy was observed between mini-cd ls and tlr / (pam csk ), tlr / (pam csk ) and tlr (odn ) agonists. synergy between cd l and tlr agonist provide the ground to use such a combination as adjuvant in vaccination strategy. however, to reach this goal, evaluation of cd l/tlr combinations on murine and human b cell activation and differentiation in antibody producing cells are under investigation. interaction of naïve cd + t cells with immature dendritic cells (idc) expressing self-peptides can result in their abortive activation (aa), which leads to the induction of cd + t cell tolerance. we have defined a phenotypic profile for cd + t cells undergoing such aa. these cells undergo limited proliferation which is associated with lack of ifn-g production, low cell surface expression of cd and cd , and high levels of expression of cd l and ly c. whereas, cd + t cells undergoing productive activation (pa), following encounter with mature dc, form effector ctl which is evidenced by extensive t cell proliferation, high levels of ifn-g, cd and cd , and loss of cd l and ly c expression. ly c is a gpi-anchored cell surface glycoprotein expressed on cells of hemopoietic origin: however, its role in peripheral tolerance induction is not understood. in this study, we show that mab-blocking of ly c in vivo and in vitro results in pa rather than aa. we hypothesize that the interaction of ly c, expressed on naïve cd + t cells, with its ligand on idcs, may be vital in controlling the induction of peripheral tolerance amongst self-reactive cd + t cells. objectives: organophosphorus compounds (opcs) are commonly used in the manufacture of insecticides and pesticides. exposure to opcs is associated with neurological toxicity but the effect on the immune system remains ill-defined. in this study, we used a subchronic exposure model to investigate the effect of the organophosphorus compound, paraoxon, on the murine immune system. methods: balb/c mice were injected i. p. daily with saline (control group) or paraoxon (experimental group) for weeks. during the treatment, animals were weighed and blood was collected weekly for determination of acetylcholinesterase activity in red blood cells. at the end of treatment, mice were sacrificed and spleen cells analyzed by flow cytometry. spleen cells were also cultured in the presence or absence of mitogens and supernatants were analyzed for cytokine content by elisa. for in vivo survival studies, mice were treated as described above and then orally infected with a virulent strain of s. typhimurium. animal survival was followed for up to days after infection. results: daily injection of paraoxon induced g % reduction in acetylcholinesterase activity by the end of the first week of treatment, a level which was thereafter maintained during the remaining weeks of treatment. mice exposed to paraoxon exhibited g % reduction in the rate of body weight gain over the treatment period in comparison with control group. at the end of treatment, ex vivo analysis of spleen cellularity and function revealed no significant differences between control and experimental groups. to analyze the status of the immune system in vivo, mice were infected with a lethal dose of a pathogenic strain of s. typhimurium and followed for survival. unexpectedly, paraoxon-treated mice exhibited a significant degree of resistance with % of mice surviving the infection compared to % in control group. protection in paraoxon-treated group was dependent on the reduced acetylcholinesterase activity as it was abrogated by coadministration of a reactivator of cholinesterase. conclusion: our data demonstrate that a reduction in the level of acetyl cholinesterase rendered mice more resistant to a virulent infection. this suggests a hitherto novel function of the neurotransmitter acetycholine in modulating the immune response to infection. t cell-dependent (td) and t cell-independent (ti) igg autoantibodies have been described in the context of the autoimmune disease systemic lupus erythematosus (sle). however, their different roles in autoimmunity are unknown. here we show that ti antigens induce anti-inflammatory igg antibodies and protect from antigen-specific immune pathology. administration of antigen-specific anti-inflammatory igg antibodies was sufficient to mediate this effect independent of the igg inhibitory receptor fcgammariib. ti but not td igg autoantibodies were further associated with inhibition of pro-inflammatory th and th cells and disease in mice deficient for fcgammariib, a spontaneous model for sle. the data suggest a novel immune regulatory function for ti immune responses through the generation of anti-inflammatory igg antibodies. objective: class i phosphoinositide -kinases (pi k) constitute a family of enzymes that generate -phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (tyr) kinase-associated receptors or g protein-coupled receptors (gpcr). the class i pi k are divided into two types: class ia p /p heterodimers, which are activated by tyr kinases, and the class ib p g (p gamma) isoform, which is activated by gpcr. although the t cell receptor (tcr) is a tyr kinase-associated receptor, previous studies showed that p g deletion affects tcr-induced t cell stimulation. mice lacking p g show a partial defect in t cell differentiation, activation and survival. p g participates in signaling pathways that regulate pre-tcr dependent differentiation and cd +/cd + t cell lineage commitment. in the mrl/lpr mouse model of systemic lupus erythematosus, administration of a pi kg-specific inhibitor causes a reduction in the number of cd + memory t cells that mediate renal injury. similarly, pi kg deletion in p pi k transgenic mice also reduces the numbers of cd + memory t cells. there is therefore evidence that pi kg has an important function in tcr-mediated t cell activation, although the mechanism by which pi kg regulates this process is not well understood. we studied the specific role of p g in t cell activation. methods: we studied whether the tcr activates p g and the consequences of interfering with p g expression or function on t cell activation. results: we found that after tcr engagement, p g interacts with and forms a complex with ga q/ , lck and zap . tcr stimulation activates p g, which affects -phosphorylated polyphosphoinositide levels at the immunological synapse. we show that tcr-stimulated p g controls rac activity, f-actin polarization, and the interaction between t cells and antigen-presenting cells (apc). we show that p g deletion affects the activation of many pathways downstream of tcr crosslinking, as well as the interaction between t cells and apc; these findings could explain the defective activation of p g-/-t cells. our observations clarify the activation mechanism and mode of action of p g in the control of t cell activation, confirming a crucial role for p g in tcr-induced t cell activation. we investigated mechanisms controlling central location of lytic granules and kinetics of their release within immune synapses formed by cytotoxic t lymphocytes (ctl). we show that cytolytic granules in ctl can be delivered to the secretory domain via two different pathways -"short" and "long". the choice between these pathways is regulated by the kinetics of early tcr signaling which depends on the strength of tcr/pmhc/co-receptor interactions. meanwhile, the molecular hardware used to deliver the granules remains the same. we conclude that the difference in temporal and spatial coordination of the two principal events, i. e., granule movement toward microtubule organizing center (mtoc) and the mtoc polarization, accounts for two different pathways of granule delivery to the secretory domain that influence efficiency of ctl cytolytic response. our findings reveal a mechanism of well-documented flexibility in t cell responsiveness that is derived from differential use of the similar set of immune receptors, signaling proteins and intracellular effector molecules. objectives: in addition to specific immune cytokines, lymphocyte activation and immune response are modulated by universal mediators like acetylcholine. nicotine was shown to suppress both cellular and humoral immune responses. previously we found that two nicotinic acetylcholine receptor (nachr) subtypes, a b and a , expressed in mouse b lymphocytes regulate their development within the bone marrow. the aim of the present study was to evaluate the roles of these two nachrs in b lymphocyte activation. methods: b lymphocytes were magnetically separated from the spleens of c bl/ j mice. they were stained with fluorescently labeled igm-, cd -or cd specific antibodies in the presence/absence of unlabeled nachr subunit-specific antibodies to be examined by flow cytometry. b lymphocyte activation was studied by h-thymidine incorporation upon stimulation with anti-cd and nachr-specific agonists or antagonists. the antibody response of mice immunized with cytochrome c with or without a nachr antagonist methyllicaconitine (mla) was studied by elisa. results: antibodies against a or b nachr subunits inhibited binding of igm-and cd -specific antibodies but facilitated that of cd -specific antibody. in contrast, antibody against a subunit prevented binding of anti-cd but not of anti-igm or anti-cd suggesting that a nachrs are located close to cd , while a b ones are close to bcr/cd . consequently, anti-cd -induced b lymphocyte proliferation was increased by mla much stronger than by a b -specific antagonist dihydro-b-erythroidine. it was also increased when cells were incubated with the inhibitor of acetylcholine synthesis hemicholine- . in contrast, proliferation of b lymphocytes from mice consuming nicotine was significantly weaker than that of control mice. mice co-injected with cytochrome c and mla responded with igm antibodies faster than those injected with cytochrome c alone, while the secondary / igg responses were similar. the cd -mediated b lymphocyte proliferation, but not the igm-igg switch or memory b cell activation, is negatively controlled by either endogenous acetylcholine or consumed nicotine through a nachrs. therefore, acetylcholine may be regarded as an auto/paracrine regulator of lymphocyte activation.this work was supported by philip morris usa inc. and philip morris international. binding of cd + t h -lymphocytes to antigen presenting cells or of cd + cytotoxic t-lymphocytes (ctl) to their target cells lead to a tight contact between these two cells, called immunological synapse (is). formation of the is induces calcium signaling, rearrangement of the actin cytoskeleton, and the recruitment of various molecules to the is, all of which are crucial for t-cell functions such as cytokine release or target cell killing. objectives: using primary human t-lymphocytes, none of the proteins involved in either calcium influx, cytokine release, actin cytoskeleton rearrangement nor in killing of target cells can be analyzed by knock-out strategies. for testing protein functions, down-regulation by rnai technology is thus an important tool. we used short interfering rnas (sirnas) to analyze the role of proteins involved in calcium influx and proliferation (stim and trpc ), and to analyze snare proteins which were shown to accumulate at the is and are good candidates to play a role in cytotoxic granule fusion and exocytosis to kill target cells. methods: to validate down-regulation of different mrnas quantitative rt-pcr was used. down-regulation of proteins was confirmed by immunocytochemistry, western blotting and various functional assays depending on the potential role of the protein of interest (calcium imaging, proliferation, cytokine release, killing assay). results: transfection efficiency of sirnas in t-lymphocytes was about %. down-regulation of stim was confirmed by qrt-pcr and by calcium imaging, but only for early time points following activation of cd + t h -lymphocytes, probably because of stability problems. to increase stability of sirnas within t-lymphocytes we used modified sirnas published by mantei et al. (eji, ) . we show that these sirnas down-regulate various snare proteins in ctls more efficiently than non-modified sirnas. the optimal sirna concentrations for transfection in primary human t-lymphocytes was found to be - pmol, which is lower than the concentrations reported in other cell types. conclusions: following optimization, down-regulation of mrnas by sirna is a powerful tool to investigate the role of different proteins involved in the activation of t-lymphocytes in primary human cells. chemical modifications increase the lifetime and efficiency of the sirnas in primary human t-lymphocytes. stress-inducible heat shock protein (hsp ) has gained plenty of attention because of its potent adjuvant capability to induce antigen-specific cd + cytotoxic t-lymphocyte (ctl) and cd + t-helper cell (th ) responses. in this study, we investigated the behavior of t-cell subsets stimulated with endotoxin-free recombinant hsp with respect to proliferation, cytokine expression, cytotoxicity against allogeneic b-lymphoblastoid cell line (b-lcl) and k cells as well as targetindependent cytotoxicity. cd + cells exhibited a strong increase in proliferation after stimulation with hsp , with rates of up to %. in the presence of target cells, a -fold up-regulation of granzyme b mrna was observed after stimulation of cd + t-helper cells with hsp in combination with il- , - and - . the target cell-independent secretion of granzyme b by cd + cells was greatly augmented after stimulation with hsp plus il- or il- , - and - . in this study, we have shown that hsp is capable of inducing a cytotoxic response of t-helper cells in the absence of lps or any other pamps. the granzyme b secretion and the cytolytic activity of cd + t cells is induced in a target-independent way, whereas the cytotoxic activity of cd + and cd + t cells can be further enhanced in the presence of the target cells. our data provide novel insights into the role of extracellular hsp on t-cell immune response concerning the induction of target-independent t-helper cell cytotoxicity. jun n-terminal kinases (jnk) have been shown to play controversial role in regulation of cell fate. cd , which is responsible for germinal centre formation in lymph nodes, trigger jnk activation. the role of other b cell co-receptor molecules that may be involved in antigen-driven differentiation were not clarified. the aim of this study was to find out whether cd receptor contributes to jnk activation in mature human b cells. protein expression and phosphorylation were studied by western blot analysis. protein associations were evaluated by immunoprecipitation and gst-pull down assays. hpk overexpression in a model system was achieved by transfection. pjnk / expression in primary hrs cells was assessed by immunohistochemistry. ligation of cd on resting (dense) and activated (buoyant) human tonsillar b cells lead to jnk , but not jnk activation. cd ligation on primary tonsillar b cells also resulted in jnk activation. however, bcr crosslinking did not affect the level of jnk / phosphorylation. cd -mediated jnk activation was independent from sh d a/sap adaptor protein expression, and was demonstrated for all studied b-lymphoblastoid, burkitt's lymphoma and hodgkin's lymphoma (hl) cell lines of b cell origin. we were searching for serine/threonine kinase that could coprecipitate with cd and link this receptor with jnk pathway. using immunoprecipitation and gst-pull down assays we found that hematopoietic progenitor kinase (hpk ) was associated with cd in primary b cells as well as in b cell lines. cd -hpk association was independent from cd tyrosine phosphorylation and sh d a expression. overexpression of hpk in a model system significantly enhanced cd mediated jnk phosphorylation. it is known that tnf family receptors such as cd , cd , rank trigger survival signals in hrs cells. we observed the expression of pjnk / in hrs cells of primary classical hl. cd could be involved in sustained jnk activation in primary hrs cells, and this may reflect the role of cd receptor as well as other receptors in the regulation of hrs survival. overall, it was shown that jnk is activated via cd in primary b cells and in all studied cell lines of b cell origin. serine-threonine kinase hpk is involved in cd -mediated jnk activation. objectives: cd has been shown to act as a negative regulator of tcr signaling during thymocyte development. however, the molecular mechanisms involved in this process remain elusive. one potential key molecule involved in the downmodulation of tcr signaling is c-cbl, a ubiquitin ligase that physically associates with cd upon tcr crosslinking in thymocytes. the objective of this study was to determine which sequences within the cytoplasmic tail of cd are involved in c-cbl phosphorylation and association. methods: el thymoma cell line was stably transfected with wild-type human cd or hcd cytoplasmic tail mutants: cd .k stop (maintaining only a pseudo itim); cd .h stop (lacking the distal s and y in the carboxy-terminal region); cd . ¿ e -l stop (lacking the pseudo-itam, putative site for c-cbl association). phosphorylaton of y in c-cbl was analyzed, which is required for vav recruitment and c-cbl dependent degradation by the proteasome. stable clones were stimulated with anti-murine cd in combination or not with anti-human cd biotinylated antibodies and phosphorylation of c-cbl was detected by flow cytometry after intracellular staining anti-phospho c-cbl (py ) antibody. murine thymocytes were used as positive control. data was analyzed using flowjo software. unpaired two-tailed student t test was used to calculate statistical significance (p x . ). in murine thymocytes, co-crosslinking of cd with cd induces an increase in c-cbl phosphorylation compared to cd alone. analysis of the el- transfectants showed that mutants cd .k stop and cd .h stop lost the ability to costimulate cd -mediated phosphorylation of c-cbl. in contrast, cd . ¿ e -l stop mutant, was able to efficiently costimulate cd -mediated c-cbl phosphorylation, similarly to the hcd wt. our results indicate that the absence of the pseudo itam in cd does not interfere with c-cbl phosphorylation in response to cd plus cd crosslinkiing on the other hand, sequences present in the carboxy-terminal region of cd appear to be important for c-cbl phosphorylation. therefore, c-cbl phosphorylation might not require physical association with the cd cytosplasmic tail, but rather, may indirectly associate with cd through the interaction with other sh -sh domain-containing molecules, that may be recruited to cd through its carboxy-terminal region. l. kolly , s. narayan , j. tschopp , a. so , n. busso chuv, rheumatology, lausanne, switzerland, unil, biochemistry, epalinges, switzerland apoptosis-associated speck-like protein containing a caspase recruitment domain (asc) is an adaptor protein that is essential for the recruitment of pro-capase- into inflammasomes and thus plays a key role in regulating capase- -dependent il- b and il- production. despite recent evidence implicating asc in adaptive immunity against infections, hyperresponsiveness and vaccination, the cellular and molecular basis for asc involvement in adaptive immune responses remains largely unexplored. to investigate the impact of asc on t cell activation and subsequent effector function. asc +/+ and asc -/-t cells or purified cd + and cd + t cells were activated in vitro through anti-cd stimulation and their proliferative potential and cytokine profiles characterized. proliferative responses by asc -/-t cells were significantly inhibited two-fold following tcr-cd ligation when compared to asc +/+ t cells. furthermore, cytokine analysis revealed that anti-cd activated asc -/-t cells predominantly displayed a more th phenotype, producing more il- ( vs. pg/ml; asc +/+ vs. asc -/-t cells respectively; p= . ) and less ifn-g ( , vs. , pg/ml; asc +/+ vs. asc -/-t cells respectively; p = . ). when asc +/+ and asc -/-t cells were purified into cd + and cd + t cell fractions and activated individually using anti-cd , no inhibition in proliferation was observed amongst activated asc -/-cd + and cd + t cells. interestingly, the activated asc -/-cd + t cell fraction produced significantly more il- when compared to activated asc -/-cd + t cells and asc +/+ cd + and cd + t cells (asc -/-cd + t cells = pg/ml il- ; asc -/-cd + t cells = undetectable il- ; asc +/+ cd + t cells = pg/ml il- ; asc +/+ cd + t cells = undetectable il- ). cd + and cd + t cell mixing experiments revealed that asc -/-cd + t cells are able to inhibit the proliferative ability of asc -/-cd + t cells, asc +/+ cd + and cd + t cells in vitro and that this suppression appears to be mediated by a soluble factor secreted by activated asc -/-cd + t cells. collectively, these results demonstrate that the absence of asc drives cd + t cells towards a suppressor cell phenotype, suggesting that asc might play an important role in determining the fate of cd + t cells. various members of the eicosanoid family derived from arachidonic acid participate in inflammatory reactions and may act as potent regulators of the immune response. in particular, e-series prostaglandins, pge and pge suppress some t-cell functions including proliferation, activation and cytokine production. pge signals through four types of gpcrs called the ep receptors. at low concentrations, pge is believed to be necessary for t cell function, whereas at higher concentrations, pge inhibits t cell proliferation. these effects are largely governed by various cell specific stimuli and tissue microenvironment. objectives: to delineate, compare and contrast the effects of pge and ep receptor antagonists on t cell activation. methods: flow cytometry, proliferation assays, migration assays. we have observed that pge diminishes expression of early, intermediate and late t cell activation markers. in contrast, pre-treatment of cd + t cells with ep receptor antagonists was found to impair cell surface expression of cd , cd , cd and ox but not cd . suppression of t cell proliferation by pge has already been widely studied. however, blocking ep receptors in cd + t cells by the use of ep antagonists prior to activation surprisingly caused a defect in t cell proliferation. migration of cd + t cells to the chemokine sdf- b was also found to be reduced due to pre-treatment with ep antagonists. in order to study the physiological relevance of these findings we studied the trafficking of basal and activated t cells to regional lymph nodes during inflammation in the presence and absence of ep receptor antagonists. this model revealed that the use of ep antagonists causes a reduction in the amount of cd + cd + adoptively transferred t cells in the regional lymph node following the induction of a local inflammatory response. conclusions: in our study we show for the first time that ep receptors are required for expression of activation markers and activating proliferation in murine cd + t cells. our results also suggest that considering pge -mediated camp signaling in cd + t cells, it will be absolutely necessary to distinguish between transient increases, which have potentiating effects, and sustained increases, which have inhibitory effects in t cell activation. objective: our objective was to investigate how ros affect the different stages of t cell activation. because activation is initiated by changes in intracellular calcium concentration, we addressed whether and how ros affect calcium signalling. the experimental results were obtained using a combination of fluorescence microscopy, patch-clamp, t-cell activation assays and molecular biology. results: we show by direct measurement of ros that t-cells are exposed to high concentrations of oxidants when they are in close vicinity of activated phagocytes. the effect of ros on calcium signalling in jurkat t-cells as well as in primary naï ve and effector cd + human t-cells was examined. oxidation affects several ca + signalling pathways by altering the activity of ip receptors, trp channels and store operated ca + channels in a concentration dependent manner. interestingly, calcium signalling is differentially affected in naï ve and effector t cells. thiol reducing agents were able to significantly reduce the effects of oxidation implicating thiol oxidation as a major player in the regulation of ca + signalling in t-lymphocytes. cysteins are the main carrier of thiol groups in proteins and we show that orai ion channels contain reactive cysteine groups that mediate ros effects on the calcium influx pathway. conclusion: ros regulate the calcium dependent t-cell activation in a complex way, affecting all three major calcium signalling pathways. by mutational analyses of the orai proteins, we are able to pinpoint molecular targets of regulation. the activation of t cells during an immune response is a crucial but tightly regulated event. to make the grade, the t cells upregulate costimulatory but also inhibitory receptors upon antigen recognition. this enables the t cell to be stimulated for proliferation to keep pace with pathogens infection, but also to become dampened upon successful defense against the pathogens via negative feedback mechanisms. in this study we present data of the signaling mechanisms underlying the potent t cell inhibitory receptor cd (emmprin, basigin) , a member of the ig-family. previous studies reported that lymphocytes from cd knockout mouse possess enhanced mixed lymphocyte reactions and cd monoclonal antibodies can interfere with t cell activation. these observations already pointed to a negative crosstalk of cd signals with the t cell antigen receptor or co-stimulatory signals. consistent with these studies, we found that rna interference (rnai) with cd in jurkat t cells augments the secretion of the t cell growth-factor interleukin- (il- ) upon t cell activation. this up-regulation is at least partially due to an increased activity of the nuclear factor of activated t cells (nfat), which resulted in an enhanced il- promoter activity. by reconstituting the rnai-mediated knockdown with various truncated rnai-resistant forms of cd , we identified the immunomodulatory sub-domain of cd . supported by the gen-au program of the austrian federal ministry of science and research. mirnas play a critical role in the control of hematopoiesis. the goal of this project is to determine whether mirnas function also during the antigen-induced activation of mature b lymphocytes. therefore, we determined mirna profiles in primary splenic b cells before and after polyclonal activation with either lps (simulates t cell-independent activation) or a combination of anti-igm, anti-cd and il (simulates t cell-dependent activation). microarray assays identified about mirnas in unstimulated b cells. of these were downregulated and one was upregulated upon stimulation. in silico analyses with various mirna target prediction programs revealed an interesting and promising set of transcripts whose translation/stability could be controlled by mirnas during the antigen-induced activation phase of mature b cells. among these targets are bcr signalling molecules and transcription factors that control proliferation, igh class switch as well as differentiation in antibody-secreting plasma cells. one of these transcripts codes for the interferon regulatory factor- (irf- ). the graded expression of this important transcription factor has been shown to coordinate isotype switching with plasma cell differentiation. first results indicate that the expression kinetic of irf- transcripts differs from that observed for irf- protein abundance after b cell stimulation. further analysis identified the irf- transcript as a target whose expression is obviously fine-tuned by a mirna upon antigen stimulation. we are in the process to biochemically verify potential targets for each of the differentially regulated mirnas and determine the effect of ectopic and retrovirally mediated expression of mirnas on b cell differentiation. the work was in part supported by the izkf erlangen, the dfg graduiertenkolleg gk and the dfg forschergruppe for . objective: transforming growth factor-b (tgf-b) signals through type i (tgfbri) and type ii (tgfbrii) tgf-b receptors and receptor regulated smad proteins. tgf-b exerts predominantly anti-proliferative and pro-apoptotic effects which are frequently lost in cancer. the mechanisms of resistance against tgf-b have not been fully elucidated. our aim is to describe how b cell lymphoma cells respond to tgf-b compared to normal peripheral b cells, to create an overview of the different signaling pathways involved, and to characterize the mechanisms behind the loss of sensitivity to tgf-b. methods: proliferation assays were performed on different b-cell lymphoma cell lines and normal peripheral b cells to screen for tgf-b-induced effects. western immunoblotting analysis was conducted to characterize protein expression and phosphorylation related to tgf-b signaling pathways. facs analysis was used to measure tgf-b receptor surface levels. cells were treated with demethylating agents to examine changes in gene expression levels. s. manthey , f. hauck , i. berberich , f. berberich-siebelt , gk -immunomodulation institute for virology and immunobiology, university of wuerzburg, würzburg, germany, university of wuerzburg, department of molecular pathology, würzburg, germany the transcription factor ccaat/enhancer-binding protein b (c/ebpb) can not act only as a transcriptional activator but also as a transcriptional repressor. in murine cd + t lymphocytes, the transcription factor is predominantly expressed in t helper (th ) compared to t helper (th ) cells. in contrast, by binding to the c-myc promoter(s), c/ebpb represses c-myc expression thereby arresting t cells in the g phase of the cell cycle. both, transactivation and repression depend on the n-terminal transactivation domain of c/ebpb. blimp- encoded by prdm is a transcription factor necessary for terminal differentiation of b cells to plasma cells. furthermore, blimp- is expressed in differentiated effector t cells where it is higher in th than th cells. the regulation of the blimp expression is not fully understood. interestingly, we found that c/ebpb can bind to the prdm promoter and activates blimp- expression in t cells. as c/ebpb is also expressed in b cells, we hypothesize that this transcription factor might as well influence the expression of blimp- in b cells. so far, we were able to show a similar expression profile of c/ebpb and blimp- in b cells using cre recombinase. moreover we found a new putative blimp- isoform lacking exon . currently, we analyze the expression of c/ebpb and blimp- in primary b cells and b cell lines after various stimulations. to get more insights into the function of c/ebpb in b and t cells, we are generating mice carrying a b as well as a t cell-specific deletion of c/ebpb. engagement of antigen receptors on lymphocytes leads to rapid increases in intracellular free calcium concentrations via phosphorylation of phospholipase c gamma (plcy) and plays an important role in activation of cells. by screening cvid patients with a flow cytometric assay we demonstrate that calcium flux is significantly reduced in b and t cells isolated from the peripheral blood of patients in the group ia of the freiburg classification as compared to non-ia patients and healthy donors (hd). ia patients are characterized by the expansion of an unusual cd low b cell population in which calcium mobilization is strikingly lower than in other b cell subsets. common subpopulations like naï ve and mz-like b cells as well as cd + t cells but not transitional b cells or cd + t cells also revealed significantly decreased calcium peaks. the cytometric data correspond to a semiquantitative rt-pcr assay and functional data showing reduced induction of the calcium dependent macrophage inflammatory protein- a (mip- a), and abrogated activation and proliferation, respectively. preliminary data on b cell receptor (bcr) mediated phosphorylation of plcy revealed constitutively high background levels in cd low b cells of ia patients. since phosphorylation in the other b cell populations as well as calcium flux upon ionomycin were the same for patients and healthy donors, we postulate an abrogated amplification or altered inhibitory pathway targeting the signalling events downstream of plcy and upstream of internal store release, thus resulting in defective calcium signalling. the underlying mechanism yet remains to be elucidated and is part of our work in the future. c. balas , v. courtois , k. de luca , r. sodoyer sanofi pasteur, marcy l'etoile, france the presence and relative abundance of cytokines at different stages of infection is relatively well documented, but their involvement in immune status, pathogenesis or disease progression is still unclear. a potential explanation to the difficult interpretation of the results obtained might be related to the intrinsic weakness of the analytical techniques. for instance monitoring of the expression level of cytokines, such as il- , il- or il- could lead to misinterpretation if molecular isoforms are not detected by antibodies currently used to measure them. the analysis of the human transcriptome is a way to access the subset of genes involved in the immune response upon infection by various pathogens. such an analysis might be completed and enriched by the analysis of the relative expression of some cytokine splice variants. methods: genetic tools (primers and qpcr probes) capable of discriminating and quantifying alternatively spliced messenger rnas from il- , il- and il- . furthermore, the recognition by several commercial antibodies of the different cytokine isoforms (expressed as recombinant proteins) has been investigated. the genetic tools have been validated on in vitro models as well as on biological samples (please refer to the abstract no a- - - ). conclusion: implication of such kind of analysis in diagnostic application and disease progression survey will be discussed. in a different context, the same kind of analysis could be applied to the monitoring of the immune response upon vaccination or more generally for new antigens or adjuvant screening. parasitic helminths affect about one third of the world population. therefore the mechanisms, which are involved in the persistence or the expulsion of the parasite, are of special interest. from other parasitic infections it is known, that the regulatory receptor cytotoxic t lymphocyte antigen- (ctla- ) plays a crucial role during infections. here, we use the strongyloides ratti infection of mice as an experimental system to investigate the role of ctla- during nematode infections. we employed a quantitative real-time pcr (qtpcr) analysis to quantify the migrating larvae (il ) in the tissue and the released eggs and first stage larvae (l ) in the feaces. the cytokine response of lymphocytes, prepared from the spleen and the mesenteric lymphnodes (mln) upon stimulation with polyclonal a-cd and s. ratti antigen was determined. additionally the humoral response was analysed in the primary and the secondary infection. to investigate the role of ctla- during the infection, a neutralysing antibody (a-ctla- ; f ) was administered intraperitoneally ( mg) two hours before subcutaneous infection with s. ratti il . the in vivo neutralisation of ctla- -signalling by applying a-ctla- during s. ratti infection led to an altered cytokine response, compared to infected mice treated with a control antibody. we detected an increase in th cytokines, such as il- and il- and a reduction of the proinflammatory cytokines ifn-g and il- . the investigation of the humoral response showed a remarked increase of the igg -titer in the serum during secondary infection in mice that had been treated with a-ctla- during primary infection. furthermore, the blockade of ctla- resulted in a diminished worm burden as indicated by reduced release of l in the faeces. these results suggest that the blockade of ctla- during s. ratti infection induces an activation of the appropriate effectors of the immune system that are beneficial for the host defence. in particular the transition of the t cell cytokine profile towards a th response supports this hypothesis and might be the reason for the reduced worm output in the primary infection. the strong increase of igg during secondary infection also reflects the induction of a potent th response. objectives: cd is a class b scavenger receptor, which has been shown to be involved in the pathogenesis of atherosclerosis as well as in the clearance of apoptotic cells by macrophages. this clearance is important in regulating the immune system to avoid autoimmune reactions, as seen in systemic lupus erythematosus (sle). it was recently described that cd is highly expressed also on the marginal zone b cell subtype. we therefore set out to investigate the role of cd on the regulation of b cell in the setting of apoptotic cell clearance and autoimmune activation. we used a mouse model for sle where apoptotic cells were injected repeatedly in order to study the auto-reactive antibody response that follows. elisa was used to measure antibody levels and flow cytometry to study cell activation as well as cd expression. cd knock-out (ko) and wild type mice were used. results: preliminary in vivo data show a tendency for a higher antibody response towards ds-dna and the common self-antigen pc in cd ko compared to heterozygous mice. since reduced levels of cd are expressed in heterozygous mice we are currently repeating this experiment using wild type mice as controls for comparison. in support of the in vivo findings, the immunosuppressive effect of injected apoptotic cells seen in wild type mice after in vitro stimulation of splenocytes with lps is gone in cd ko mice. after one injection of apoptotic cells, cd ko b cells are activated while wild type b cells are not. after four injections a break of tolerance is seen and apoptotic cells do no longer have an immunosuppressive effect and we show that cd on b-cells are involved in setting this threshold. conclusion: our data suggest that cd is involved in the early regulation of b cell response towards apoptotic cells and production of autoreactive antibodies. it does so by being involved in regulation of the tolerance effect exerted by apoptotic cells. successful t cell immunity requires lymphocytes to be at the right time at the right place. the co-receptor cd acts as a major check-point of immune responses, but the mechanism by which cd controls peripheral t cell responses is unknown. the consequences of cd signaling on murine th cell migration were analyzed using chemotaxis assays in vitro and radioactive cell tracking in vivo. the genetic and serological inactivation of cd in th cells reduced migration towards ccl , cxcl and ccl . crosslinking of cd together with cd and cd stimulation on activated th cells increased expression of the chemokine receptors ccr and ccr , which in turn enhanced cell migration. sensitive liposome technology reveals that mature dendritic cells but not activated b cells were potent at inducing surface cd expression and cd -mediated migration-enhancing signals. importantly, migration of cd positive th lymphocytes in in vivo experiments increased, as compared to cd negative counterparts, showing that indeed cd orchestrates specific migration of selected th cells to sites of inflammation and antigenic challenge in vivo. these data show that cd signaling does not just silence cells, but selects individual ones for migration. this novel activity of cd adds to the already significant role of cd in controlling peripheral immune responses by allowing t cells to localize correctly during infection. it also suggests that interference with cd signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge. here we analyzed the role of cd signaling on the longevity of cd null t cells. using a sensitive staining method for cd , we show that human cd + cd null and cd + cd null t cells rapidly express surface cd . serological inactivation of cd using specific fab fragments or blockade of cd ligands using ctla ig in cd + cd null and cd + cd null t cells reduces the number of non-apoptotic cells in a fas/fasl-dependent manner. cd crosslinking on activated cd null cells prevents activation-induced cell death (aicd) as a result of reduced caspase activity. apoptosis protection conferred by cd is mediated by pi 'k dependent activation of the kinase akt resulting in enhanced phosphorylation and thereby inhibition of the pro-apoptotic molecule bad. we show that signals triggered by cd act directly on activated cd null t lymphocytes and, due to its exclusive expression as a receptor for cd /cd on cd null t cells, prevention of cd mediated signaling is likely a major target mechanism taking place during therapy with ctla ig. objectives: cd is a transmembrane protein tyrosine phosphatase (ptp) expressed in all nucleated leukocytes. it activates src family kinases (sfks) by dephosphorylating inhibitory tyrosines in their c-terminal tails. in cd -/mouse t cell signaling and development is severely impaired, while other leukocyte populations seem much less affected. at least in part, it is due to the activity of another transmembrane tyrosine phosphatase cd (ptprj, dep- ) which acts as a positive regulator of sfk in cd -/-b cells and macrophages and can compensate for cd deficiency in these cells. indeed, combined deficiency of cd and cd in mice results in defective macrophage and b cell signaling and development, a phenotype much more severe than the loss of either protein alone. naïve murine t cells do not express cd and its expression is increased only after activation. accordingly, no defects in t cell development and signaling in cd -/mice were reported so far. however, in human t cells the role of cd may be different since naive human t cells express cd at a level comparable to b cells. using cd -/-/cd -/human t cell line (jurkat-derived js- cells) we tested the ability of cd to complement cd deficiency in t cells. we used retroviral transduction to express human cd or cd in js- cells and tested their ability to reconstitute major signaling pathways. we also employed substrate trapping mutant of cd to identify direct substrates of this phosphatase. in agreement with previously published data, defective t cell receptor (tcr) signaling was observed in js- cells. expression of wild type cd or cd in js- cells resulted in more rapid calcium mobilization, enhanced tyrosine phosphorylation, and increased cd upregulation after tcr cross-linking. moreover, the carboxy-terminal tyrosine of lck, major t cell sfk, was hypophosphorylated in js- cells expressing cd when compared to control cells. finally, cd substrate trapping mutant expressed in js- cells interacted with lck in vivo suggesting that lck is a direct substrate of cd in js- cells. the results suggest a level of redundancy between cd and cd in human t cells not appreciated so far. during the past decades, great efforts have been made to get insights into the complex process of antigen-induced t cell activation and the underlying signal transduction pathways. the t cell antigen receptor signaling cascade is initiated by phosphorylation of itam-tyrosine residues through the t-cell specific src protein tyrosine kinase family member lck. during t cell activation, lck is supposed to undergo structural changes from a closed inactive to an open active conformation followed by phosphorylation of the itam-motifs. in order to resolve conformational changes of lck in living cells with high spatio-temporal resolution, we designed biochemically active conformational-sensitive förster resonance energy transfer (fret) biosensors using cyan and yellow fluorescent proteins inserted at special positions of the complete kinase backbone. for the live-fret imaging and biochemical assays we complemented lck-deficient jurkat t cells (jcam . ) with the biosensors. by introducing point mutations affecting the two major regulatory tyrosines tyr and tyr we found a dramatic decrease and increase, respectively, of intramolecular fret efficiency compared to the wild type biosensor. these results correspond to unfolding of the biosensor to its active conformation on the one hand and condensation of the kinase structure to its inactive form on the other hand. thus, our biosensor is able to detect phosphorylation modifications of key residues. however, we could not detect any overall change in fret and thus conformation of membrane-associated lck molecules during t cell activation indicating that other mechanisms, presumably reorganization of localization, underlie lck regulation. furthermore, we observed a contribution of intermolecular fret, which indicated homophilic interaction of lck. indeed, by performing single molecule analysis and native d immunoblotting we found lck dimers and higher order oligomers. together, these advanced imaging studies in the live cell context provide a novel picture of the function and regulation of this key kinase in signaling via the t cell antigen receptor. it has been reported that mitochondria accumulate under the immunological synapse (is) in response to tcr (t cell receptor) stimulation. this process seems to be required to allow proper tcr-induced calcium influx in t cells in contact with antigen presenting cells (apcs), because mitochondria can sequester calcium and thus keep crac (ca + release-activated ca +) channels open. however, antigen-induced calcium signaling is very fast, and clearly much faster than mitochondrial translocation toward the is. thus, we speculated that other signals are involved in recruiting the organelles to the contact region between t cells and apcs. we found that the adhesion molecule lfa- (leukocyte function-associated antigen ) induces localization of mitochondria at the is. this process is antigenindependent and is enhanced by the presence of chemokines in the t cell environment. however, tcr triggering stabilizes mitochondria at the synapse and it is important to sustain their recruitment in time. our data suggest that, by recruiting mitochondria to the cell-cell contact region, lfa- prepares and facilitates tcr signaling. we are performing experiments to understand the signalling pathways involved in mitochondria translocation at the is. burkitt lymphoma (bl) is a high grade b cell malignancy (non-hodgkin lymphoma (nhl)) derived from germinal center b cells, that harbours a chromosomal translocation juxtaposing the protooncogene myc next to the regulatory elements of one of the immunoglobulin loci. however, the precise contribution of myc to the pathogenesis of this tumour is poorly understood. based on the definition of a distinguishing gene expression signature for the molecular burkitt lymphoma (mbl) with myc as one hallmarking signature gene (hummel et al. ) we describe a non-viral vector based approach (vockerodt et al. ) to express myc in primary human gcb cells from pediatric tonsils. comparative whole genome gene expression profiling was performed in independent preparations. our data reveal a global change in gene expression in lymphoma precursor cells by myc giving new insight into potential changes of the gene expression program of gcb cells on the accidental way to bl in addition as a first step the function of selected signature genes in bl is accomplished. in a representative cell line with a mbl signature and with a non-mbl signature rnai directed inhibition of elements of the cd signaling cascade was conducted. after activating this particular signaling cascade (cd ) we analysed respective gene expression profiles of ikks, trafs and mapk deficient cells. based on these different rnai-mediated ge-profiles a comparison between both lymphoma types is performed. first attempts are made to reconstruct the topology of the respective signaling pathway by using the nested effects bioinformatic model, which has been described recently (markowetz et al. ). a rat thymic epithelial cell (tec) line (r-tnc. ) was established from a long-term tec culture. this line was characterized as a type of rat cortical tec with nursing activity (tnc). very little is known about molecular mechanism of the tnc/thymocyes interaction. in our previous studies we investigated molecular mechanisms involved in the binding and emperiopolesis of resting thymocytes by r-tnc. cell line in vitro. it was found that a number of adhesion molecules, such as cd , cd , cd , cd a, cd , cd , cd was involved in these processes. objectives: a main goal of this study was to define the adhesion molecules involved in the interaction between r-tnc. line and activated thymocytes. methods: experiments was performed on inbred ao rats. monoclonal antibodies (mabs)-mediated modulation of thymocyte binding and emperiopolesis was tested by adhesion and engulfment assay, respectively, using a coculture of cona and il- activated syngeneic thymocytes and unstimulated or ifn-g stimulated r-tnc. cells. we found that both the adhesion ( min and h) of activated thymoytes were partially blocked by mab to cd and cd molecules (ifn-g unstimulated and ifn-g stimulated r-tnc. cells). early adhesion was inhibited by mab to cd , abtcr, mhc class i molecule (ifn-g stimulated r-tnc. cells) and cd molecule (ifn-g unstimulated r-tnc. cells). after prolonged incubation, significant inhibition was obtained using anti-mhc class i mab (ifn-g unstimulated r-tnc. cells). almost all mabs which were inhibitory in the binding assay were inhibitory in the engulfment assay ( h), namely mab to cd , cd , cd , cd molecule (ifn-g unstimulated and ifn-g stimulated r-tnc. cells) and mhc calss i and mhc class ii molecule (ifn-g unstimulated r-tnc. cells). our results also suggest the involvement of cd a/cd dependent -cd independent pathway in adhesion and cd a/cd dependent -cd dependent pathway in emperiopolesis. the obtained results imply that adhesion, deadhesion and emperiopolesis of activated thymocytes by r-tnc. cell line are tightly regulated processes in which multiple adhesion molecules are involved. the crucial roles of cytokines in shaping t cell responses have been documented in both healthy and disease conditions. interleukin- (il- ), a recently described cytokine, has been shown to exhibit both pro-and anti-inflammatory properties. il- favours naï ve cd t cell differentiation into th cells to the detriment of th or th differentiation. the il- receptor (il- r) is a heterodimer composed of tccr, which confers ligand specificity, and gp , a signal transducing chain that is utilized by several other cytokines. il- has been demonstrated to promote cytotoxic lymphocyte functions of mouse cd t cells, but the potential impact of il- on human cd t cells has not been elucidated. our goal is to investigate the impact of il- on human cd t cell functions. we used peripheral blood mononuclear cells (pbmc) from healthy donors, either exvivo or after short term in vitro activation to perform our analyses. we first assessed whether the il- r is detectable on ex-vivo t cells using flow cytometry. we observed a greater proportion of cd than cd t cells expressing the complete surface il- r (gp +tccr). however, we detected high amounts of intracellular tccr in both, cd and cd t cells, but only polyclonal activation (anti-cd ) of cd t cells led to an actual increase of il- r surface expression. purified cd t cells from healthy donors were shortly stimulated in vitro and then analyzed using flow cytometry-based functional assays. il- activated stat and stat signalling with rapid kinetics in both cd and cd t cells, indicating the capacity of il- to signal through these molecules. addition of il- to anti-cd activated cd t cells led to a significant dose dependent increase of proliferation (as measured by cfse-based assay) and ifn-gamma and granzyme b production (determined by intracellular staining). these results demonstrate a pro-inflammatory impact of il- on human cd t cells. defects in immune regulation could result in the breakdown of immune tolerance leading to development of multiple sclerosis (ms). the pd- /pd-l pathway is associated with production of the immunoregulatory cytokine il- , the suppression of t lymphocytes proliferation by inhibition of akt phosphorylation (pakt), and the elicitation of apoptosis of antigen-specific cells; an impairment in this pathway could play a pathogenetic role in ms. we analysed by flow-cytometry the surface expression of pd-l and pd , as well as myelin basic protein (mbp)-stimulated il- production, pakt inhibition, and apoptosis (annexin v), in ms patients with relapsing-remitting disease. twenty-six patients were diagnosed as being affected by acute disease (ams); had a diagnosis of stable disease (sms). results showed that: ) pd-l -expressing cd + and cd + cells are reduced in ams compared to sms individuals (p= . ); and ) pd expression is increased in cd + t cells of sms individuals and is comparable on cd + t cells of ams and sms patients. this is associated with a significant decrease in il- production by mbp-stimulated cd + and cd + cells of ams patients (p= . ). additionally, cd + anexin v+ (av+) cells were diminished and cd + pakt+ cells were higher in ams compared to sms patients, while similar percentages of cd +av+ and cd + pakt+ were observed in both groups of individuals. data herein show that the impairments of the pd-l /pd- pathway seen in ams patients result in a reduced mbp-specific il- production by cd + and cd + cells as well as in a reduced apoptosis (annexin v) and an augmented proliferation (pakt) of mbp-specific cd + t. the pd /pdl pathway plays an important role in the pathogenesis of multiple sclerosis. monitoring of the expression of these proteins could be a novel diagnostic tool. anti- - bb in cd cells. this difference could be due to down regulation of cd by activated lymphocytes and possible preferential response of cd cells to anti- - bb costimulation. moreover, increase in ifn-g concentration in costimulated cultures also may enhance the suppressive function of mscs which again could explain the inability of costimulation in proliferation recovery. likewise, reducing tgf-ß by costimulation is not sufficient to abolish suppressive effect of mscs. in overall, these results suggest that lack of costimulation expression by mscs is not the mechanism of msc suppression and other mechanisms are involved. cytotoxic t lymphocytes (ctls) kill target cells by secretion of cytotoxic components contained in lytic granules at the contact zone between the target cell and the ctl, the immunological synapse (is). t cell receptor (tcr) enrichment at the is is one of the early and key events of is formation. objectives: soluble nsf attachment receptor (snare) proteins are required in almost all fusion events in cells. in the present study we tested if the snare protein syntaxin (stx ) is part of the is and whether it serves as a key player of is formation and/or the fusion process itself. methods: pcr-techniques, cell transfection, immunocytochemistry and different microscopic techniques like confocal microscopy and total internal reflection microscopy (tirf) were used on primary human ctls to test the function of stx . rna interference technique was also used to down regulate stx expression in primary human ctls. results: we identified stx in ctls by pcr and immunocytochemistry. stx accumulates at the is after ctl/target cell contact. when stx function was blocked by overexpression of a dominant negative stx mutant (deletion of the transmembrane region), functional studies with tirf showed a reduced accumulation and fusion of lytic granules at the is. furthermore, confocal studies showed a loss of tcr accumulation at the ctl/target contact side. conclusion: these results imply that the snare protein stx is present at the is and moreover is required for is formation in ctls. the observed block of lytic granule release is probably caused by disturbing an upstream process such as vesicle transport, recycling or sorting. objectives: despite the years history of mouse t h and t h subpopulations, relatively little is known about the differences in their signaling mechanisms and the membrane organization of critical receptors and signal transducing molecules. we have developed mouse t h hybridomas to study these differences between polarized t h cells. the in vitro established hybridomas were first characterized as t h , t h or t h phenotypes, based on their cytokine production (il- , ifng or il- ). a comparative analysis of t-bet, ifng and il- mrna levels was also done on quiescent and activated t h hybridomas. in the present study, the ca +response, membrane raft expression/organization, k + -and ca + -ion channel expression/function and sensitivity to apoptosis (aicd) were compared in these hybridomas. expressions and molecular localizations were investigated by flow cytometry and confocal microscopy, respectively. ion channnels were functionally analyzed by patch-clamp technique. apoptosis was analyzed using three markers (mitochondrial membrane potential, caspase activation, dna fragmentation) and flow cytometry. results: expression level of plasma membrane rafts/gangliosides (assessed by cholera toxin b-staining) showed the following rank: t h g t h g t h , although the membrane cholesterol level (detected with anti-cholesterol ab, ac ) was similar in the three cells. in connection, tcr displayed stronger colocalization with rafts and appeared more polarized in t h cells upon activation than in t h cells. t h cells produced a more sustained calcium response with higher amplitude than t h cells to the same tcr-mediated triggering signal. interestingly, this does not coincide with the expression of cav . and kv . ion channels, major functional determinants of the sustained calcium influx. t h cells expressed the highest levels of these two ion channels. there were also marked differences in their sensitivity to activation induced apoptosis (aicd) as assessed by three different markers of apoptosis. the results suggest that a different membrane compartmentation/organization rather than the differential expressions of certain receptors, ion channels and/or other upstream signaling molecules of these t h hybridomas may be responsible for the observed differences in their functional characteristics. objectives: bone morphogenetic proteins (bmps) belong to the tgf-b superfamily, which plays a central role in controlling cellular processes like proliferation, differentiation, apoptosis and migration. whereas tgf-b is well established as one of the most potent negative regulators of hematopoietic cells, the role of bmps in b lymphoid cells remains more elusive. in this study we investigated the effects of bmps on mature human b-cells. methods: b cells were isolated from peripheral blood of healthy donors using cd -dynabeads. cd + isolated cells were facs sorted into cd + cd naïve b or cd + cd + memory b cells. dna synthesis was measured by h-thymidine incorporation, immunoglobulin (ig) levels in cell supernatants were measured by elisa and phospho-protein levels were measured by western immunoblotting analysis. results: all bmps significantly suppressed anti-igm-induced proliferation of cd + cd naï ve b cells, of which bmp- and - were most efficient ( % suppression). similarly, all bmps suppressed cpg-induced proliferation of cd + cd + memory b cells by - %. to induce differentiation, both naï ve and memory b cells were stimulated with cd l and il- . this increased the production of igm, iga and igg - -fold compared to medium control, whereas addition of bmps inhibited the production of all ig classes. all bmps highly induced phosphorylation of smad / / in cd + b cells. the mechanisms for how bmps mediate their inhibitory effects are currently being explored in more detail. conclusion: bmps have prominent inhibitory effects on anti-igm-and cpg-induced proliferation of naive and memory human b cells, respectively. they also suppress cd l/il- -induced production of igs in mature human b cells. s. gutenberger , k. warnatz university medical centre freiburg, freiburg, germany background: signals through the b cell receptor (bcr) and co-receptors are essential for the survival, differentiation and effector function of b cells. the stimulation of the bcr initiates several independent but interrelated signaling pathways. one important pathway leads to the activation of mitogen activated protein kinases (mapk) and especially the phosphorylation of extracellular signal-regulated kinases and (erk / ). in a subgroup of patients with common variable immunodeficiency (cvid) we have previously demonstrated intrinsic defects in the activation of b cells revealed by the insufficient cd upregulation and proliferation after b cell receptor (bcr) stimulation. therefore we assessed signaling pathways downstream of the bcr in order to identify defects in the activation of b cells. methods: pbmc of hd and cvid patients were stimulated by anti-igm. different igm expressing b cell subsets were analyzed separately for erk / phosphorylation by intracellular flow-cytometry using phospho-specific antibodies to erk / . to increase the signal intracellular phosphatases were inhibited by h o . as markers of activation and initiation of proliferation, cd and ki expression were measured after days of in vitro stimulation. k. theil , p. aichele immh university freiburg, immunology, freiburg, germany type i interferons are homone-like molecules that are produced early after viral and bacterial infections. they signal via the type i interferon receptor (ifnar) and have pleiotropic effects on different cells of the immune system. their best known function is the antiviral activity. to test the direct effect of type i interferons on cd t cells in vivo we adoptively transferred lcmv glycoprotein specific tcr transgenic p cd t cells that are deficient in type i interferon receptor (ifnar-/-) into wild-type b -recipient mice and compared their expansion with wild-type (wt) p t cells after viral infection. we could demonstrate a severe impairment in the capacity of p t cells lacking type i ifnr (ifnar-/-) to expand after lcmv infection. following infection of recipient mice with recombinant vaccinia virus, recombinant vsv (vesicular stomatitis virus) or recombinant listeria monocytogenes expressing lcmv glycoprotein, p t cells expansion was considerably less dependent on type i ifnr expression. therefore direct type i ifn signalling is essential for cd t cell expansion and survival only after lcmv infection. our experiments showed that the lcmv generated cytokine milieu is responsible for the failure of expansion of ifnar-/-t cells during lcmv infection. a suitable model for elucidating the impact of the lcmv generated cytokine milieu is the transfer of p t cells into h mice. h mice ubiquitously express the lcmv immunodominant glycoprotein-epitope gp - . therefore the antigen-presentation can be uncoupled from the lcmv induced cytokine milieu when the h mice are infected with lcmv . . this is a lcmv variant that has got a point mutation in gp - and consequently cannot be recognized by the p t cells. s. frischbutter , r. baumgrass deutsches rheuma-forschungszentrum, signal transduction, berlin, germany antigen-specific stimulation of t helper cells induces activation of the main transcription factors nfat, nf-kb and ap which are important for expression of cytokines such as il- , ifng and il . it is known that the immunosuppressive drug cyclosporin a (csa) blocks the activity of the ser/thr phosphatase calcineurin and thereby the activation of the transcription factor nfat. however, we and others observed that this drug also inhibits the activation of nf-kb. to detect targets of calcineurin within the nf-kb pathway we analyzed phosphorylation and degradation levels of different nf-kb signaling proteins in the presence of csa and other calcineurin inhibitors. we found that phosphorylation of the signaling protein bcl- was prolonged in cells treated with inhibitors. our data do not indicate an enhanced bcl- phosphorylation but rather an inhibition of bcl- dephosphorylation. furthermore, calcineurin and bcl- co-precipitated with each other. interestingly, this interaction was observed only in t cell receptor-but not in tnfa-stimulated cells. in our proposed model, we hypothesize that calcineurin interacts with the carma/bcl- /malt signaling complex and dephosphorylates bcl- and, thus, promotes nf-kb activation. therefore, calcineurin is not only a hub for nfat but also for nf-kb activation. a. t. fulop , j. lamoureux , c. fortin université de sherbrooke, medicine, sherbrooke, canada objectives: aging is accompanied by a decrease in immune functions, called immunosenescence. the exact cause is still not known. changes in t cell subpopulations, thymic involution were invoked. we have demonstrated that the signal transduction is altered with aging. in the present work we studied the negative regulatory molecules in the t cell signaling to explain the altered activation of t cells with aging leading to decreased clonal expansion. methods: healthy young and elderly subjects were studied. lymphocytes were separated by fycoll-hypaque. the molecules participating in the negative control loop of lck were studied by western blot and confocal microscopy. the surface expression of ctla- has been studied by facscan. the translocation of the molecules in the membrane lipid rafts (mlr) was also studied by western blot. the activity of phosphatases was also determined. results: we found that the phosphorylation of pag was altered with aging explaining the decreased release of csk from mlr and the decreased lck activation. the activation of fynt was also altered. the phosphatase activity studies showed an increase in their activities with aging. the ctla- expression was higher after stimulation in t cells of elderly. there was differences between cd and cd t cells with aging. conclusion: these results suggest that the negative regulation is preponderant in t cells with aging on the positive activation and as such explaining the defect in t cell functions with aging. this opens new therapeutical avenues in the future. in contrast to other members of the tumour necrosis factor superfamily, fas ligand (cd l) contains a cytosolic proline-rich domain (prd) that enables interactions with sh and ww domain proteins. since fasl surface expression is regulated by adam -mediated ectodomain shedding and fasl might be subsequently released into the cytosol by regulated intramembrane proteolysis (riping) through the secretase-like enzyme sppl a, we are interested in defining interactions involving the generated intracellular fragment of fasl. employing a monoclonal antibody directed against the intracellular domain of fasl, we observed that previously described fasl-interacting proteins of the pch family selectively bind to the full length molecule but not to n-terminal fragments (ntfs). in order to identify other sh domain proteins that potentially interact with the riped fasl prd, we used a sh domain phage display library containing all sh domains expressed in humans. the screen confirmed several previously identified interactions but also revealed numerous new and interesting candidate binding proteins includig non-receptor tyrosine kinases and adaptor proteins or enzymes implicated in membrane, organelle, and actin cytoskeleton dynamics. selected interactions were verified biochemically and by laserscanning microscopy in transfected cells. it could be demonstrated that tec kinases known to be involved in immune receptor-associated signal transduction as well as members of the snx family, which are crucial regulators of endocytic and endosomal dynamics and trafficking, join the list of known fasl-interacting proteins. of note, in contrast to pch proteins, the snxs bound both ntfs and unprocessed fasl, indicating that individual interactors might influence different facets of fasl biology. in conclusion, the present data provide substantial evidence for a selective binding of individual interaction partners of fasl to the full length protein or ntfs. this more detailed glance at the fasl interactome will facilitate focussed strategies to clarify unanswered questions regarding reverse signalling and functional conse- optimal t cell activation requires the engagement of the t cell receptor (tcr) by the specific mhc/antigen complex and costimulatory signals as the interaction of b family members on antigen-presenting cells with cd on t cells. remarkably, whereas classical glucocorticoids (gcs) effectively suppress solely tcrtriggered t cell activation in vitro, additional cd co-stimulation leads to gc-resistance. in this study, we compared the non-steroidal selective glucocorticoid receptor agonist (segra), compound , with classical gcs regarding their suppressive effect on cd -costimulated t cells. human primary t cell subpopulations and jurkat cells were stimulated in vitro with plate-bound anti-cd and anti-cd , and proliferation, cytokine secretion as well as phenotypic activation parameters were determined. remarkably, a clearly improved inhibition of ifn-gamma secretion was observed in cd -costimulated human memory/effector cd + t cells by compound than by classical gcs. interestingly, apoptosis and activation antigen expression were similarly regulated. improved inhibition of lymphokine secretion by compound was also seen after pma / ionomycin stimulation of human primary t cells and jurkat cells. when investigating the in vivo effects of compound and prednisolone in acute and subacute dnfb-induced contact hypersensitivity models in mice, we observed comparable efficacy for inhibition of t cell-dependent skin inflammation when treating before hapten challenge. in contrast, however, when treating around hapten sensitization markedly stronger effects were demonstrated for compound than prednisolone. when evaluating possible mechanism for the increased activity of compound in inhibition of t cell activation we got hints for a specific inhibition of the calcineurin pathway by compound which was not prevented by the partial gc receptor antagonist, ru- , in vitro. moreover, in vivo we observed less induction of il- beta and tnf-alpha by pre-treatment with compound than with prednisolone. our data indicate that the non-steroidal segra, compound , may represent a promising drug candidate for the treatment of t cell-dependent inflammatory diseases where therapy with classical gcs is hampered by t cell resistance. influenza a infection of b mice elicits robust cd + t cell responses, with virus-specific cells showing a distinct pattern of cytokine production: tnfa+ cells always express ifng; and il- + cells are contained entirely in the ifng+tnfa+ subset. interestingly, the co-expression of ifng and tnfa varies for different epitope specificities. almost all ifng+ pa -specific cells also express tnfa, but only about half of the ifng+ np -specific cells co-express tnfa. this was originally linked to the avidity of the responding population for the specific peptide/mhc complex, with the ifng+tnfa+ phenotype representing cd + t cells with higher avidity and a more differentiated phenotype. however, the same cytokine pattern is seen in adoptively transferred cd + t cells expressing a clonal tcr, implying avidity alone cannot control development of cytokine profiles. co-expression of ifng and tnfa by adoptively transferred cfse-labelled ot-i cells following infection with influenza a virus expressing ova - peptide shows a close correlation with division in vivo. early after antigen encounter ( - divisions) the vast majority of cells express only tnfa. after - divisions cells begin to co-express ifng and tnfa. the emergence of an ifng+tnfa-phenotype increases with subsequent divisions ( - divisions), indicating cytokine profile is closely linked to cell cycling, as described previously for both b cells and cd + t cells. titration of adoptively transferred ot-i cells, which controls the level of expansion in vivo, reveals that more cd + t cells develop an ifng+tnfa-phenotype with increased expansion. thus we conclude that while tcr avidity and co-stimulation can impact the differentiation of cd + t cells, expansion plays a very important role in the regulation of cd + t cell effector function. in addition to its chemo-attractant function, sdf- a (stromal-cell derived factor- a, cxcl ) has been described to costimulate cd + t cell during tcr triggering. our objective is to clarify the mechanism regulating this costimulatory activity. tcr-driven proliferation of human cd + t cells was increased by immobilized sdf- a to a level similar to that obtained with the costimulatory molecule cd . as visualized by real time confocal microscopy, t cells entering in contact with sdf- a formed a tether and displayed an active scanning activity. since sdf- a induced a similar activity in t cells stimulated with a sub-optimal dose of anti-cd mabs, it is conceivable that the sdf- a-driven scanning may favour productive tcr engagement. to test this hypothesis, we are studying the effect of sdf- a on tcr internalization, calcium mobilization, mapk activation and actin cytoskeleton reorganization. we are also studying the role of sdf- a in the context of cd + t activation by antigen-presenting cells secreting sdf- a. this study should help us to better define how sdf- a modulates cd + t cell activation beyond its chemo-attractant function. background: propolis, an ancient herbal medicine, is well known for the management of respiratory diseases. caffeic acid phenethyl ester (cape), an active component in propolis, is known to have anti-tumor, anti-inflammatory, and antioxidant properties. in this study, the effect of cape on the functions of t cells, which play the major role in chronic airway inflammation of asthma, was evaluated. method: cd + t cells isolated from human peripheral mononuclear cells by automacs were stimulated with anti-cd and anti-cd antibodies and cape for days. cytokine levels were dertermined by elisa and lymphoproliferation was analyzed by h-thymidine incorporation method. signaling pathway of t cells was studied by western blot. result: it was found that cape significantly inhibited ifn-g and il- production and lymphoproliferation in cd + t cells stimulated by anti-cd /cd . cape could inhibit nuclear factor-kb (nf-kb) activation, but not mitogen-activated protein kinase (mapk) family phosphorylation in t cells. cape could also inhibit akt phosphorylation. conclusion: these results indicated that cape inhibits cytokine production and lymphoproliferation of t cells which might be related to the nf-kb and akt signaling pathway. this study also provided a new insight into the mechanism of cape in immunology and the rationale for propolis in the treatment of allergic disorders. objective: upon activation, cd t cells express a variety of molecules on their surface, such as mhc-class ii, cd , cd , cd , whose ligands are constitutively expressed on resting t cells. whereas these molecules are physiologically expressed on antigen presenting cells, their function on t cells is not understood. we tested the hypothesis that activated cd t cells might induce t cell proliferation and differentiation from cd resting t cells through interaction of activationinduced surface molecules and their constitutively expressed ligands. methods: cd t cells from the peripheral blood of healthy donors were co-cultured with fixed activated t cells from the same donor. after days of co-culture, the phenotype of the resulting cells was analyzed by assessing their surface molecules and production of cytokines. results: cd memory t cells but not naive t cells proliferated in response to contact with activated t cells. these cells showed a mild activated phenotype assessed by the expression of cd , cd , and cd . analysis of the cytokine profile of these cells revealed the differentiation of il- -and ifn-g-double-producing cells in response to contact with th effector cells, and il- -producing cells in response to contact with th effector cells. the levels of produced cytokines were, however, significantly lower than those produced by activated cells in response to anti-cd /cd stimulation. whereas neutralization of ifn-g or il- during culture did not diminish the frequency of the arising cytokine-producing cells, separation of the responder cell population from effector cells by a transwell system led to a significant decrease of cytokine secretion. blocking particular receptor/ligand interactions by neutralizing antibodies against hla-dr, cd , cd , and cd could not prevent cytokine production induced by t-t cell interaction. however, simultaneous addition of all antibodies significantly inhibited cytokine production to - %. conclusion: interaction of cd memory t cells with activated t cells resulted in the production of the cytokines il- , il- , and ifn-g. given the immunomodulatory capacity of il- and il- , these findings might indicate a novel potential negative feedback mechanism to control t cell-driven immunity. a. nasir , s. thompson , j.j. murphy king's college london, division of immunology infection and inflammatory disease, london, united kingdom the murine bcl leukaemia cell line can be induced to undergo plasmacytoid differentiation in vitro with cytokines il- and il- and this is characterised by a marked reduction in proliferation and production of large amounts of secreted igm. these cells were observed to express significant levels of the zinc fingercontaining protein zfp l by western blot analysis. this protein is reported to act in post-transcriptional regulation of gene expression by binding to au rich elements (ares) of mrnas of certain genes and consequently promoting mrna degradation. at a cell functional level, zfp l has been described to have roles in apoptosis, proliferation and differentiation in different cellular contexts. cytokine-induced bcl differentiation was observed to be associated with downregulation of zfp l protein. in an attempt to determine whether zfp l downregulation was directly linked to bcl differentiation, a zfp l shrna expressing lentivirus (psicor) was employed to knockdown zfp l expression. this reagent downregulated zfp l expression very effectively . shrna infected cells proliferated less well than either control virus infected cells or wild-type cells with or without cytokines. zfp l shrna infected cells also produced more secreted igm per cell than either control virus infected cells or wild-type cells in the presence or absence of cytokines. these results are consistent with a role for zfp l downregulation in promoting bcl plasmacytoid differentiation. vidual lysates of peripheral blood lymphocytes (pbl) of patients with igg multiple myeloma and healthy controls were investigated for the expression of sialic acid (sa), galactose (gal) and n-acetylglucosamine (glcnac), the sugars known to specify the glycoforms of human serum igg. the degree of glycosylation and signaling status of all isolated myeloma igg bcrs were correlated and compared with the glycosylation of the igg paraproteins isolated from sera of the same patients. it was shown that bcr igg in myeloma is more heavily sialylated when compared with normal controls, that the increased sialylation of igg bcr is associated with higher levels of tyrosine phosphorylation (signaling activity) of the igg bcr supramolecular complex and that bcr igg and serum igg paraprotein from the same patient differed in all cases in the levels of terminal sugar expression. the results suggest that the development of the malignant clone in mm from postswitch b cells expressing igg bcr at their surfaces to plasma cells secreting igg paraprotein may be followed by permanent glycosylation changes in the igg molecules. caused by thapsigargin-induced release of calcium from the endoplasmic reticulum was insensitive to tpen. conclusion: the signal with fluorescent probes for the detection of calcium ions in response to thimerosal is entirely due to zinc release, and no indication for a calcium signal was detected. in light of these observations, zinc may also contribute to calcium signals caused by mercury containing compounds other than tms, and a potential involvement of zinc release in the immunomodulatory effects of these substances should be considered. although best known for its pro-apoptotic function, it seems clear now that cd (fas, apo- ) also exerts anti-apoptotic effects associated with costimulation and the induction of proliferation. we investigated effects of fas co-ligation during tcr/cd /cd -triggered activation of freshly isolated human t-lymphocytes. to this end, tcr-triggered cells were incubated in presence or absence of different ligand concentrations of anti-apo mab, faslfc or faslstrepfc fusion proteins, or leucin zipper (lz-)cd l. interestingly for all ligands tested, we could clearly demonstrate a correlation between ligand concentration and t cell response: low doses drastically augmented proliferation in the sense of costimulation, whereas high doses completely blocked tcr-induced cell proliferation without inducing cell death. the positive costimulatory effect of fas at low concentrations is associated with elevated il- and ifng production, upregulation of activation markers, adhesion molecules and cell-cycle regulating cdks and cyclins. in addition, we observed an increased activation of important signalling molecules including mapk and caspases. using pharmacological inhibitors, we demonstrate that fas is internalized upon ligation. we also observed an increased tcr internalisation following fas co-incubation potentially resulting in the generation of larger signalling platforms that allow optimal t cell activation. in stark contrast, most fas ligands at high concentrations almost completely inhibited cell proliferation of tcr-triggered lymphocytes. in this context, crucial events associated with t cell activation, i. e. tyrosine and erk / phosphorylation, the expression of various activation markers, the il- production and caspase activation were almost completely abrogated. these findings highlight that fas-triggering accelerates or blocks t cell activation, depending on the strength of the stimulus. in addition, we provide further evidence for an anti-apoptotic function of fasl during signal initiation in human t lymphocytes. sponsored by the dfg (sfb ) and the medical faculty kiel (to oj) it has been shown that glycosylation of cell surface proteins controls critical t cell processes, including homing, thymocytes maturation, activation, and cell death. plant lectins have been long used to study changes in cell surface carbohydrate structures, to identify leukocyte cell subsets, and as surrogates for authentic t cell activation stimulus. the galb , galnac-specific lectin from amaranthus leucocarpus (all) shows a differential binding pattern to murine thymocytes and peripheral cd + and cd + t cells. in addition, mitogenic stimulus increase -fold the all binding to cd + t cells. previous studies in human pbmc showed that all binds to human cd + t cells and all-binding increased after a mitogenic stimulus using total cell cultures as murine studies. these data suggest that all detects selectively activation-related changes in cd + t cell surface carbohydrate but none study has been performed to examine the all effect on human t cell activation. to examine the effect of all on human t cell activation, we analyzed the anti-cd -dependent activation of purified cd + t cells from pbmc in presence or absent of all by measuring proliferation using cfda-se staining, expression of the surface activation marker cd and calcium influx by flow cytometry. results showed that all did not induce significantly t cell proliferation or cd expression, but enhanced the anti-cd -dependent proliferation and cd expression of purified cd + t cells. analisis of calcium influx showed that all enhanced anti-cd dependent calcium influx. our findings indicated that all alone does not affect t cell activation but suggested that all induces a costimulatory effect on human cd + t cells by up-regulating t cell activation mediated by anti-cd stimulus, as further studies have to be performed to elucidate all-induced costimulatory effect. financed in part by papiit-unam (in ) a. the adaptor protein lat (linker for activation of t cells) has a prominent role in the transduction of intracellular signals elicited by the tcr/cd complex. upon tcr engagement, lat becomes tyrosine-phosphorylated and thereby recruits to the membrane several proteins implicated in the activation of downstream signaling pathways, leading to tightly equilibrated programs of activation and survival or induced cell death. the balance between cell survival and cell death is critical for normal t cell development and activation, and is maintained by signals through lymphocyte antigen receptors and death receptors such as cd receptor. it has been previously demonstrated that cd ligation in t cells induces the proteolytic cleavage of several adaptor proteins, including gads, slp- , slap- and lat. given the dual role of lat as a transducer of activation and negative signals in t cells, we have analyzed the role of the lat cleavage in t cell functions and studied the proteases responsible for this cleavage. objective: the study is designed to explore preliminarily the need of t cells for cytokines during the culture in vitro, which are associated with the activation, proliferation and apoptosis of t cells, and by detecting the expressions of il-rs, co-stimulatory molecules and apoptotic receptors/ligands onto human peripheral blood lymphocytes (hpbls). the results may lay a theoretic and experimental basis for developing the condition media qualified especially to t cell culture. methods: pbls were isolated , and cultured in different media. both immunocytochemistry staining and cell enzyme linked immunosorbent assay (celisa) were used to detect the expressions of il- r, il- r, il- r, il- r, il- r, il- r, cd , cd , cdw ( - bb), cd (fas) and cd (fasl) on hpbls in different cultured time, i. e. d, d, d, d, d, d, d and d. using typan blue staining, the living cells, dead cells and total cells of each cultured group were counted, then their cell growth curves were drawn out. to evaluate the cellular activity, growth situation and cell cycle of t cells, both mtt and fcm analysis were also performed separately. . the expressions of several membrane immune molecules on the lymphocytes in different cultured conditions. ) the expressions of membrane immune molecules before cultured. ) expressions of the membrane molecules on hpbls during culturing. % fbs rpmi group ( group), il- group, pha group... ( ) mtt assay. ( ) proliferative times and growth curves of hpbls... . during cultured in vitro, there are expression changes of the il- rs (il- ra, il- ra, il- rg, il- r, il- r, il- r, il- r), co-stimulatory molecules (cd , cd , - bb) and apoptosis associated molecules (fas/fasl) on hpbls in different time and cultured media. the expression patterns of the most molecules checked are similar in group, il- group and pha group, but the rests are different. . our data also suggest that the hpbls cultured in cd mcab+cd mcab+il +il a group has a great proliferative potential compared with the other groups. using this condition medium, may have a practical prospect to tumor therapy. . celisa will become probably an effective test to detect the expressions of membrane receptors or molecules quantitatively on a large scale. f. beceren-braun , r. tauber zentralinstitut für laboratoriumsmedizin und pathobiochemie, berlin, germany l-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates tethering of leukocytes to the endothelial surface during inflammation. apart from its role in adhesion, l-selectin functions as a signal transduction molecule. crosslinking of l-selectin with antibodies or ligand binding to the receptor have been shown to elicit a wide range of cellular responses. in addition to process signals coming from outside of the cell, the intracellular part of l-selectin (lscyto) is also able to conduct intracellular signals, e. g. activates tyrosine kinase p lck and the ras/rac signalling pathway ( ) followed by mitogen-activated protein kinases ( ) and c-jun n-terminal kinase ( ), which leads to an enhanced binding of l-selectin to soluble ligands ( ). in our previous work we described an association of lscyto with isozymes of the pkc family which phosphorylate the receptor on serine residues ( ). here we show that the protein phosphatase a inhibitor phapii is a novel direct interacting partner of the lscyto. we propose a model in which the l-selectin mediated signalling is regulated by the interaction of pkc, pp a and phapii: phapii binds to the unphosphorylated lscyto. upon l-selectin crosslinking lscyto is phosphorylated, pha-pii dissociates and inhibits the phosphatase pp a. in addition we have started structural analysis to investigate ligand binding induced conformational changes of the cytoplasmatic domain of l-selectin. v. heissmeyer , e. glasmacher helmholtz center munich, molecular immunology, munich, germany during self-antigen recognition, roquin dependent posttranscriptional downregulation of icos prevents t cell help to b cells and autoantibody production. the molecular mechanism by which roquin interferes with icos translation remained unclear. we have identified two critical regions in roquin. the amino-terminus is required for rna binding and can be functionally replaced by conserved sequences from its paralog mnab. the carboxy-terminus mediates p body localization and has specialized in roquin for efficient repression of icos in t cells. using knockout cells of dicer or ago - genes, we prove that roquin mediated repression of icos occurs in the absence of mirisc formation. instead, roquin function required intact p bodies, and was impaired after knockdown of lsm and rck or expression of dominant-negative gw . interestingly, roquin activity is blocked through induced mirisc formation implicating the mutual regulation of different mechanisms of posttranscriptional gene silencing in immune responses. s objectives: upon encountering their antigens, naï ve t cells are activated and driven to clonal expansion and differentiation into armed effector cells. according to the two-signal hypothesis, the induction of an optimal cd + t-cell immune response requires both antigen-specific and co-stimulatory signals. in contrast, stimulating naïve cd + t cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. thus, cd + t cells require additional signals for full activation and further differentiation into effector cells. methods: in this study, we adopted an in vitro approach to dissect the cellular and molecular requirements for cd + t-cell activation and differentiation. naïve cd l hi cd lo cd + t cells were sorted and stimulated by anti-cd and anti-cd antibodies. results: firstly, we show that the activation and differentiation of cd + t cells require il- provided by activated cd + t cells at the initial priming stage after stimulation. secondly, this critical il- signal is delivered through il rbg of cd + cells and is independent of il- ra. besides promoting cell proliferation, il- stimulation increases the amount of ifng and granzyme b produced by cd + t cells. conclusion: therefore, our studies demonstrate that a full cd + t-cell response is elicited by a critical temporal function of il- released from cd + t cells, providing mechanistic insights into the regulation of cd + t cell activation and differentiation. most antigenic peptides recognized by cd t lymphocytes are produced through degradation of intracellular proteins by the proteasome. however, some antigenic peptides are produced by a proteasome-independent pathway, which is poorly characterized. mage-a - is a tumor antigenic peptide presented by hla-a and widely used for vaccination of melanoma patients. we observed that proteasome and tppii inhibitors failed to block presentation of the antigen by tumor cells. however, processing of this peptide occurred in the cytosol because tap inhibition prevented its presentation. to characterize the cytosolic peptidase producing mage-a - we setup an in vitro digestion assay using a -mer precursor peptide encompassing the sequence of the antigenic peptide. we observed that only the cytosolic fraction was able to produce the antigenic peptide from this precursor. this production was abolished by treating the cytosolic fraction with o-phenanthroline, a broad-spectrum inhibitor of metallopeptidases. this inhibitor also blocked the presentation of mage-a - by tumor cells. by electroporating hla-a cells with a precursor peptide blocked at the c-and the n-terminus, we could exclude the involvement of exopeptidases in the processing of this peptide, and conclude to a major role of a cytosolic metalloendopeptidase. one such enzyme is insulin-degrading enzyme (ide). we observed that depletion of ide abolished the capacity of a cytosolic fraction to produce the antigenic peptide. furthermore, recombinant ide was able to produce the peptide in vitro from the precursor peptide. lastly, silencing of ide with sirna reduced presentation of the peptide by tumor cells. with tppii, ide is the second example of a proteasomealternative pathway in the production of class-i restricted peptides. antigen-specific t cell based tumor immunotherapy, though extensively studied, has only been of limited clinical success so far. immune escape, due to impairment of hla dependent tumor epitope presentation is believed to be one major reason for this failure. to identify novel mechanisms by which tumors can become refractory to immune elimination, human melanoma cells of different donors expressing the transmembrane mart- /melan-a tumor antigen were exposed to two or three rounds of brief co-culture with mart- /melan-a - specific cytotoxic t lymphocytes (ctls). immune selected melanoma cell clones, being resistant to lysis by mart- /melan-a - ctls due to impaired epitope processing were further investigated. our results show that in addition to previously described immune evasion mechanisms like down regulation of mhc class i and mart- expression, the ifn-gamma independent endoplasmic reticulum associated degradation (erad) pathway is crucial for mart- /melan-a - epitope generation. moreover, deregulation of several erad components is essentially responsible for the observed immune escape of the immune selected melanoma cells. in support, re-expression of down-regulated erad components in ctl-resistant melanoma cells completely restored immune recognition by mart- /melan-a - ctls. thus, our studies demonstrate for the first time that erad not only plays a central role in the production of cd + t cell epitopes from membrane proteins but also contributes to tumor escape mechanisms by cancer immunoediting. studies of t cell responses to hen egg lysozyme suggest that several conformers of peptide-mhc class ii complexes can be generated for a single peptide epitope and that distinct cd t cell repertoires known as type a and type b recognise these different conformers (lovitch and unanue, immunol rev : - , ) . type a t cells recognise peptide-mhc complexes generated from intact proteins after intracellular antigen processing under h -m (dm) control, where as type b t cells respond to synthetic peptides in the absence of dm editing, but fail to respond to processed intact protein. type b t cells escape thymic deletion in mice (petersen et al., immunity : - , ) , with implications for autoimmunity. so we studied whether type a and type b recognition patterns occur in t cell responses to autoantigens such as the rheumatoid arthritis (ra)-associated proteoglycan aggrecan, and whether naturally occurring extracellular ligands that activate type b t cells are found in inflamed joints. lymph node cells from aggrecan-immunised balb/c mice proliferated in response to intact aggrecan and to the immunodominant peptide - , whereas peptide-immunised mice responded to peptide, with low or absent responses to intact aggrecan. t cell hybridomas generated from - peptide-immunised mice either recognised peptide only (the majority) or peptide and intact aggrecan (the minority), a pattern consistent with type a and type b t cell recognition. responses to staggered and alanine-substituted peptide sets showed that type a and b t cell hybridomas recognized the - epitope in the same register, consistent with this peptide epitope binding to mhc in distinct conformers. type b t cell hybridomas recognised aggrecan fragments in supernatants from cartilage degraded by stimulation with proinflammatory cytokines that induce raassociated aggrecanases. our data suggest that inflammation generates extracellular peptides that activate type b t cells. we are also characterising human type b t cell responses as well as searching for type b t cell ligands in synovial fluid from ra patients. we propose that extracellular cartilage degradation generates ligands that induce autoreactive type b t cell responses which participate in the pathogenesis of autoimmune arthritis. a to cope with mhc i antigen presentation hcmv encodes for several post-translational strategies which have been extensively studied in transfected cells. in this study we analysed the plc in naturally hcmv-infected cells and monitored the composition of the plc throughout hcmv replication. metabolic labeling experiments revealed the absence of tapasin incorporation into the plc. in contrast, western blot analysis demonstrated only a slow decline of tapasin steady state levels in infected cells, suggesting a blocked synthesis rather than degradation. tapasin mrna levels were found to be continuously downregulated during infection, however, the tapasin transcripts were stable and long-lived. taking advantage of a novel method, in which newly transcribed rna is selectively labeled and analysed (dölken et al, ), we found, after an initial induction at hrs p. i., a strong inhibition of tapasin transcription at hrs p. i. furthermore, also reduction of tap and tap transcription was observed contrasting to the elevated levels of erp and mhc i transcripts. importantly, ectopic expression of tapasin restored the incorporation of tapasin into the plc in hcmv-infected cells. the data indicate that hcmv controls mhc i antigen presentation also on a transcriptional level and show for the first time the regulation of tapasin transcription as a viral immune evasive function. most peptides presented by mhc class i molecules are produced by the proteasome during degradation of intracellullar proteins. two main proteasome types have been described, differing in their content of catalytic subunits. the standard proteasome comprises catalytic subunits ß , ß and ß , which are replaced by their ifng-inducible counterparts ß i, ß i and ß i in the immunoproteasome. the thymoproteasome represents a third proteasome type, where catalytic subunit ß i is replaced by a thymus-specific subunit ß t. the standard proteasome is present in most tissues, the immunoproteasome in found in cells exposed to ifng and in dendritic cells, while the thymoproteasome is found exclusively in the thymus. we produced a panel of novel antibodies that recognize subunits ß i, ß i, ß i and ß in their native form. using these antibodies for successive immuodepletions performed on tumor lysates, we identified two new proteasome types that are intermediate between the standard proteasome and the immunoproteasome, i. e. they contain only one or two the three catalytic subunits of the immunoproteasome. one comprises ß ,ß and ß i (single intermediate proteasome), and the other comprises ß i, ß and ß i (double intermediate proteasome). we quantified these intermediate proteasomes in a series a tumor lines of various origins, and found that they represent - % of the total proteasome content of those tumor cells. they are also present in dendritic cells, where they represent about % of the proteasome content. we characterized the activity of these intermediate proteasomes, not only on fluorogenic substrates but also on actual antigenic peptides recognized by anti-tumor ctl. with respect to antigens known to be processed differently by the standard and the immunoproteasome, the intermediate proteasomes often behaved like the immunoproteasome. importantly, we identified two tumor antigens that are processed exclusively by either the single intermediate proteasome ( tapasin is a multi-functional protein dedicated to mhc-i biosynthesis; it serves as a structural component in the so called mhc-i peptide loading complex (plc), as a chaperone putatively acting as an active peptide editor and mhc-i quality control mechanism, as an er retention signal for immature mhc-i, and as a chaperone stabilizing tap expression and increasing tap-performance. furthermore, tapasin has been found outside the er, where it has been suggested to regulate retrograde transport of escaped immature mhc-i back to the er from the trans-golgi compartment. the role of tapasin as an active peptide-editor has been debated and we here set out to study the effect of tapasin on binding of peptides of both high-and low-affinity to a human mhc-i allele (hla-a* ) using protein interaction-and peptide-competition assays. specifically we wanted to in detail compare the binding of two peptides of the same affinity. at high concentrations all of the tested hla-a* binding peptides (tap-transported high-affinity peptide (ttp-ha), signal-peptide of high affinity (sp-ha), tap-transported mediumaffinity peptide (ttp-ma)) induced dissociation of hla-a* from tapasin, but only ttp-ha dissociated hla-a* from tapasin at lower concentrations. using peptide-competition assays against ttp-ma, a peptide of lower affinity, we could show that ttp-ha, one of the two peptides of equally high affinity was a significantly more efficient competitor than peptide sp-ha. however, analysis of mhc-i peptide loading in the tapasin-negative cell line lcl- . -a showed no competitive advantage of ttp-ha compared to sp-ha supporting a role for tapasin as a selective facilitator of mhc-i peptide binding. in conclusion, we here show that peptides of different affinities dissociate hla-a* from tapasin in a dose-dependent manner, and that tapasin facilitates ttp-ha, but not sp-ha replacement of a lower-affinity peptide (ttp-ma). together these data strongly suggest a role for tapasin as a selective facilitator of peptide binding to mhc-i. importantly, this study implies that criteria in addition to peptide-affinity determines whether tapasin will promote peptide binding to hla-a* . m. basler , , c. lauer , m. groettrup , biotechnology institute thurgau, kreuzlingen, switzerland, university of constance, division of immunology, department of biology, konstanz, germany two lmp -dependent antigens have been described that relied on the 'structural presence' of lmp in the proteasome but not on the activity of lmp . here we have investigated processing of the h- d b -restricted uty - epitope of the male minor antigen uty reported to be lmp -dependent. using splenocytes from lmp -/-, lmp -/and mecl- -/mice we found that the uty - epitope requires lmp and lmp but not mecl- . curiously, a selective lmp inhibitor did not interfere with uty - presentation. objective: we investigated why the deletion but not the inhibition of lmp interferes with uty - presentation. we hypothesized that the 'structural' requirement for lmp is based on replacement of the caspase-like activity of b in the proteasome. methods: it was determined if t a mutants of lmp and/or b can rescue the uty - epitope. we used a b -selective inhibitor to determine if the inhibition of the caspase-like activity of b preserves the epitope. finally we determined by mass spectrometry if the uty - epitope embedded within a mer precursor peptide is differentially cleaved by lmp -deficient and proficient immunoproteasomes in vitro. results: we found that t a mutants of lmp and b rescue presentation of uty [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . also inhibition of cells with a b -selective inhibitor preserves uty [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] presentation. an aspartate in position of the uty - sequence wmhhnmdli is preferentially used as a cleavage site by lmp -deficient but not half as frequently by lmp -proficient immunoproteasomes. the generation of the uty - epitope relies on the replacement of the caspase-like activity of b by lmp because the b activity destroys the uty - epitope. this is the first example for the 'structural' requirement of lmp for generation of an epitope. eliminating the activity of their constitutively expressed homologous subunits may explain the requirement for immuno-subunits of the proteasome also for the generation of other antigens. thus we have discovered a so far unrecognized mechanism how lmp and perhaps also lmp and mecl- exert their function in antigen processing. a. linnemann , a. musiol , r. lindner hannover medical school, cell biology, hannover, germany, hannover veterinary school, graduate school for biomedical sciences, hannover, germany objectives: mhc i molecules are constitutively endocytosed and recycled to the cell surface. this process is required for the turnover of aged molecules and for some forms of cross-presentation of exogenous peptides on mhc i. in fibroblasts, mhc i is known to internalize via a clathrin-independent, arf -regulated pathway that is highly sensitive towards the cholesterol-sequestering drug filipin. although this observation suggests that membrane rafts are involved in the internalization of mhc i, no evidence for an association of mhc i with membrane rafts has been found in this cell type. methods: a novel detergent extraction protocol was used to investigate the association of mhc i with membranes rafts. endocytosis of mhc i was measured with a biotinylation-based biochemical assay and with a cell biological assay employing confocal laser scanning fluorescence microscopy. for characterization of mhc i internalization pathways, dominant negative mutants of gtpases (dynamin and arf ) were overexpressed in t fibroblasts. we show that antibody-mediated oligomerization of mhc i in t fibroblasts shifted this molecule from soluble fractions to detergent-resistant membranes. this change in detergent resistance coincided with a switch to a novel internalization pathway: oligomerized mhc i internalized faster and more completely and arrived at different endocytic organelles. the two mhc i internalization pathways differed in their sensitivity towards dominant negative arf : endocytosis of oligomerized mhc i was not affected, whereas non-oligomerized mhc i endocytosed more slowly and changed its subcellular distribution. unlike transferrin receptor internalization, none of the mhc i endocytosis pathways was affected by overexpression of dominant negative dynamin suggesting internalization mechanisms independent of clathrin, caveolin and rhoa. conclusion: we propose that mhc i switches from an arf -regulated to a novel, arf -independent internalization pathway in response to a change in membrane environment induced by oligomerization of mhc i. since mhc i is one of the cellular receptors for sv virus and since sv binding triggers mhc i oligomerization, this novel pathway may be involved in sv uptake. antigen cross-presentation in dendritic cells is a complex intracellular membrane transport process, but the underlying molecular mechanisms remain to be thoroughly investigated. in this study, we tested the effect of sirna-mediated knockdown of rab gtpases, the key regulators of membrane trafficking, on antigen cross-presentation. twelve rab gtpases were identified to be associated with antigen cross-presentation, and among which rab b, c were found to be colocalized with mhc class i molecules at perinuclear tubular structure. tracing with fluorescence protein tagged beta -microglobulin demonstrated that the mhc class i molecules were internalized from plasma membrane to rab b and rab c postitive compartment. moreover, the recycling ligand transferrin was enriched in the rab b or c positive vesicles. furthermore, the rab b, c positive compartment were colocalizd with a fraction of rab a at a juxtaposition of phagosomes. together these data demonstrate that rab b and rab c positive vesicles is involved in and may constitute the recycling compartment of exogenous antigen crosspresentation. introduction: while the proteasome is thought to generate most of the hla peptidome, other proteases were also proposed to be significant for this process. both t cell based assays and proteasome inhibitors were used in the past to follow presentation of specific model hla peptides. the hla-peptidomes presented at the cell surface depend on the rate of peptide generation within the cells, their transport from the cytoplasm and loading in the er, binding stability at the cell surface and retrograde uptake of the hla molecules back into the cytoplasm. objectives: the role of the proteasome in hla peptide presentation was evaluated using proteasome inhibition, while following the turnover rates of the entire hla peptidome. the peptidomes of both the authentic membranal and a recombinant soluble form of the hla molecules were collected for analysis at different time points after the inhibition of the proteasomes. the turnover rates of the hla-peptides were followed using pulse-chase analysis with stable-isotope labeled amino acids concurrently with epoxomicin treatment. the hla molecules were immunoaffinity purified and the peptides were analyzed by capillary chromatography and orbitrap tandem mass spectrometry. both the endogenous membranal and soluble mhc molecules were studied in parallel from the same cells. peptides were identified by their ms/ms fingerprints and the turnover rates were determined by the shift from the 'light' to the 'heavy' leucine of each peptide. results: a few thousands hla-peptides were identified, and for a large portion of them, the turnover rates could be defined. proteasome inhibition did not affect the complexity of the hla peptidomes or reduced significantly the amounts of membranal hla molecules. many peptides were labeled relatively rapidly with heavy leucine, indicating that the hla peptidome contains also the products of newly synthesized and rapidly degrading proteins. the source proteins of the hla peptides seemed to have similar biological functions and cellular origins in both the inhibited and untreated cells. the centrality of proteasomal degradation in hla-peptide presentation is put into doubt and the role of the proteasome in the generation of each peptide and each cleavage site can be defined. epstein-barr virus (ebv) is a ubiquitous y-herpesvirus, infecting over % of adults worldwide. it can cause mononucleosis and several lymphomas and carcinomas, reflecting the tropism of the virus for b-lymphocytes and epithelial cells. ebv persists for life despite the presence of virus-specific adaptive immunity, indicating that it has evolved strategies to counter the host immune response. one such strategy is the persistence of the virus in the latent phase of its life cycle, where expression of viral proteins is minimized. however, for ebv replication and dissemination to occur, it must enter the lytic phase. here, over viral proteins are expressed, creating many potential antigens for presentation to cytotoxic t -lymphocytes. ebv can circumvent possible eradication by cd + t lymphocytes during the lytic phase by interference with antigen processing and presentation through hla class i in the infected cell. the viral proteins bnlf a and bglf have been shown to achieve this by impairing peptide-loading of hla class i and inducing the degradation of mrnas encoding hla molecules, respectively. a third ebv lytic phase protein, the g-protein coupled receptor (gpcr) bilf , has now been found to down-regulate cell surface hla class i expression (zuo et al, plos pathogens ). this represents a novel function for a virally-encoded gpcr. bilf is expressed early in the ebv lytic cycle and is localized predominantly at the cell surface. there it can interact with hla class i molecules, resulting in their internalization and lysosomal degradation. this has a profound effect on the ability of cytotoxic t-lymphocytes to recognize cells displaying antigens derived from ebv proteins. interestingly, bilf displays a differential effect on distinct hla class i haplotypes. furthermore, we have shown that the intracellular c-terminal tail of bilf is required for its effect on hla class i expression. however, the ability of the gpcr to activate intracellular signaling pathways is dispensable in this regard. thus, by reducing the cell surface expression of hla class i molecules, ebv bilf can hinder the recognition of virally-infected cells by cytotoxic cd + t lymphocytes, thereby facilitating the evasion of adaptive immune mechanisms. t lymphocytes mature in the thymus, generating a non-dangerous t cell repertoire. for the adquisition of tolerance, thymocytes suffer positive and negative selection processes. during t cell maturation, tcrs contact with different mhc-peptide complexes on the surface of pressenting cells, allowing tolerization against self proteins. to obtain a non-self-reactive t cell repertoire, it is of most importance that pressenting cells in the thymus express a repertoire of mhc-peptides complexes representative of the proteins that t cells will found in periphery, including tissue restricted antigens (tras)-derived peptides. in the last decade, transcription of tras in thymus has been well-reported. furthermore, the expression of many genes codifying for tras are dependent.on the expression of the autoimmune regulator (aire). aire is mainly expressed in medullar thymic epithelial eells (mtecs), which are involved in negative selection. so far no systematic study have been made to describe the peptide repertoires associated to hla molecules in the thymus. in addition, although many data of tras transcription in thymus have been reported, much less work has been performed at biochemical level, and to our knowledge, no hla ligand arising from any tra have been reported in thymus. in this report we present the results of analyzing the hla-dr-associated peptide repertoire from whole tissue samples of different human thymi by mass spectrometry. we describe natural ligands, including two peptides derived from semenogelin- , a tissue restricted antigen expressed mainly in the prostate, and present in semen. using qpcr we demonstrate that semg is transcribed in thymus from both male and female individuals. finally, we detected the semg mrna expression in a fraction enriched in stromal cells, but not in the thymocyte fraction of the thymi. the proteasome is the major protease complex for non-lysosomal protein degradation in eukaryotic cells, which generates most peptides for mhc class i antigen presentation. vertebrates express two sets of catalytic subunits, constitutive (beta , beta , beta ) and immuno-subunits (beta i, beta i, beta i). deficiency in beta i results in profound reduction of mhc class i expression, demonstrating the significance of this subunit for efficient antigen presentation. currently, this is attributed to the specific proteolytic activity of the beta i subunit, its role in the maturation of immunoproteasomes or both. however, re-expression of catalytically inactive beta i subunits is capable to rescue antigen presentation suggesting that the proteolytic activity of this subunit is not limiting in this process. here, we show that following infection with listeria monocytogenes induction of beta i expression increases the cellular proteasome content in the infected organs. our results indicate that this is due to the high chaperone activity of its propeptide which drives proteasome neosynthesis and thus enhances the overall proteasome quantity. further, mhc class i antigen presentation on beta i-deficient cells could be restored by treatment with d t, which increases the amount of proteasomes independent of beta i via induction of mixed proteasomes containing beta i, beta i and beta . consequently, not the lack of the specific proteolytic activity of beta i or immunoproteasomes, but the reduced proteasome quantity in beta i deficient cells is the major limiting factor for mhc class i cell surface expression. . we have previously shown that lc, in contrast to ddc, do not express cell surface tlr , and , which results in their inability to respond to both gram-positive and gram-negative extracellular bacteria in terms of maturation into immuno-stimulatory cells and production of inflammatory cytokines. therefore, the question remained what the role is of lc in class ii mhc-mediated activation of anti-bacterial t cells. we determined the capacity of ddc and lc to internalize and process whole bacteria and present bacterial antigens to cd + t cells. in vitro generated lcs and ddcs were cocultured with gfp-expressing bacteria and subsequently analysed by clsm and facs for their uptake capacities. furthermore we investigated their capacity to stimulate autologous bacteria-specific t cell lines as a measure for antigen presentation. results: we found that lc are principally able to internalize bacteria, but far less efficient than ddc. moreover, visualisation of bacterial uptake by em revealed different uptake mechanisms by lc and ddc. both in lc and ddc internalized bacteria were detected in the endosomal and lysosomal compartments of the mhcii processing route. nevertheless, presentation of bacterial antigens by lc on mhcii was inefficient compared to that of ddc, as indicated by a low capacity to activate autologous bacteria-specific cd + t cells. the presence of exogenous tlr and tlr ligands did not overcome the differences between lc and ddc, indicating that the impaired capacity to internalize and process bacteria and activate bacteria-specific t cells is not due to the lack of tlr signalling or insufficient expression of co-stimulatory molecules, but could be an intrinsic characteristic of lc. conclusion: we propose that the epidermis of the skin is an immune-privileged site where lc play a minor role in anti-bacterial immunity and may play a role in inducing tolerance to the bacterial skin flora by steady-state presentation of antigens from commensal skin bacteria. e. james , i. bailey , t. elliott university of southampton, cancer sciences division, southampton, united kingdom regulatory t cells (tregs) play a pivotal role in the suppression of tumour specific t cell responses. depletion of tregs in balb/c mice results in a robust immunity to the normally poorly immunogenic ct colon carcinoma. this response is long lasting and mediated by both cd and cd t cells. importantly, the treg depleted ct specific immunity is cross-protective; capable of mediating rejection of tumour lines of different histological origins (a , c , bcl , renca) implying a broader repertoire of response. we have characterised one of these cross-protective antigens, gsw , which is h -d d restricted. analysis of the generation of gsw in ct revealed that the peptide is susceptible to over-processing by the er-resident aminopeptidase eraap. inhibition of eraap in ct cells substantially increased the amount of gsw present, observed by increased t cell responses to the tumour in vitro and hplc analysis. this increase was in spite of an overall reduction of mhc class i molecules at the cell surface. to investigate whether the increase in immunogenicity following knockdown of eraap would protect mice, we generated stable eraap knockdown (kd) ct and immunised balb/c mice. greater than % of mice injected with eraap kd ct were found to reject the tumour. analysis of t cell responses revealed the presence of gsw -specific t cells, however, these responses were small ( . - %). this compared to a much larger response to ct (˚ %). preliminary results indicate that the majority of the t cell responses (non-gsw -specific) in these mice are directed toward unstable peptide/mhc complexes, possibly indicating presentation of n-terminally extended peptide antigens. this highlights manipulation of the peptide repertoire as a potent tool for the generation of t cell responses in vivo. minor histocompatibility antigens play important roles in the outcome of stem cell and organ transplantation as they are involved in the development of graftversus-host-disease and in the graft-versus-tumor reactivity in hla-identical stem cell transplantation [ ] . the di-allelic hla-a restricted minor histocompatibility antigen ha- locus codes for the highly immunogenic ha- his and the non-immunogenic ha- arg nonapeptides, differing in one amino acid. the only difference that could explain the absence of the ha- arg immunogenicity was the estimated numbers of cell surface presented copies i. e. /cell for ha- his and less than / cell for ha- arg [ ] . as ha- his/arg is hematopoietic system specific and shows additional expression on epithelial cancer cells while absent on the normal epithelial cell counterpart, the ha- his allele is currently used for boosting the graft-versus-tumor responses after hla matched ha- mismatched stem cell transplantation. to elucidate the mechanisms underlying the differential cell surface presentation of the ha- allelic peptides, we investigated the impact of the ha- his/arg polymorphism on molecular and cellular processes involved in the intracellular generation and stable cell surface presentation of hla class i-bound peptides. therefore, proteasome-mediated digestion experiments, tap translocation analyses, and hla-dissociation assays with ha- his and ha- arg peptides were performed. moreover, the crystal structures of hla-a in complex with either ha- his , ha- arg or a ha- variant with a citrulline residue at position were determined in order to obtain atomic level insights into the conformation of the hla-a /ha- peptide complexes. our results exclude a role for antigen processing in preventing ha- arg to be presented at the cell surface and both the structural and hla-dissociation data clearly show that the lack of cell surface expression essentially results from an increased instability of the ha- arg allele in the hla-a peptide binding groove [ ] . they provide a rationale for the lack of ha- arg peptide immunogenicity essential for the choice of tumor peptides for stem cell based immunotherapeutical application. proteasomes play an important role in mhc class i antigen processing. exposure of cells to proinflammatory cytokines such as tnfa or ifng leads to the expression of three facultative catalytic proteasome subunits (i. e. immunosubunits) that replace the constitutively expressed subunits in the cellular proteasome population. immunoproteasomes generate many pathogen-derived cd t cell epitopes with high efficiency and thereby shape the specificity of the pathogen-specific cd t cell response. on the other hand, immunosubunit expression is not essential for development of cd t cell-mediated protective immunity, thus the physiological relevance of these cytokine-induced proteasome subunits remains unclear. we observed that mice that lack the immunosubunits lmp (ib ) and mecl- (ib ) develop a variety of autoimmune responses, including a latent form of t d (or insulin dependent diabetes mellitus, iddm), following irradiation and bone marrow reconstitution. iddm development in these mice is characterized by inflammation of the islets of langerhans, glucose intolerance and increased water consumption, and is dependent on the presence of cd but not cd t cells. a cd t cell epitope, encoded by the islet beta cell-expressed "islet-specific glucose- -phosphatase catalytic-subunit-related protein" (igrp) mrna, was identified as an important target of the cd t cell response. this epitope, like many other known diabetes-associated epitopes, binds its presenting mhc class i molecule with low affinity. as t cells specific for low affinity binders most likely can escape central and peripheral tolerance while t cells specific for high affine binders do not, we postulate that inflammation-induced immunoproteasome expression primarily functions to replace self-peptides that are derived from tissue-associated antigens and bind mhc class i molecules with low affinity, by a higher affine peptide species towards which t cell tolerance exists. thus, the inducible proteasome subunits may play an important role in immune regulation, by removing the targets of potential auto-immune cd t cells that enter inflamed tissues. endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express hla-ii molecules with compact conformation and are therefore expected to stably bind autologous peptides. the role of these molecules is not known but they could be involved in the maintenance and regulation of the in situ autoimmune response. to study in situ t cell responses without characterizing self-reactive t cells, we have identified natural hla-dr-associated peptides from autoimmune organs that will help finding peptide-specific t cells in situ. here we report the first analysis of hla-dr natural ligands from ex-vivo graves' disease-affected thyroid tissue. using mass spectrometry, autologous peptides were identified from hla-dr-expressing cells, including thyroid follicular cells, some corresponding to predominant molecules of the thyroid colloid. most interestingly, eight of the peptides derived from a major thyroid autoantigen, thyroglobulin. cell-free in vitro binding assays were performed with the thyroglobulin peptides and some other thyroid-eluted peptides as controls, to identify to which hla alleles were these peptides associated in vivo. all but two of the thyroglobulin peptides showed low binding with the corresponding alleles. the two peptides with relatively high binding affinity were presented in the context of dr and dr . analyzing the digestion patterns used for the generation of the thyroid peptides, a preferentially cleavage after a lys and arg was observed for all of them, independent of the restricting allele. our data demonstrate that although the hla-dr-associated peptide pool in autoimmune tissue mostly belong to abundant ubiquitous proteins, peptides from autoantigens are also associated to hla-dr in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response. the t cell response generated following herpes simplex virus type (hsv- ) infection is known to be crucial in the clearance of replicating virus and in limiting the severity of infection. despite this, the relative contributions of cd + and cd + t cells in hsv- immunity have yet to be clearly elucidated. to better understand the role of hsv- -specific cd + t cells in immune control we have identified a amino acid epitope derived from glycoprotein d of hsv- . following flank infection, gd-specific cd + t cells were first detected in the draining brachial and axillary lymph nodes (ln) -days post-infection (pi), peaking at day and declining thereafter. gd-specific cd + t cells were first recovered from the spleen, skin and dorsal root ganglia (drg) at day pi and peaked at day . while hsv-specific t cells were first observed in the draining ln at day pi, hybridoma assays showed ex vivo presentation of the gd epitope by brachial ln cells as early as days pi, with peak activity days pi before declining to background by day . however presentation of the gd epitope was much more prolonged in vivo as proliferation of transgenic gdspecific cd + t cells was observed up to days post-infection in the brachial ln. ex vivo analyses suggest that only cd c + cells were involved in gd antigen presentation at days , and post-infection. subdivision of dendritic cells (dcs) populations indicated that both skin-derived dcs and cd a + dcs can present the gd antigen to cd + t cells at day pi, whereas by day pi the skin-derived dcs were the predominant population presenting the gd epitope. together these data show that following hsv- infection, antigen presentation is initiated rapidly and persists well after clearance of replicating virus. furthermore, we present evidence that different dc populations have distinct roles in the presentation of viral antigens and that they may vary during the course of infection. complementary zippers induced complete dimer formation, whereas identical zippers impaired stable interactions of the tagged peptidases. we also verified that the zippers did not influence the substrate "preferences" of the respective erap. our results from in vitro digestions suggest that the stabilised heterodimer is significantly more efficient in the production of a model epitope than the mix of monomeric erap and erap unable to form dimers. this observation is not due to mere thermodynamic stabilisation but involves positive cooperative effects in the heterodimers. conclusion: allosteric interaction of erap /erap in heterodimeric complexes enhances the global efficiency of precursor peptide trimming in the human er. during the biogenesis of class i molecules, newly synthesized heavy chains fold and acquire disulfide bonds while interacting with the lectin-chaperone calnexin (cnx) and its associated thiol oxidoreductase erp . upon assembly of the heavy chain with b m, the class i molecule enters a peptide loading complex (plc) that consists of the tap transporter, tapasin, the calnexin homologue calreticulin (crt) plus associated erp . both crt and erp are required for efficient assembly of peptide-loaded class i molecules and their subsequent expression at the cell surface. we examined functional sites on crt and erp to gain insights into their mechanisms of action in class i biogenesis. for crt, its lectin function is thought to be crucial for its association with class i molecules. however, when crt mutants lacking lectin function were expressed in crt-deficient cells, they completely complemented all class i biosynthetic defects. thus polypeptide-based contacts either mediated through erp or directly between crt and the heavy chain are sufficient to effect the chaperone and quality control functions of crt in class i biogenesis. we also tested the notion that erp must be recruited by cnx or crt to function on class i molecules. we found that the rates of heavy chain disulfide formation were normal in cells lacking cnx, crt or both chaperones. furthermore, an erp point mutant that fails to bind to cnx or crt was just as effective as wild type erp in normalizing rates of disulfide formation. we conclude that erp does not require recruitment by cnx or crt and likely acts directly on class i heavy chains to promote disulfide formation. furthermore, in cells expressing the erp point mutant, class i heavy chains, crt and the tapasin-erp disulfide conjugate were present at normal levels in the plc, indicating that the interaction between erp and crt is not required for plc assembly. finally, we show that mutations that destroy the enzymatic function of erp have no effect on plc stability or class i surface expression, suggesting that erp plays a structural as opposed to catalytic role in plc function. autoimmune pancreatitis (aip) underlies - % of cases of chronic pancreatitis and is characterized by prominent lymphocytic infiltration. a strong association of aip with the hla-drb * /dqb * haplotype has been reported, but identification of the predisposing hla gene(s) has been precluded by strong linkage disequilibrium. here, we show that hla-dr* transgenic ab nod mice suffer from aip and additional pneumonitis after sublethal irradiation and adoptive t cell transfer from syngenic donors, leading to complete pancreatic atrophy. pancreas histology is characterized by destructive infiltration of the exocrine tissue with cd + and cd + t cells, b cells and macrophages. mice with complete pancreatic atrophy have reduced serum lipase activity, develop fat stools and loose weight on regular chow. hla-dr* transgenic mice (cd + t cell competent) develop aip even unprovoked, similar to ab nod mice (cd + t cell deficient), while hla-dr* , hla-dq or hla-dr* /dq (double-) transgenic controls all remain normal after same treatment. we conclude that hla-dr* fails to protect from aip, likely due to defects in the induction of cd + regulatory t cells. our results identify hla-dr* as a prominent risk factor for aip on the hla-drb * /dqb * haplotype. this humanized mouse model should be useful to study mechanisms that underlie the hla association of autoimmune diseases, but also immunopathogenesis, diagnostic markers and therapy of human aip. s. khan , c. britten , h. overkleeft , g. van der marel , k. melief , d. filippov , f. ossendorp leiden university medical center, section tumorimmunology, leiden, netherlands, leiden university, biosynthesis group, leiden, netherlands objective: we have targeted peptide antigens to dendritic cells by the use of synthetic peptides chemically coupled to synthetic tlr ligands to study the impact on mhc class i and class ii antigen presentation. the potency of the vaccine was addressed by monitoring antigen presentation, priming of t-cells and tumor protection. results: our data show that this type of targeting of peptides greatly improves antigen presentation and t-cell priming compared to free peptide. vaccination of mice with the tlr-ligand peptide conjugates induced high numbers of functional cd and cd t-cells that could protect mice for aggressive melanoma. this potency relies on tlr signaling since peptide coupled to a non-functional tlr ligand was unable to support induction of specific t-cells. these data indicate that simultaneous encounter of antigen and a maturation signal are crucial for optimal t-cell activation by dendritic cells, and show the potency of tlr-l peptide conjugates as a vaccine modality. y. shi , , x. hu , , a. kawana- tachikawa objectives: nef protein of human immunodeficiency virus (hiv) holds some important immunodominant ctl epitopes. two overlapping -mer and -mer epitopes (rypltfgwcf (nef - ) and rypltfgw (nef - )) were found to be presented by hla-a* and some immune escape mutants of these two epitopes have also been found in some patients, e. g. y f, y w, t c, f l, w r, f r, f y etc. or their combinations. it's important to study the molecular basis of the peptide being displayed on the cell surface, through which we can analyze the mechanism of immune escape of hiv. methods: refolding method was used to attain the soluble protein pmhc. crystals are grown using hanging drop vapor diffusion method and x-ray diffraction technology is used to determine the structure. we have determined six peptide-mhc(pmhc) structures containing nef - (wild type) and its four mostly common immune escape mutants (y f, t c, y f&t c, f l), and also nef - (wild type). we found that there was little difference between the nef - (wild type) and nef - (y f) when they were displayed in the peptide-binding groove of mhc molecule, except water molecule distribution near the anchor residue y or f . interestingly the central bulge region of the peptide was becoming very flexible for the nef - (t c) and nef - (y f&t c), which may affect the binding of peptide and the recognition of t cell receptor. for nef - (f l), the side chain of l was more flexible compared to the nef - (wild type). alignment of the nef - and nef - showed that the nef - became flat and the side chain of f was not solvent-exposed due to shortening of the length of the peptide. conclusion: as the peptide nef - was featured, while the peptide nef - was featureless, so the different topology of these two epitopes indicates that they have different tcr repertoire diversity in hiv-specific responses. different immune escape mutants of nef - was using different strategies to avoid the killing of host ctls, which indicates that the therapy strategy based on the cellular immune response should be diversity. for the in vivo or ex vivo activation of antigen-specific t cell responses long synthetic peptides are used to activate both cd + and cd + t cells. in this study we investigated the efficiency and mechanism of cross-presentation of these long synthetic peptides in mhc class i. we observed a large variation in the effectiveness of activation of specific t cells by the extended peptides corresponding to different epitopes, indicating a difference in the efficiency of processing and presentation of these peptides. for the hla-a restricted cmvpp derived nlv epitope specific t cells were most efficiently activated by n-terminally extended variants of the minimal epitope, while the use of c-terminally extended variants resulted in a - log reduction of activation efficiency. this pattern was seen for / epitopes tested in different hla restrictions. furthermore, for all epitopes tested, extending both the c-terminus and n-terminus led to - log less efficient activation of the specific t cells, compared to the minimal peptide. exchange of the c-terminal sequence of the c-terminal extended hla-b restricted cmvpp rph peptide with the c-terminal extended nlv peptide led to the enhancement of t cell activation by the exchanged nlv peptide, indicating a role of the extended peptide sequence in the efficacy of processing and presentation of the peptide. tap-deficient t cells loaded with extended nlv peptides efficiently activated nlv-specific t cells, indicating that the route of presentation was tap-independent. addition of lactacystin did not affect activation of specific t cells, illustrating that crosspre-sentation was proteasome-independent. primaquine reduced the activation of specific t cells by extended nlv peptides, but not by the minimal nlv -mer peptide, suggesting that cross-presentation was dependent on endosomal recycling. these data suggest that long synthetic peptides can be processed by peptidases in endocytic compartments and presented by recycling mhc class i molecules. not all immunogenic epitopes that have been selected in vivo for efficient processing and presentation by the classical pathway may be presented efficiently by cross-presentation. therefore, a rational design of peptides is crucial for efficient activation of cd + t cells in approaches of vaccination, adoptive transfer and immune monitoring. antigenic peptides presented by mhc class i molecules are fragments that are usually excised from intracellular proteins while these are degraded by the proteasome. recently, three antigenic peptides were found to result from the splicing of segments that are not contiguous in the parental protein. for two of these peptides, splicing was found to occur in the proteasome by a mechanism of transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by a free peptide fragment. one of them is derived from melanocytic protein gp and requires excision of a four-amino acid intervening segment. the other peptide is derived from protein sp , and requires splicing in the reverse order of two segments initially separated by six amino acids. the first spliced antigenic peptide described was derived from fibroblast growth factor- (fgf- ) and was recognized by human cytotoxic t lymphocytes directed against kidney cancer cells. it is made of two spliced fragments, which are initially separated by a long segment of amino acids. the splicing mechanism of this peptide has not been worked out. the length of the intervening segment made the transpeptidation model more difficult to account for the splicing of this peptide. we therefore evaluated the role of the proteasome in the splicing of this peptide. we observed that the spliced fgf- peptide was produced in vitro after incubation of proteasomes with a -amino acid long precursor peptide. we evaluated the mechanism of the catalytic reaction by incubating proteasomes with several peptide precursors in a pair wise manner. the results confirmed the transpeptidation model of splicing. we further compared the production of the fgf- spliced peptide by cells transfected with mutant constructs encoding fgf- proteins where the intervening segment was shortened from amino acids to , or residues. we observed an increase in the production of the spliced peptide that was proportional to the reduction in length of the intervening segment, as predicted by the transpeptidation model. finally, using the spliced gp peptide model, we observed that splicing did not occur at a significant level between fragments of two distinct proteins in the cell. the polymorphic residues within the peptide binding cleft of hla class i molecules not only diversify the range of peptides presented to cytotoxic t lymphocytes but also influence the pathway of antigen presentation. in order to acquire high affinity peptides, some class i allotypes, such as hla-b* , are heavily dependent upon tapasin and other molecules comprising the peptide loading complex (plc). other class i molecules, like hla-b* , appear to largely bypass this complex but are consequently loaded suboptimally with peptide. hla-b* and b* are naturally occurring allotypes that differ by only a single amino acid, making this difference in behaviour all the more remarkable. we have previously speculated that such tapasin-independent class i molecules may have been selected in response to viral inhibitors that target the plc, such as the human cytomegalovirus us protein. to address this hypothesis, us was stably coexpressed in b lymphoblastoid cell lines expressing hla-b* or hla-b* . in the presence of us , the surface expression of hla-b* was substantially reduced whereas hla-b* expression was relatively unaffected. although us was able to form complexes with both hla class i allotypes, only hla-b* was retained intracellularly in an immature form whereas hla-b* was transported to the cell surface. accordingly, in the presence of us , hla-b* , but not hla-b* , constitutively presented a hla-b restricted alloantigen to reporter t cells, suggesting that us binds hla-b* without interfering with peptide loading. us has been reported by others to bind the plc but surprisingly we have not detected such us -plc complexes in our system. rather, in the presence of us we identified a pool of class i molecules distinct from the plc and only present in us expressing cells, implying that us may act independently of the plc. these findings demonstrate how hla class i polymorphism not only impacts upon the t cell repertoire and diversifies determinant selection, but also serves to evade the impact of viral inhibitors on antigen presentation. c. massa , b. seliger martin-luther-university halle-wittenberg, institute of medical immunology, halle, germany in the attempt to optimize vaccine dc, modifications have been proposed both in the antigen loading and in the maturation protocols. for dc loading "whole antigens" are now preferred to peptides. therefore, it is important to consider not only the costimulatory properties of the vaccine dc, but also their antigen processing abilities. this is even more important since there is the trend to stimulate dc with tlr ligands combined with ifn-y in order to induce dc not only able to correctly migrate, but also secreting the bioactive il p . since ifn-g is known to influence the expression of multiple proteases involved in antigen processing, aim of this study was to compare the various maturation cocktails for the consequences on the antigen processing capabilities of the dc in parallel to their costimulatory potential. for this purpose monocyte-derived dc were stimulated for h with the gold standard of maturation (tnfa, il b, il and pge ) or a combination of ifn-g and different tlr ligands. the dc obtained exhibit a similar expression of costimulatory and adhesion molecules together with the ability to induce proliferation of allogeneic pbmc, but differ for the pattern of proteases expression as evaluated by real time pcr. with the exception of the downregulation of the tripeptidyl peptidase ii (tppii), no dramatic differences were observed for endo-and aminopeptidase between immature and "gold standard" mature dc. in response to the "ifn-gcontaining" cocktails there was a similar tppii downregulation, but also the induction of many other enzymes. the cytosolic leucine aminopeptidase- (lap ) had a more than -fold increase in transcription levels, whereas the mrna expression of the aminopeptidases of the endoplasmic reticulum erap and erap and of the immunoproteasome subunits lmp and lmp was enhanced between and -fold under these culture conditions. with regard to the different tlr ligands used in combination with ifn-g, there was a reproducible higher mrna induction in the presence of the tlr ligand mpla in comparison to the tlr- and / ligand polyi:c and r . these data suggest that the maturation cocktail of dc may alter the peptide repertoire presented by hla class i surface antigens. it has been suggested that mast cells might serve, under certain circumstances, as antigen presenting cells for t cells. however, whether cognate interactions between mast cells and class ii restricted cd + t cells actually occur, is still an open question. we addressed this question using peritoneal cell-derived mast cells (pcmc) as an antigen presenting cell model. our results show that in vitro treatment of pcmc with ifn-g and il- induced surface expression of mature mhc class ii molecules and cd . when ifn-g/il- primed pcmc were used as antigen presenting cells for cd + t cells they induced activation of effector t cells but not of their naive counterparts as evidenced by cd up-regulation, induction of proliferation and cytokine production. confocal laser scanning microscopy showed that helper ot-ii t lymphocytes form with pcmc functional immunological synapses, characterized by pkcq enrichment and ifn-g polarized secretion towards the antigen-presenting mast cells. finally, upon cognate interaction with ot-ii t cells, mast cells lowered their threshold of activation via fceri. our results show that mast cells can establish cognate interactions with class ii restricted helper t cells, implying that they can actually serve as resident apc in inflamed tissues. h the vast majority of peptide ligands presented by mhc class i molecules is thought to be produced by cytosolic degradation of source proteins by the proteasome. although, next to cytosolic and nuclear proteins, proteins targeted to the endoplasmic reticulum (er) can also be degraded through this pathway following retrograde transport into the cytosol, antigen processing of er proteins remains little characterized. studying processing and presentation of er-targeted and cytosolic forms of proinsulin (pi), an autoantigen playing a pivotal role in triggering of cellular autoimmune responses in type -diabetes, we found that er-targeting of this model antigen has profound effects not only on how pi is degraded, but also on regulation of its synthesis. as expected, proteasome inhibition inhibited degradation of cytosolic pi as well as presentation of the epitope insulin b - to specific cd + t cells. in contrast, prior exposure of cells to proteasome inhibitors strongly reduced production of er-targeted pi (pre-pi) through induction of er stress, both in cells infected with a recombinant vaccinia virus and in cells transfected with a tetracycline-regulated expression system. experiments using conditions permissive for pre-pi expression showed that er-targeting modified proteolytic processing of pi for mhc class i presentation. these experiments suggested that two proteolytic pathways contribute to degradation of er-targeted pi, with their relative contribution depending on the stability of the protein. while degradation of unmodified pre-pi was partially dependent on the proteasome, removal of one or several disulfide bridges increased the role of the proteasome in processing of pre-pi for presentation, while introduction of a site for n-glycosylation had the opposite effect. these findings imply that er-targeting together with structural features can have profound effects both on antigen production and on the pathway of proteolytic antigen degradation and presentation. cd + t cell immune response to exogenous antigens relies on cross presentation by dendritic cells (dcs) in secondary lymphoid organs. recently, in several infectious murine models, it has been shown that in addition to dc located in tissues, de novo differentiating dc participate in the protective th immune response. the role of de novo differentiating dc in cross presentation is however poorly documented, and difficulties of human immunology prevent the accurate identification of the apc subsets patrolling for exogenous ag. a prerequisite for cross presentation is a moderate ag degradation rate in the endocytic pathway, allowing the generation of antigenic epitopes and their binding to mhc molecules. this prerequisite is of special importance considering dc precursors (such as monocytes), which are not yet dcs and may take up antigen before differentiating into dcs. the objective of our in vitro study is to evaluate whether ex vivo purified human blood monocytes are able to cross present long antigenic peptides to cd + t cells and whether they are able to sustain this cross presentation while differentiating into dcs. we have previously shown the unique property of dendritic cells to maintain for several days the capacity to stimulate cd + tumor-specific t cell clones when pulsed with long antigenic peptides (that need to be processed before presentation to cd + t cell clones, faure, , eur j immunol ( ): - ). in the present study, we address the question of the mechanisms of long peptide cross-presentation by blood monocytes along the course of their in vitro differentiation into dcs. we have shown that despite their high degradative capacity, ex vivo purified monocytes pulsed with long peptides are able to stimulate cd + t cells after their in vitro differentiation into dc, days following their antigenic pulse. the delineation of apc subsets able to sustain ag cross-presentation and t cell stimulating potential might be of clinical relevance in immunotherapy using synthetic long peptides. viral genomes contain alternative reading frames (arfs) encoding for mhc-i restricted epitopes (arf-epitope). in the siv/macaque model, ctl responses directed against arf-epitopes participate in controlling viral replication. we previously described that hiv- genome contains arfs within gag, pol and env genes encoding for a panel of hla-b* restricted epitopes. qprsdthvf (q vf/ d) is one such epitope but its parental epitope qprsnthvf (q vf/ n) has a significant higher frequency among hiv- isolates. strikingly, q vf/ d-or q vf/ n-specific ctls recognize apcs infected with hiv strains encoding for q vf/ d (e. g. hiv lai ). in contrast, hiv strains (e. g. hiv nl-ad ) encoding for q vf/ n do not activate ctl responses raising the possibility that q vf/ n epitope is not presented by infected cells. we asked whether introducing mutations within q vf might be a mean for the virus to escape ctl responses directed against this arf-encoded epitopes. we dissected the mechanism responsible for the lack of q vf/ n mhc-i presentation. we modified hiv lai to introduce a d to n mutation in q vf. introducing this single amino-acid mutation abrogated ctl recognition indicating that this asparagine (n) alters q vf mhc-i presentation. we performed in vitro proteasomal digestions of mer peptides encompassing q vf/ d or q vf/ n and cleaved polypeptides were analyzed by mass spectrometry. the asparagine (n) in q vf/ n is a preferential proteasomal cleavage site. thus suggesting that proteasome cleavages within q vf/ n might be responsible for its lack of mhc-i presentation. we then sought in hiv-infected patients for the presence of proviruses encoding for q vf/ d or q vf/ n, and ctls responses directed against these epitopes. far thus, two out of three donors tested recognized the q vf/ d peptide. we cloned and sequenced hiv- genomes from the three donors. surprisingly, out of hiv proviral genomes isolated from pbmcs of q vf/ d reactive donors, we could not find any virus bearing the q vf/ d sequence. the isolated hiv sequences either encoded for q vf/ n or had a stop codon within the epitope. in contrast, viruses encoding for q vf/ d were isolated from pbmcs of the q vf/ d nonreactive patient. altogether, our data suggest that ctls exert a selection pressure on viral arfs. hiv- seems to escape immune surveillance by introducing mutations altering processing of arf-derived epitopes. i. e. flesch , y. wang , d.c. tscharke the australian national university, biochemistry and molecular biology, canberra, australia vaccinia virus (vacv) was the live vaccine used to eradicate smallpox and some strains are now being used as vectors for recombinant vaccines. cd + t cells recognizing viral peptides in association with mhc class i molecules on infected cells play a crucial role in the defence of viruses. despite the large number of possible mhc class i-peptide combinations, cd + t cells only recognize a small number of epitopes, a phenomenon called immunodominance. using recently defined cd + t cell epitopes for vacv in mice, we have investigated how heterozygosity of mhc class i molecules influences immunodominance patterns in h- bxd f mice compared with their inbred parent strains. we find that the immunogenicity of vacv peptides defined using inbred mice is variable in f progeny, with some peptides being almost equally immunogenic in f and inbred mice, while others elicit responses that are reduced by more than % in f mice. during acute infection as well as memory responses, the dominance hierarchy in inbred mice did not predict the epitopes that would be poorly immunogenic in f mice. in line with these findings, a multiepitope construct expressed by a recombinant vacv was less immunogenic in f mice than would be predicted from its performance in parent strains. in terms of mechanism, we find evidence of altered tcr repertoires including in the case of one epitope, the loss of many diverse tcr vb clones and outgrowth of cd + t cells with a restricted vb usage in f mice. these data have implications for our interpretation of experimental vaccine work done in inbred mice and for our understanding of how mhc diversity can alter the range of epitopes that are immunogenic in outbred populations. objective: tlr ligands are being exploited as potential adjuvants, and have impact on the antigen processing and presentation by dendritic cells (dc). therefore we aimed to study the efficacy of a tlr agonist, s-[ , -bispalmitoyiloxy-( r)-propyl]-r-cysteinyl-amido-monomethoxyl polyethylene glycol (bppcysmpeg), a synthetic derivative of the mycoplasma macrophage activating lipopeptide (malp- ), as an adjuvant for cross-priming against cellular and soluble antigens. malp- has been characterized as an effective mucosal adjuvant and synthesis of bppcysmpeg further improved solubility and pharmacokinetic features of the adjuvant. methods: dc isolation, in vitro and in vivo t cell stimulation, intracellular cytokine staining, in vivo cytotoxicity assays. results: systemic administration of bppcysmpeg induced maturation of cd + and cd -dc in the spleen resulting in enhanced cross-presentation of intravenously co-administered soluble antigen in mice. in addition, administration of bppcysmpeg and cell-associated ova resulted in generation of an effective ctl response against ova in vivo in a t-helper cell-dependent manner, but independent of ifna. delivering antigenic peptides directly linked to bppcysmpeg led to superior ctl immunity as compared to giving antigens and adjuvants admixed. in contrast to other tlr ligands such as cpg, systemic activation of dc with bppcysmpeg did not result in shutdown of antigen presentation by splenic dc subsets, although cross-priming against subsequently encountered antigens was reduced. we provide evidence that bppcysmpeg stimulation of dc via tlr / results in the generation of an effective ctl response and that delivering antigenic peptides linked to bppcysmpeg is a promising strategy for vaccination. while bppcysmpeg-matured dc retain their antigen uptake and presentation capabilities, cross-priming against subsequently encountered antigens is inhibited, indicating that mechanisms beyond down-regulation of macropinocytosis and phagocytosis contribute to shut-down of cross-priming after tlr-mediated dc maturation. altogether our study promotes synthetic lipopeptides as potential adjuvant for specific applications (e. g. viral infections, cancer) for the reason that they can be chemically engineered to carry specific antigenic peptides which allows targeting of antigens and simultaneous activation. tumor immunevasion. to verify whether the loss of erap expression could confer a survival advantage on tumor cells and enhance tumor progression, we stably knocked down expression of eraap (murine erap ) in a murine t lymphoma cell line, rma. we used a method that allows an efficient and continuous expression of mirnas that directly silence eraap and obtained several eraap-deficient rma clones with different levels of eraap expression (up to % of reduction at the protein level). microsomal aminopeptidase activity and mhc class i surface expression were decreased in all clones proportionally to eraap expression. moreover, low expression of eraap affected the stability of mhc class i molecules as evaluated after acid and brefeldin a treatment. de-regulated er peptide trimming also drastically affected the tumor formation of rma cells and host survival. eraap-deficient rma clones with different levels of eraap, and % as compared to control rma cells, were injected s. c. in the flank of c bl/ syngenic mice, and analysed tumor growth. all mice injected with control rma cells developed a tumor but survived up to days after injection. all mice injected with rma clone with a % level of eraap expression developed a tumor and died within days after injection. surprisingly, any animal injected with rma clone with a % level of eraap died or showed a visible tumor. thus, knockdown of eraap expression appears differently to affect the immunogenicity of rma cells, depending on the eraap silencing level. hemophilia a is an x-chromosome-linked bleeding disorder caused by the absence or dysfunction of clotting factor viii (fviii). treatment consists of regular administration of fviii, but is complicated by the formation of inhibiting antibodies against fviii. both genetic and treatment-related factors play a role in the etiology of inhibitor development in patients with hemophilia a. the development of inhibitory antibodies in hemophilia a patients has been shown to be a cd + t-cell driven process. therefore, in order to better understand the process of inhibitor formation, we aim to identify the epitope specificity and phenotype of t cells against fviii in hemophilia a patients using mhc class ii tetramers. cd + t-cell responses of two monozygotic twins with severe hemophilia a were analyzed. one of these subjects developed a high titer inhibitor ( bu/ml) following intensive factor viii (fviii) treatment. high dose immune tolerance therapy together with anti-cd therapy resulted in eradication of the inhibitor. in contrast, his twin brother developed a low titer inhibitor ( . bu/ml) which declined rapidly after tolerance induction. fundamental differences in the twins' antibody responses were further suggested by elevated and persistent igg levels in the subject with the high titer inhibitor. in order to gain a better understanding of processes leading to inhibitor formation versus tolerance, we investigated drb * -restricted t-cell responses of the high titer inhibitor subject, using fluorescent mhc class ii tetramers loaded with -mer synthetic fviii peptides to stain epitope-specific cd + cells.cd + t-cells from the high-titre inhibitor subject recognized three peptides corresponding to the fviii a domain: fviii - , fviii - and fviii - , as well as the c domain peptide fviii - , but not any c domain peptides. the c domain peptide contains a sequence that was reported as a promiscuous t-cell epitope (jones td et al., j thromb haemost. : - , ). analysis of t cells from the lower titer inhibitor subject is expected to reveal differences in the epitope specificity and phenotypes of t cells that may underlie the discordant immune responses of these twins to infused fviii. m. forloni , s. albini , m.z. limongi , l. cifaldi , d. fruci ospedale pediatrico bambin gesù, rome, italy neuroblastoma (nb) is a pediatric tumor that derives from neural crest. the most aggressive forms are characterized by amplification of the mycn oncogene and severe reduction of hla class i expression. mycn has been claimed to hinder hla class i expression through affecting the expression of the transcription factor p nf-kb subunit. since in many human tumors the expression of hla class i molecules is positively co-ordinated with that of er aminopeptidases, erap and erap , we wondered whether in nb cell lines mycn may impair expression of these aminopeptidases. to explore this possibility, nb cell lines that differ in mycn expression were quantified for expression of mycn, erap , erap and hla class i heavy chains by western blotting and for surface hla class i expression by flow cytometry. we found that mycn negatively correlates with expression of hla class i, erap and erap . this negative correlation was confirmed in a nb cell line expressing a tetracycline repressible mycn transgene. then, by the use of tnfa (a nf-kb nuclear translocation stimulator), sulfasazine and ikba mutant (two nf-kb nuclear translocation inhibitors) and knockdown of p nf-kb subunit, we demonstrated that nf-kb is involved in erap and erap expression in nb cell lines and that mycn does not affect nf-kb expression. furthermore, we showed that mycn and nf-kb are recruited to the promoter regions of erap and erap and that mycn affects the recruitment of nf-kb binding to these promoter regions. in conclusion, the present results indicate that an enhanced mycn level, linked or not to mycn amplification, represses erap , erap and hla class i expression in nb cell lines by affecting the recruitments of nf-kb binding to their promoters. s. brosch , s. tenzer , h. schild , e. von stebut-borschitz uniklinik mainz, mainz, germany infection of inbred mouse strains with the intracellular protozoan parasite leishmania major either leads to self-healing cutaneous disease (resistant phenotype; e. g. c bl/ mice) or systemic disease (susceptible phenotype; balb/c mice) depending on the genetic background of an individual. healing of leishmania infections is based on th immunity, whereas ifng secretion of both cd + th and cd + tc cells is critically important for protection by inducing oxidative radicals in macrophages, which enables them to kill the parasite. stimulation of antigen-specific effector t cells is driven by l. major-infected dendritic cells (dc) in an il- -dependent manner. proteasome/immunoproteasome-dependent antigen processing is necessary for clearance of viral or intracellular parasitic diseases to induce effective cd + t-cell responses via the mhc class i. here, we analysed the role of the ifng inducible immunoproteasome for the priming of cd + t cells in l. major infections. using an in vivo model, we show that the functional knock-out mouse in the chymotrypsin-like catalytic domain of the immunoproteasome lpm (lmp -/-) does not exhibit an altered course of infection (lesion development, parasite loads, cytokine profiles) in intradermal, low dose infections with l. major mimiking natural transmission of the parasite as compared to wild type c bl/ mice. in addition, ex vivo co-cultures with infected dc from either lmp -/or wild type mice together with antigen-specific t cells from infected wild types showed no differences in tc cell ifng secretion and the dc restimulatory capacity of cd + t cells. furthermore, significant differences in the proliferation of antigen-specifically restimulated (with soluble leishmania antigen; sla) cd + t cells, isolated from low dose infected c bl/ wildtype or lmp -/mice, were not detected. in summary, our data indicate that despite the fact that cd responses in l. major infections are important for disease outcome, processing of antigen and thus priming of cd + t cells against l. major is independent of the lmp subunit of the immunoproteasome. studies have defined an essential requirement for autoantigen-specific b cells as antigen presenting cells in rheumatoid arthritis. however, the cellular mechanisms involved in antigen processing and presentation of joint-derived autoantigens by b cells are unknown. in this study we have developed a system to investigate how antigen-specific b cells recognise and present the proteoglycan aggrecan, a major component and candidate autoantigen of joint cartilage. we have utilised these cells to characterise the mechanisms by which aggrecan-specific b cells could induce autoimmunity. we have constructed plasmids encoding an aggrecan-specific b cell receptor and have transfected them into the b cell line a , generating b cell lines that specifically recognise and target aggrecan for presentation to t cells. in addition, we have established conditions for a panel of aggrecan-specific t cell hybridomas to recognise aggrecan pulsed b cells following fixation, to allow the kinetics and mechanisms of aggrecan processing to be studied. we used inhibitors of mhc class ii transport, endosomal ph and enzymes involved in aggrecan degradation. we found that aggrecan-specific b cell lines presented the major arthritogenic cd + t cell epitope ( - ) from the g domain of aggrecan , times more efficiently than non-specific b cells and over times more efficiently than the macrophage line j . however, despite this highly efficient aggrecan capture, processing and presentation of the - epitope took at least hours, comparable to the time required for presentation of aggrecan by j . treatment of aggrecan-specific b cells with ammonium chloride to raise endosomal ph or brefeldin-a to disrupt golgi transport inhibited presentation of the - epitope, suggesting a requirement for low endosomal ph and presentation by newly synthesised mhc class ii. interestingly, aggrecan presentation by antigen-specific b cells was also reduced by phenanthroline, an inhibitor of the aggrecan-degrading metallo-proteinases that are found in abundance in the arthritic synovium understanding the mechanisms of antigen processing and presentation by autoantigen-specific b cells may explain their role in the pathogenesis of diseases such as rheumatoid arthritis. tapasin is an mhc-dedicated chaperone that facilitates peptide loading and optimization of the peptide cargo of mhc class i molecules within the peptide loading complex (plc). class i molecules differ in their dependence on tapasin for efficient cell surface expression, dependence that is determined by the nature of amino acids at positions , and at the peptide binding groove. position also determines the strength of tapasin binding and influences peptide specificity, but its precise effect is probably context dependent. the mhc class i antigen b is strongly associated to ankylosing spondylitis (as) and other spondyloarthropathies. hla-b subtypes differ in their dependence of tapasin for cell surface expression and incorporation into the plc. tapasin also modulates b folding but not maturation and although tapasin optimizes the constitutive peptide repertoire of b* , peptide loading is relatively independent of this chaperone. we analyzed the effect of b subtype polymorphism on tapasin binding and the correlation of this feature with the affinity of the peptide repertoires, the maturation kinetics and the folding efficiency of b subtypes. the association of b heavy chain with tapasin was analyzed in c r cells transfected with hla-b subtypes and mutants by pulse-chase analysis and co-immunoprecipition with the monoclonal antibody pasta- , which recognizes human tapasin. we also analyzed the global thermostability, as a measure of the stability of the peptide cargoes, and the optimization of the b peptide repertoire with thermostability assays, by pulse-chase analysis and immunoprecipitation with the me monoclonal antibody that recognizes b properly folded b /peptide complexes. the formation of fully assembled b molecules was analyzed by pulse-chase analysis and immunoprecipitation either with the monoclonal antibody hc , which recognizes mhc class i free heavy chains (hc), or with me . maturation was analyzed by pulse-chase analysis, immunoprecipitation with me and treatment with endoglycosidase h (endo h). hla-b polymorphic positions other than , both at the a and c/f pockets modulate tapasin binding and the optimization of the peptide cargo. the stability of the peptide repertoires critically influences the folding efficiency of b subtypes. from as early as the initial phases of infection, hiv is coated with complement (c) fragments and following seroconversion, the circulating virus forms immunecomplexes with igg and complement. recent in vitro experiments revealed differences with respect to productive infection of immature dendritic cells (idcs) with differentially opsonized hiv. the opsonization pattern of hiv may additionally have profound consequences for the outcomes of the antigen-presenting capacity of dcs and their ability to mount an adequate immune response. in this context, we compared the impact of differential hiv-opsonization on the antigen-presenting capacity of dcs and found that c-opsonized hiv triggered ctl responses, while igg-coated virus did not. these in vitro generated ctls showed an enhanced ifn-g secretion and recognized the help independent ctl epitope slyntvatl. c-generated ctls also degranulated upon stimulation with specific hiv peptides and were able to elicit antiviral activity against hiv-infected cd + t cells. our results indicate that c-opsonization of hiv drives the virus towards the mhc class i pathway in dcs, thereby promoting a more efficient stimulation of naïve cd + t cells. this ctl-stimulating property of c could be exploited when searching for a novel approach against hiv. igg isolated from patients with high titers of anti-ccp antibodies showed a cross-reactivity with hcit peptides. vaccination experiments supported a triggering role of hcit for the development of arthritis in mice model. conclusions: diamination process is significantly increased in patients with ra while carbamylation is suppressed. production of specific antibodies against diaminated residues in ra patients may have a modulating role for the development of autoimmune arthritis. the classical pathway of mhc class i antigen presentation involves cytosolic degradation of viral proteins by the proteasome. peptides generated entry the endoplasmic reticulum through the transporter associated with antigen processing (tap). previous reports have shown that viral epitopes are presented to ctl independently of tap in smaller viruses. we hypothesized that presentation of vacv by mhc class i might proceed by alternative pathways. the aim of this study was to characterize these alternative pathways in tap-deficient mice. our results show that ctl derived from c bl/ mice immunized with vacv, recognized tapdeficient dendritic cells infected with the virus. approximately % of vacv global presentation in the context of h- b was independent of tap. in addition, vacv infection induced a virus-specific ctl response in mice deficient in tap. dendritic cells (dc) initiate robust ctl immunity via the presentation of antigen-derived peptides by surface major histocompatibility complex class i molecules (pmhc). two major dc subtypes have been described, cd + and cd -dc, which differ in their mhci antigen presentation capacities. cd + dc are the major dc subset responsible for cross presentation (presentation of exogenous antigen by mhci), while cd -dc display little cross presenting capacity. here, we examined the mhci antigen presentation pathway of cd + and cd -dc in more detail. first, turnover (half-life) of total mhci at the cell surface of cd + and cd -dc was determined. surprisingly, cd + dc exhibit rapid surface mhci turnover compared to cd -dc (following culture in the presence of brefeldin a). following activation of dc with cpg, mhci levels at the surface of both cd + and cd -dc were stabilized and no longer underwent rapid turnover. this suggests that cd + and cd -dc differ in their regulation of surface mhci turnover and that this is subject to regulation by antigen-associated signals. second, we examined the ability of cd + and cd -dc to generate pmhci complexes containing cross presented antigen. we utilized the model antigen ovalbumin (ova) and an antibody that can detect h- kb loaded with the ova-derived peptide, siinfekl. cd + and cd -dc isolated from ova-expressing mice (actin-ova transgenics) displayed abundant kb-siinfekl complexes at their cell surface. in contrast, in response to exogenous soluble ova protein, only the cd + dc, but not the cd -dc, displayed kb-siinfekl complexes at the cell surface. similarly, when dc were pulsed with ova-coated splenocytes, kb-siinfekl complexes were only detected on the surface of cd +, and not cd -dc. this data further validates the role for cd + dc as the major cell type responsible for cross presentation and provides insight into the mechanisms that prevent other dc subsets from accessing the important cross presentation pathway. objectives: the action radius of matrix metalloproteinases or mmps is not restricted to massive extracellular matrix (ecm) degradation but extends to the proteolysis of secreted cytokines and membrane-bound receptors and adhesion molecules. although many instances exist in which cells disintegrate, often in conjunction with induction of mmps, the intracellular mmp substrate repertoire or degradome remains relatively unexplored. the aims of the present study were to identify novel intracellular mmp targets and to answer the question whether the proteolytic modification of intracellular proteins alters the immunogenicity of released intracellular contents. methods: multidimensional degradomics technology was developed by the integration of broadly available biotechniques and applied to thp- cytosol using gelatinase b/mmp- as a model enzyme. in the first dimension, ion exchange chromatography separated the thp- proteins by their net charge and/or isoelectric point (pi) followed by cleavage of the proteins by mmp- . in the second dimension, potential substrates were separated by molecular weight on sds-page. to evaluate the effect of proteolysis by mmp- on the immunogenicity of the intracellular protein pool, mice were immunized twice with thp- cytosol in complete freund's adjuvant. lymph node t cells were isolated and stimulated with mmp- -cleaved or intact thp- cytosol. proliferation was assessed by measuring incorporated hthymidine. results: - mmp- candidate substrates were isolated, of which were identified, revealing many novel mmp- (candidate) substrates from the intracellular matrix (icm), such as actin, tubulin, stathmin,... about / of the identified substrates were described as systemic autoantigens in one or multiple autoimmune conditions. remarkably, a significantly lower t cell proliferation was observed in the presence of cleaved vs. intact cytosol. conclusion: multidimensional degradomics technology is a valuable tool for high-throughput identification of novel mmp substrates. proteolysis by mmp- decreased the immunogenicity of the intracellular contents, suggesting that mmps may contribute to the dampening of inflammation by the clearance of toxic and immunogenic burdens of intracellular (matrix) proteins released after extensive necrosis and tissue injury. a preference for hla-a versus -b molecules; e - k did not detectably associate with hla-c molecules under identical conditions. this locus specificity may provide a functional advantage to ads by inactivating t-cell receptors, while avoiding activation of nk receptors. finally, we showed that residue in hla-a and residue in e - k are highly critical for association of both proteins. this defines a putative interaction surface between e - k and class i molecules. conclusions: our studies provide novel insights into the functional relationship between e - k and the class i antigen presentation pathway. moreover, because soluble e - k can differentiate between polymorphic gene products encoded in the mhc, our results may contribute to define paradigms for how class i substrate specificity is established for er retention. overall, our studies represent an important step towards a molecular understanding of the strategy evolved by ads to establish life-long persistence in host cells. objectives: the transporter associated with antigen processing (tap) belongs to the abc transporter superfamily and is a heterodimer consisting of the two subunits tap and tap . tap transports peptides yielded by proteasomal degradation from the cytosol into the endoplasmic reticulum (er) and is thus a key element of the mhc class i antigen processing machinery (apm). methods: target-specific tap knock downs were generated by shrna technology. the resulting transfectants were subsequently analysed regarding mhc class i surface expression using flow cytometry, whereas mrna and protein expression levels of tap and tap were analysed by rt-pcr and western blots, respectively. furthermore, the protein stabilizing effect of tap on tap was investigated in the presence of two distinct proteasome inhibitors. results: previous findings obtained with rare tap mutants suggested that the lack of tap protein expression is associated with a strong reduction of tap protein levels, which could be restored by tap gene transfer, whereas no such regulation is found vice versa. to investigate this stabilizing effect of tap on tap different shrna plasmids specifically targeting tap or tap , respectively, were stably transfected into constitutively tap and tap expressing hacat keratinocytes and colo melanoma cells. in both cell types the shrna-mediated tap and tap inhibition resulted in a significant downregulation of the respective transcript and protein expression levels. the knock down of tap caused not only an almost complete loss of tap , but also a strong decrease of tap protein expression. in contrast, the tap knock down exhibited no influence on the tap expression. specific inhibition of the proteasome prevented the degradation of tap in the tap knock down variants. the results of our study emphasize that an unidirectional stabilisation of tap on tap protein expression is not restricted to rare tap mutants, but rather suggest a common regulatory mechanism for the tap complex. uv injury profoundly affects the skin immune homeostasis by promoting strong inflammation and cellular immuno-modulation. in this study, we characterized the inflammatory cell subsets that emigrate in the epidermis the days following uv exposure. therefore, the buttock skin of healthy volunteers was exposed twice to . minimal erythema dose of uv. blister roofs were then collected before and , and days after uv-exposure from un-exposed and exposed skin and the resulting epidermal cells were analysed by flow cytometry. we demonstrated that, along with the rapid activation and migration of langerhans cells (lc), uv skin radiation exposure promotes the infiltration into the epidermis of a monocytic cd + cd + and of a macrophagic cd -cd + cell subsets that emerge and days post exposition, respectively. more importantly whereas classical cd a hi cd + lc are the unique dendritic cell (dc) subset found in the epidermis of unexposed skin, we detected two new subsets of epidermal dc namely cd a low cd and cd a low cd + that emerged and days post irradiation, respectively. these two distinct populations of epidermal dc (edc) differ from classical epidermal lc by their activation/maturation profile as assessed by the strong expression of cd and hladr. finally, days post-exposure, we observed that lc represented almost the only haematopoietic cell population in the epidermis. these results suggesting that the uv-recruited edc and monocytic/macrophagic subsets participate to the progressive recovery of the epidermal immune cell network homeostasis. i. bailey , e. reeves , t. elliott , e. james university of southampton, school of medicine, cancer sciences division, southampton, united kingdom there is accumulating evidence that cd + cd + regulatory t cells (treg) play an important role in anti-tumour immunity by preventing effective t cell responses to tumour antigens. tregs have also been shown to inhibit development of organ-specific autoimmune diseases suggesting they inhibit immune responses to tissue-specific self-antigens. the depletion of tregs prior to challenge with the murine colorectal tumour, ct , stimulates a robust, protective t cell response which is also protective to challenge with other tumours of different histological origins, such as b cell lymphomas and a renal cell carcinoma. this cross protection has not been seen with other tumour cell lines. we have identified a ct -derived cross-protective antigen, gsw , which was found to be encoded within the ectotropic murine leukaemia virus (emv- ) envelope protein, gp . this protein has previously been shown to encode ct -specific cd and cd antigens, implicating it as a 'hot-spot' for ct tumour antigens. interestingly, we have identified a truncated version of gp which may be responsible for generation of gsw . expression studies have revealed increased gp expression in ct compared to other tumour cell lines, indicating the ability to cross-protect is related to the quantity of antigen (gsw ) generated. the current knowledge of hla class ii antigen presentation and peptide binding is mainly based on studies of hla-dr molecules. they contain a large hydrophobic p pocket, which can accommodate large hydrophobic amino acid residues and is the most important pocket in selecting and binding peptides, while p , p and p tune the peptide repertoire that can be presented by individual hla-dr alleles. the same rules and requirements do not necessarily exists for peptide binding to hla-dp molecules, however. the present study adresses this issue. we have expressed and affinity purified soluble recombinant hla-dp molecules from drosophila melanogaster cells and studied its binding of a number of peptides known to bind hla-dr, -dq or dp molecules. unexpectedly, the immunodominant epitope in multiple sclerosis (ms), the myelin basic protein derived peptide, mbp - , bound to hla-dp with high affinity ( - nm). binding studies of mbp - derived peptides containing single alanine substitution at each position revealed that only three of the peptides (f a, f a and k a) were affected, and only by a - fold reduction in affinity for hla-dp . the observation that none of the substitutions resulted in a complete loss of binding to hla-dp indicates that ) hla-dp binding to peptides does not depend on a large hydrophobic residue accomodated in p , or ) mbp - can bind in more than one register. we will present data addressing this issue. the hla-dp peptide binding capacity was increased at neutral as compared to acidic ph, and by the presence of n-butanol, a small organic mhc loading enhancer (mle). in summary, the hla-dp molecule binds the immunodominant epitope in ms, mbp - , possibly in more than one register. additional studies are required to resolve the hla-dp peptide binding properties, and to determine whether expression of hla-dp affects the disease course in ms patients. results: depletion of mncs for cd + cells abrogated the tg-induced cytokine production and proliferation of cd + t cells, indicating a primary role for monocytes as apcs. however, the encounter of t cell with antigens presumably occurs in b cell-rich compartments such as lymph nodes or lymphoid tissue in inflamed organs. to mimic the conditions prevailing there, we depleted pbmcs for g % of the monocytes (without significant loss of t-cells), and compared the tgelicited t-helper cell responses in the presence and absence of b cells. the tg-induced cd + t cell proliferation was significantly reduced in cd /cd -depleted mnc cultures, as compared to cultures depleted for cd + monocytes alone. the same applied to the production of il- , il- and tnf-a. production of ifn-g and il- was generally not observed. our data indicate that normal b cells are capable of inducing a pro-inflammatory cytokine response in mnc-cultures, where monocytes and monocyte-derived cells are not preponderant. studies addressing the relative contributions to this cytokine production, by b cells themselves and by t cells (following antigen-presentation by b cells), are in progress. j. kyosiimire-lugemwa , , p. pala , g. miiro , j. todd , p. kaleebu , , n. imami , f. gotch , mrc uganda, basic science, entebbe, uganda, imperial college, london, united kingdom, mrc uganda, entebbe, uganda background: hiv- -specific t-cell responses are preserved in hiv- infected individuals with non-progressing hiv- disease. "long term non progressors" (ltnps) were defined as art naï ve individuals infected with hiv- for g years, maintaining cd + t-cell counts g , and with minimal cd + decline over time. we tested the hypothesis that gag-specific t-cell responses are inversely correlated to disease progression whereas nef-specific t-cell responses are not. methods: art naï ve hiv- infected patients from the entebbe cohort in uganda were recruited and stratified by cd + t-cell count, cd + decline slopes, and time of enrolment, into groups - ltnp and rapid progressors (rp). all patients were women reflecting the patient base at the entebbe cohort. we measured plasma viral load, current cd t-cell count, and ifn-g, il- and il- elispot responses to pools of to peptides ( -mers overlapping by aa). peptides were based on consensus sequences of gag and nef from hiv- clades a , a and d. medians and inter-quartile ranges were calculated and comparisons between groups were performed using the mann-whitney u test. correlations were presented using spearmann's linear correlation coefficients. results: some gag-specific ifn-g and il- responses were significantly higher in the ltnp than in rp (p= . , ifn-g responses to gaga pool ; p= . , il- responses to gaga pool ). il- responses were low and not significantly different between ltnp and rp. there was a positive correlation between il- responses to gaga pool and cd t-cell counts (r = . , p= . ), but no correlation between either il- or ifn-g responses and viral load. cytokine responses to nef peptides were not significantly different between the ltnp and rp. conclusion: overall, gag hiv- specific responses were higher in ltnps than in rps confirming previous results. non-specific il- responses were high possibly reflecting baseline th responses to helminths a common environmental exposure in the study population. objectives: in human and murine tumors and in in vitro oncogene-transformed cells defects in the expression of components of the hla class i antigen processing machinery (apm) have been described, which were associated with a reduced antigenicity of these cells. so far, the molecular mechanisms of such defects have not been elucidated in detail. to investigate whether impaired apm component expression was due to altered transcription and associated with cell growth properties murine her /neuand her /neu + fibroblasts were employed. methods: using tapasin as a model molecule its cell cycle-dependent expression was analysed in a time kinetics upon serum starvation followed by stimulation with complete culture medium over time. cells were harvested at different cell cycle phases and expression of tapasin was analyzed by qrt-pcr and western blot. flow cytometry was employed for determination of the distinct phases using -aad for dna analysis and specific antibodies directed against the proliferation marker ki- , the m-phase specific phistone h as well as for the h- l d surface antigens. in addition, chromatin immunoprecipitation (chip) experiments with an antibody directed against rna polymerase ii were performed to investigate the transcriptional levels of tapasin in her /neuversus her /neu + cells. results: serum starvation and subsequent stimulation with complete culture medium led to the enrichment of cells at the g /g -, s-and g /m-phases of the cell cycle, which was associated with an altered tapasin transcription during the cell cycle. tapasin mrna level decreased during cell cycle progression, whereas an inverse protein expression was observed with low expression levels at the g /g -phase, which continuously raised and peaked within the s-phase. however, h- l d surface antigen expression was not altered in her /neucells during cell cycle progression. in contrast to her /neufibroblasts the her /neu + transfectants exhibit a decreased tapasin transcription, which was accompanied by an altered h- l d surface expression. this was confirmed by a reduced promoter activity and decreased accessibility of the rna polymerase ii to the tapasin promoter. conclusion: these findings lead to an improved understanding of immune escape mechanisms demonstrating a cell cycle dependent and oncogene-mediated tapasin regulation that may provide novel targets for therapeutic intervention. recent studies suggest that dendritic cells (dcs) are key players in shaping the respiratory syncytial virus (rsv) specific immune response. before, dcs within the airway epithelium were characterized as langerhans cells. in this study, in vitro counterparts of langerhans cells expressing langerin (cd ) and ccr were cultured from cd + stem cells under the influence of tgf-b (tgf-b-dcs) and compared to cd + derived dcs, which passed through a monocytic stage . after infection with rsv, both types of dcs generated viral rna and viral-proteins. although tgf-b-dcs expressed higher levels of viral proteins as revealed by flow cytometry and fluorescence microscopy, more than hundredfold more viral particles were released by il- -dcs. the increased expression of viral proteins is most likely responsible for the pronounced inhibition of t-cell functions by tgf-b-dcs. since there is evidence that langerhans cells are expressed in airway epithelium not before the age of one year, the results may indicate, that an inhibition of rsv replication is characteristic of a more mature answer against rsv. the occurrence of inhibitory antibodies against exogenous factor viii (fviii) remains the major concern of fviii replacement therapy in patients with hemophilia a. initiation of the immune response implies the endocytosis of fviii by professional antigen presenting cells (apcs): b lymphocytes, dendritic cells (dcs) and macrophages (mØ). the organ where the anti-fviii immune response is initiated and the type of apcs involved in this process have not been investigated. we hypothesized that the spleen, which is the principal filter for blood-born antigens, is the principal organ where apcs interact with fviii to initiate the anti-fviii immune response. we first administered radiolabeled fviii at therapeutic doses to fviii-deficient mice. fviii was found to preferentially accumulate in the spleen and liver of the mice. levels of fviii in the spleen remained stable for up to min following fviii administration, while they rapidly decreased in the liver. unlabelled fviii was then administered to fviii-deficient mice that had been splenectomized or sham operated and the anti-fviii humoral responses were compared. removal of the spleen resulted in significantly reduced levels of anti-fviii igg. using flow cytometry, fviii was found to preferentially accumulate with splenic mØ than dcs and b cells. elimination of apcs by treatment of the mice with clodronate-containing liposomes prior to fviii administration resulted in a drastic reduction of the anti-fviii igg response, as compared to control mice treated with pbs-containing liposomes. taken together, our results suggest that the spleen is the principal organ in the initiation of the anti-fviii immune response and that splenic mØ have an important part in this process. the interactions between antigen presenting cells (apc) and t-lymphocytes are a relevant current issue. the area of contact between an antigen presenting cell and a t-lymphocyte is termed immunological synapse (underhill et al, ) . the present work started, in experiments with leukocyte of healthy individuals from the finding that under certain experimental conditions, cell-cell association with closely contact between monocyte-derived macrophages and human autologous lymphocytes are produced when the cells are harvested from total leukocyte cell cultures. in this way, such cells selective forming rosettes with a central macrophage and adherent lymphocytes. objectives: as central hypothesis it was postulated that the phenomenon would be due to antigen presentation made (performed) by macrophages to lymphocytes and that would be t cells. methods: autologous total human leukocyte cultures, from samples of healthy blood donors were harvested at various times and centrifuged and performed as previously reported (cabral and novak, , ) . cytopreparations of each experiment were performed. statistical analysis: regression model. results: experimentally, it was found a) phagocytosis of autologous antigens by macrophages, stimulates the formation of rosettes, b) a linear relation between rosettes formation and culture-time occurs, (p x , ), anova for regression, c) the cell-cell approximation is very important and was performed by centrifugation of the cells to form pellets, d) the forming rosettes lymphocytes are t-cells, cd +, e) purified macrophages and lymphocytes produced few rosettes, however if antigens were added, the phenomenon was stimulated, f) if inhibitors of the antigen processing and antigen presentation, such as chloroquine or brefeldin a, were added, rosettes were not formed, g) monoclonal antibodies anti-human mhc ii precluded the formation of rosettes, h) gangliosides diminishes rosettes formation. conclusion: taken together, the findings suggest that the model of rosettes formation might be useful to study cell-cell interactions during antigen presentation in immunological synapses and other immunologic aspects on the cells involved, in short time assays. objective: to investigate if patients with multiple sclerosis (ms), without the typical increase of antibodies in csf, are less likely to develop neutralizing antibodies (nabs) against ifnb-treatment, and whether the absence of such an immunological response might reflect a difference in their antigen presenting ability due to a distinct genetic background. methods: overall, patients were obtained from the swedish multiple sclerosis registry and the swedish nab registry, and treatment information was available for of them. for of these patients hla-drb data was available. results: a significant correlation between lack of antibodies in csf and nab-negativity was found (p= . ). patients without csf antibodies were to a lesser extent nab-positive when treated with the ifnb- a preparations, whereas no differences were shown for ifnb- b. an association between hla-drb * and nab-negativity was detected (p= . ). the known associations between hla-drb * and csf-positive ms and hla-drb * and csf-negative ms were confirmed. conclusion: we show for the first time that patients without antibodies in csf have a different propensity to induce nabs compared to csf-positive patients, indicating an extended immunological difference between the two ms sub-groups. hla-drb potentially contributes to this, which indicates that it might have something to do with differences in antigen presentation. in csf-negative patients the reaction against ifnb- a molecules, possibly through a t-cell dependent pathway, is lower than for csf-positive patients. however, reaction against ifnb- b, which might also be activated through a t-cell independent pathway, shows no difference in seroprevalence between the groups. abstract withdrawn by author tyrosinase-derived epitope was confirmed by five independent assays: flow cytometry on multiple melanoma lines generated from patients, confocal microscopy immuno-staining of melanoma lines, frozen sections staining of authentic melanoma tissue from patients, cytotoxicity assays using tyrosinase-specific ctls, and finally mass spectrometry analysis of peptides isolated from a melanoma cell line. there was no correlation between the level of antigen presentation and mrna expression levels for the three antigens; however, our data suggest that tyrosinase protein stability may play a major role in the high level presentation of this antigen. measurement of the half lives of these proteins revealed a hierarchy in protein stability, with mart- and gp more stable than tyrosinase. by the use of the cofactor dopa, which stabilizes the tyrosinase protein, significant decrease of hla-tyr complexes presentation was achieved. in addition to the study of antigen presentation, these tcr-like antibodies can also actively participate in immunotherapy as targeting molecules, considering their high affinity and specificity. by generating a whole igg antibody, tumor cell lysis was achieved by antibody-dependent cell-mediated cytotoxicity (adcc). with the addition of point mutations in the fc fragment, which increased the affinity of the fc to the fc receptor, enhanced tumor cell lysis was achieved. g. schiavoni , s. lorenzi , f. mattei , f. spadaro , l. gabriele istitituto superiore di sanità, cell biology and neurosciences, rome, italy cross-presentation is a crucial mechanism for generating cd t cell responses against exogenous antigens (ag), such as dead cell-derived ag, and is mainly fulfilled by dendritic cells (dc), particularly cd a + dc. however, apoptotic cell death occurring in steady-state conditions is largely tolerogenic, thus hampering the onset of effector cd t cell responses. type i ifn are a family of cytokines induced upon infection and acting as danger signals by stimulating multiple arms of the immune response. in particular, type i ifn have been shown to promote the cross-priming of cd t cells against soluble or viral antigens, partly through the stimulation of dc. in this study we evaluate the role of type i ifn to affect dc capacity to capture and cross-present apoptotic cell-derived ag. by using uv-irradiated ova-expressing eg thymoma line, we show that type i ifn promote the ability of cd a + dc to capture apoptotic eg cells and to undergo phenotypic activation, both in vitro and in vivo. remarkably, ifn-treatment prolongs the survival of ag-bearing cd a + dc and the persistence of apoptotic eg -cell ag within the phagosomal dc compartment, a process that is known to facilitate the recruitment of ag into the mhc-i presentation pathway. accordingly, type i ifn-treatment increases cross-presentation of apoptotic eg -derived ova ag by dc, as revealed by higher expression levels of siinfekl peptide in association to mhc-i molecules on cell surface of phagocytic cd a + dc. as a result, eg -loaded dc become competent at inducing ot-i cd t cell proliferation and activation both in vitro and in vivo. our data indicate that type i ifn promote the cross-presentation of apoptotic cell-derived ag by cd a + dc and suggest that these cytokines may act as a switch signal for cross-presenting dc, thus skewing the immune response from tolerogenic to immunogenic. ( ). we have investigated the mechanisms of cross-presentation of soluble antigen in freshly purified splenic dc subsets. using biochemical methods, we show that only cd + dc efficiently transfer soluble antigen to their cytosol. the amount of antigen detected in the cytosol increased up to ten-fold after a short exposure to tlr ligands cpg, poly i:c or pam csk , and this correlated with enhanced cross-presentation. the increase in antigen accumulation within the cytosol was not due to increased uptake of antigen. measurement of the proteasome activity at different times after exposure to tlr ligands revealed that tlr signalling induced transient inhibition (maximum at two hours) of the proteasome in cd + dc but not cd -dc, thus promoting accumulation of exogenous antigen in the cytosol of cross-presenting dc. this correlated with formation of aggresome-like structures only in cross-presenting dc exposed to tlr ligand. by limiting the degradation of transferred proteins during early activation, when endogenous proteins are being stored in aggresome-like structures, this mechanism could favour the loading of exogenous antigen peptides over endogenous peptides, promoting cross-presentation. to our knowledge this is the first report of a direct, immediate effect of tlr activation on proteasome activity. exosomes are nano-sized membrane vesicles of endosomal origin which can exert both immune stimulatory and tolerance inducing effects depending on their cellular origin. they are currently being investigated for use in vaccination and immune therapy strategies, but their physiological role has not been elucidated. here we explore whether exosomes of different origin can selectively target different immune cells. we compare the binding of exosomes from human breast milk, monocyte derived dendritic cells and b cells to peripheral blood mononuclear cells. flow cytometry, confocal laser scanning microscopy and multispectral imaging flow cytometry (imagestream) reveal that exosomes derived from human dendritic cells and human breast milk preferably associate with monocytes, whereas exosomes from an epstein-barr virus (ebv) transformed b cell line selectively target b cells. our data suggest a highly selective association between cells and exosomes which can be a way to direct their functional effects. one of the hallmarks of cancer cells is the resistance to cell death. it has been suggested that cancer cells also have the capacity to evade the surveillance by the immune system. the proteasome and macroautophagocytosis are attractive effector mechanisms for the immune system, because they can be used to degrade foreign substances, including pathogenic protein, within cells. here, we investigated that dm , which is a saponin derivative isolated the stem bark of dracaena mannii induced autophagocytosis on a human lung cancer cell line. methods: dm induced cell cytotoxicity was measured by mtt assay and propidium iodide staining on a cells. we examined the morphological change using optical microscope. and gfp-lc punctation was measured using confocal. the protein expression was measured by western blot analysis and the mrna expression was measured using reverse transcription poly chain reaction (rt-pcr). results: dm was showed high cytotoxicity on various cancer cell line, especially a cells. dm treated a cells did not show regular dna fragmentation and caspases activation. we also analyzed protein expression of apoptotic marker, but protein level didn't change. as a result, we hypothesized that this non-apoptotic cell death is mediated autophagocytosis. we checked lc and beclin- protein and mrna expression and inhibitory effect of autophagocytosis inhibitor. the expression level of lc was increased concentration and time-dependently. beclin- also was increased. and then, we examined gfp-lc punctation on a / gfp-lc stable cells using confocal. a cells were formed gfp punctuation after dm treatment. to confirm these data, we measured cell death ratio using -methyladenine ( -ma), an autophagocytosis inhibitor. after -ma treatment, dm induced cell death was decreased comparing with dm treatment. conclusion: dm did not induce apoptotic cell death. but dm showed several characteristics of autophagic cell death. taken together, dm induced autophagocytosis on a cells. these finding indicated that therapeutic potential of dm by triggering autophagic cell death. s. b. rasmussen , s. r. paludan university of aarhus, department of medical microbiology and immunology, aarhus, denmark during viral infections, different pattern recognition receptors detect specific pathogen associated molecular patterns (pamp)s. in the case of herpes simplex virus (hsv), detection is, among others, conducted by toll like receptor (tlr) , which is a transmembrane receptor located in the endosomes where it detects unmethylated cpg rich dna of extracellular origin, e. g. viruses. upon binding to dna, tlr initiates downstream signalling cascades resulting in induction of antiviral cytokines, interferon (ifn)a and ifnb being some of the most essential ones. the exact route of hsv to the endosomes and thus tlr recognition is not clear-cut. the endocytosis pathway is believed to be a central mechanism in which viruses translocate to the endosome, but recently the role of autophagy in tlr mediated viral recognition has been drawn in to focus. we have found indications of an autophagy dependent ifn expression during hsv- infection. by use of an entry defect glycoprotein l and glycoprotein h deficient hsv- , we found that tlr dependent ifn regulatory factor activation was abrogated. in addition, inhibition by -methyladenine of phosphoinositol -kinase class iii, which is crucial in autophagosome formation, abrogates ifnb induction. these findings points to a role for autophagy in tlr dependent recognition. in the ongoing project we are examining how hsv- triggers formation of autophagosomes. especially the role of endoplasmatic reticulum stress and doublestranded rna-dependent protein kinase will receive attention. also the newly found involvement of ubiquitin and acetylation in autophagy execution and regulation will be investigated. j. zovko , i. berberich , gk -immunomodulation universität würzburg institut für virologie und immunbiologie, würzburg, germany members of the bcl- family control the integrity of mitochondria and thereby influence survival and death of cells. most bcl- family members can localize to intracellular membranes via hydrophobic sequences within their c-terminal portion. murine a and its human homologue bfl- are anti-apoptotic members of the bcl- family. a is expressed in small amounts in the bone marrow and immature b cells, but in high amounts in mature b cells. thus the protein seems to be important for b cell maturation. we analyzed the function of the c-terminus of a . unless the c-terminal ends of other bcl- proteins the tail of a does not function as a strong membrane anchor. nevertheless, the last amino acids of a are important for the protein. in fact, the c-terminus of a serves a dual function by being required for the instability and the anti-apoptotic potential of the protein. we show that a undergoes proteosomal degradation controlled by its c-terminus. interestingly, binding to the proapoptotic bcl- factor bimel results in increased stability of a . this is due to reduced ubiquitination of a after binding of bimel. we conclude that the cterminus of a /bfl- serves as a docking site for e ubiquitin ligase(s) that control the stability of a by targeting the protein to the proteasomal pathway. very recently, we have identified a putative e ubiquitin ligase that interacted with a in a yeast two-hybrid screen. currently, we are trying to confirm this interaction in mammalian cells. suppressors of cytokine signaling (socs), initially identified as negative regulators of cytokine signal transduction, have recently emerged as multi-functional proteins regulating inflammation, survival, differentiation, and apoptosis of immune cells. here we describe novel function of socs- in the suppression of rosmediated apoptosis and associated mechanisms using tnf-alpha induced t cell apoptosis as a model system. both in jurkat t cells and primary splenocytes, socs- is induced under tnf alpha-induced apoptosis conditions. the tnf-induced apoptosis was mediated by generation of ros, and the over-expression of socs- significantly suppressed tnf-induced ros levels and the subsequent apoptosis. such anti-apoptotic function of socs- was manifest not only by the suppression of jaks acting upstream of p mapk, but also protection of ptps which down-regulate the tnf alpha -induced jak / activities. we further show that tnf-alphainduced ptp inactivation can be prevented by socs which up-regulates thioredoxin levels. finally we present data that there is a molecular interaction between thioredoxin and ptp which is formed in response to ros-generating stimuli and sustained in socs- overexpressing cells. the results strongly suggest that socs- acts as an anti-oxidant and anti-apoptotic factor to sustain t cell homeostasis and survival under oxidative stress imposed upon inflammatory cytokines during infection or other immune scenarios. aim: inflammation is a cardinal host response to injury, tissue ischemia, autoimmune responses or infectious agents. the effects of inflammatory mediators on the glial function are of particular interest since astrocytes contribute to the local inflammatory responses in the cns by producing cytokines, chemokines, and maintain local homeostasis clearing released neurotransmitters. cxcl /sdf- a not only regulates cell growth and migration of hematopoietic stem cells but may also play a central role in brain development. moreover, it has been described that tnf-a mediates in cxcl effects on primary astrocytes, and it is clear that tnf-a participates in the pathogenesis of many neurological conditions. methods: we used the astrocytoma cell line u- , and sk-n-mc as neuroblastoma cells. detection of tnf-a mrna expression was carried out by rt-pcr. transcriptional activity was measured using luciferase reporter gene assays in transiently lipofectin transfected-cells. we performed cotransfection experiments of nfat promoter construct with a dominant negative version of nfat (dn-nfat). neuronal death was performed by mtt and tunel assays. nfat translocation was confirmed by western-blot. p and fas-l expression was carried out by western-blot. results: cxcl induced mrna-tnf-a and transcriptional activity of tnf-a promoter in human astrocytes. this cytokine by itself was not toxic when added directly to astrocytes, but when we investigated its effect on nb cultures, neuronal death increased in a direct and indirect way. surprisingly, tnf-a did not induce nf-kb activation in nb cells but it induced nfat activity. nfat translocation was confirmed by western-blot. neurotoxicity was absolutely reverted in the presence of ciclosporin. we discard p pathway as responsible for tnf-mediated toxicity since we did not find any alteration in p -ser levels in stimulated cells. in addition, we found increased fasl expression in neuroblastoma cells after h of tnf-a treatment. conclusions: cxcl induced-tnf-a promotes nfat activation in neuroblastoma cells and this event leads to increased apoptosis through an increase of fasl expression without alter p function/levels. s. y. demiroglu , r. dressel universitätsmedizin göttingen, zelluläre und molekulare immunologie, göttingen, germany objectives: intracellular hsp is part of the cellular stress response system and can inhibit specific apoptotic pathways. extracellular hsp on the other hand, is an immunological danger signal that can induce the antigen-specific activity of cytotoxic t lymphocytes (ctl). interestingly, hsp does not protect against cell death mediated by ctl. acute overexpression of hsp can even increase the susceptibility of target cells to ctl, which use the granule-exocytosis pathway to induce apoptosis (dressel et al. cancer res : ) . granzyme b is one of the main effector proteases of cytotoxic granules. therefore, we analyzed the effect of acute overexpression of hsp on granzyme b-induced apoptosis. methods: hsp was expressed in a human melanoma cell line under the control of a tetracycline-inducible promoter (ge-tet). the effect of hsp induction on granzyme b-mediated apoptosis was now analyzed after delivery of granzyme b into the cytosol of the target cells by the endosomolytic activity of adenovirus type . results: hsp did not protect the melanoma cells against granzyme b-mediated apoptosis when annexin v binding, loss of mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase- were evaluated. instead, we observed a moderate but significant pro-apoptotic effect of hsp in ge-tet cells when late apoptosis was analyzed at the nuclear level by sub g peak measurements (p= . ). in contrast, a partial protection of ge-tet cells was observed after acute hsp overexpression when apoptosis was induced by staurosporine. conclusion: acute overexpression of hsp does not seem to protect tumor cells from granzyme b-induced apoptosis; it appears even to accelerate the progression of apoptosis to dna fragmentation and nuclear condensation. it is conceivable that this hsp effect is mediated by chaperoning pro-apoptotic molecules and improving their function as it has been reported for the caspase-activated dnase (liu et al., blood : ) . thus, granzyme b might be able to kill even those tumor cells that undergo an otherwise protective stress response. the work has been supported by the grants grk and dr / - . the authors thank prof. c. j. froelich (evanston, usa) for the granzyme b and dr. t. seidler (göttingen) for the adenoviruses. tumor necrosis factor (tnf) elicits its biological activities by stimulation of tnfr and tnfr , both belonging to the tnf receptor superfamily. tnfr -mediated signal transduction has been intensively studied and is well understood, especially with respect to activation of the classical nfkb pathway, cell death induction and map kinase signaling. in contrast tnfr -associated signal transduction is poorly defined. earlier findings demonstrated that only membrane tnf, but not soluble tnf, properly activates tnfr , resulting in depletion of traf from the cytoplasm. here we confirm that tnfr induced depletion of cytosolic traf by the use of tnfr -and tnfr -specific mutants of soluble and membrane tnf. corresponding with the known inhibitory role of traf in the alternative nfkb pathway, we show that tnfr induced activation of the alternative nfkb pathway. thus, we identified activation of the alternative nfkb pathway as a tnf signaling effect that can be specifically assigned to tnfr and membrane tnf. j. c. morales , d. ruiz-magaña , d. carranza , c. ruiz-ruiz universidad de granada, instituto de biopatologí a y medicina regenerativa. centro de investigación biomédica, armilla, spain different molecular mechanisms have been involved in the resistance of tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (trail)-mediated apoptosis. epigenetic modifications commonly associated with tumor development, such as histone deacetylation, may influence the resistance of some tumor cells to trail by regulating gene transcription of trail receptors and other trail pathway related-genes. in the present study we have analyzed the effect of several histone deacetylase inhibitors (hdaci), belonging to different structural families, on trail-induced apoptosis in leukemic t cell lines. moreover, we have analyzed the activity of hdaci in normal t lymphocytes. we have found that, to a greater or lesser extent, all hdaci potentiate the induction of apoptosis in leukemic t cells by enhancing the signals triggered upon trail ligation, from the activation of the most apical caspase- . in contrast, hdaci do not sensitize primary resting or activated t lymphocytes to trail-mediated apoptosis. the analysis of the expression of several pro-and anti-apoptotic proteins involved in the trail-signalling pathway indicates that most hdaci regulate the expression of trail-r and c-flip in leukemic t cells, but not in normal cells, which may explain their selective pro-apoptotic effect on leukemic cells. zfp l is a zinc finger containing protein that is involved in post-transcriptional gene regulation. it can bind to mrnas containing adenine uridine rich (are) regions and subsequently mediate their degradation. we have previously reported a role for this protein in promotion of b cell apoptosis. one mechanism whereby zfp l may mediate cell apoptosis could be by degradation of cell survival gene mrnas. the bcl- protein is an important cell survival protein at different stages of b cell development. bcl- mrna also contains are regions that could possibly be targeted by zfp l protein. in the present study, we have tested the ability of zfp l protein to bind to a bcl- mrna are probe and to degrade bcl- mrna. recombinant bacterially expressed zfp l protein was shown to bind specifically to a bcl- are probe by rna electrophoretic shift assays (remsas). furthermore, remsas using cell lysates of ramos burkitt lymphoma b cells stimulated to express high levels of endogenous zfp l also provided evidence that endogenous zfp l in b cells could bind to the bcl- are. in order to examine whether zfp l binding to bcl- are resulted in bcl- mrna degradation, actinomycin d rna degradation assays were carried out on murine embryonic fibroblast (mefs) cells from zfp l knockout mice and wild-type mice using quantitative real-time pcr analysis. bcl- mrna was expressed in both wild-type and knockout mefs. the half-life of bcl- mrna was found to be extended in knockout mefs compared to wild-type mefs suggesting that zfp l does play a role in degradation of bcl- mrna. overall, our data are consistent with a role for zfp l in degradation of bcl- mrna which could be a mechanism for the reported role of this protein in induction of b cell apoptosis. the epstein-barr virus (ebv) is a common human herpes virus, which can predominantly infect two types of human cells: lymphoid cells and epithelial cells. its infection is associated with several human malignancies (hodgkin's lymphoma, burkitt's lymphoma, nasopharyngeal carcinoma), where it expresses limited subsets of latent proteins among which the latent membrane protein lmp . since lmp is able to transform numerous cell types, it is considered as the main oncogenic protein of ebv. the principal mechanism of lmp function is based on mimicry of activated member of the tnf receptor super family (tnfr), by its ability to bind a similar sets of adapters and to activate overlapping signalling pathways like nfkb, c-fos-jnk, pi -kinase...involved in the regulation of cellular processes. we previously generated two unique model, a monocytic (te ) and lymphocytic (nc ) immortalized by ebv and which expressing type ii latency program. here we developed original dominant negative (dn), by generating a fusion between gfp and tes or tes (transforming effectors site) derived from the c-terminal intracellular part of lmp , in inducible vectors. then, we generated cell lines conditionally expressing these dns. we showed these dns not only inhibit survival processes resulting to the impairment of nfkb and akt pathway but increase apoptosis in this cell lines. we demonstrated that this pro-apoptotic effect is due to i) the depletion of lmp 's specific adapters and ii) the recruitment of theses adapters by dns interestingly allowed generation of apoptotic complex involved tradd, fadd and caspase- . using this nc tumorigenic model in scid mice, we showed that induction of the dn lmp -tes prevent development of tumours and mouse death. these dominant negative derived from lmp could be used to develop therapeutic approaches in malignant diseases associated with epstein-barr virus, but also in inflammatory pathologies. recent studies indicate that suppressors of cytokine signaling (socs) proteins play, in addition to their action as cytokine signaling inhibitors in the immuneinflammatory response, multiple roles in cell survival, differentiation, and apoptosis in diverse cell systems. since tumor cells often exhibit aberrant expression of socs genes, which may be involved in determining resistance to anti-tumor therapies, we have investigated the role of socs isoforms during dna damageinduced apoptotic response and cell cycle changes in various tumor cell types. by using tumor cell lines transduced for over-expression or knock-down of distinct socs isoforms, it is found that socs and socs differentially affect apoptosis and cell cycle changes induced by dna damaging agents in a cell type-specific manner. in t lymphocytic leukemia cell line jurkat, socs exhibited anti-apoptotic effect in response to ionizing radiation, hydrogen peroxide, and etoposide by inducing suppression of p mapk activities, while socs promoted apoptosis with an increase in p activities. in contrast, both socs and socs display proapoptotic effect in rko colon cancer cell lines upon exposure to gamma radiation or ros-generating agents. notably, effects of socs proteins on cell cycle changes induced by dna damaging agents were rather similar in that over-expression of either socs or socs induced a slight decrease in g or s phase cells and a prominent increase in g /m cells, regardless of their distinct effects on apoptosis. the analysis of cell cycle regulator proteins, however, revealed that different mechanisms are operating to regulate cell cycle via distinct cyclins and cdk inhibitors affecting g /s transition and g /m arrest induced by socs or socs . socs promoted dna damage-induced p induction and g /m cyclin b expression, while socs induced decrease in g cyclin e expression. the results suggest that socs isoforms potentially modulate growth of tumor cells exposed to dna damage via complex network involving apoptotic response and cell cycle regulation in a cell type-specific manner. the heat shock protein (hsp ) is a highly conserved a widely expressed molecular chaperone. it is known to regulate the activity of several protein kinases or the proper folding of client proteins. hsp has also been identified as an important regulator of cellular survival. besides these intracellular functions, extracellular hsp can initiate cross presentation or immune responses. apoptotic cell death occurs permanently in multicellular organisms, without initiation of an immune response. however the mechanisms which prevent an inflammatory response to apoptotic cells are not understood to date. hsp is released during necrotic cell death and proinflammatory effects of extracellular hsp have been observed. thus, we asked whether apoptozing cells cleave hsp during apoptosis or how hsp is disposed by these cells. we induced apoptosis either in activated or resting primary human cells and analyzed the hsp protein content. we observed that hsp is degraded during apoptosis resulting in the formation of a fragment of about kda. this fragment was to be observed exclusively in activated cells, while it was not detected if resting cells were induced to undergo apoptosis. analyzing the isoforms of hsp (hsp alpha and hsp beta) we could show that the kda fragment is formed after degradation of the alpha isoform of hsp . further, we were able to show, that hsp cleavage is dependent on caspase activity and most probably mediated by calpain. analyzing the cytokine response of monocyte derived phagocytes to apoptotic cells in presence or absence of exogenous hsp and caspase inhibitors. we observed a rather proinflammatory cytokine profile, if cleavage of hsp was inhibited or if exogenous hsp was added. these results demonstrate that cleavage of hsp represents a mechanism preventing the release of proinflammatory molecules from apoptozing cells. activity. mifc integrates the advantages of flow cytometry and fluorescence microscopy in one system, the imagestream. upon induction of autophagy, cytosolic lc -i is processed to lc -ii, which then remains associated with the autophagosome until its degradation upon fusion with the lysosome. an increase in steadystate levels of autophagosomes can be due to enhanced autophagy or decreased lysosomal activity. mcf- gfp-lc cells were therefore incubated in starvation medium for hours, +/-bafilomycin, which potently inhibits lysosomal activity. classical gating strategies allowed the detection of cell populations of interest, which were further analyzed on single cell levels. we conclude that imagestream-based analysis provides an improved method in terms of objectivity, sensitivity and significance, to quantify autophagic activity. our results clearly show the need for discrimination between "steady-state" levels of autophagosomes and "current flux" of fully functional autophagy, i. e. quantification of autophagic flux. jnk seems to mediate the bcl- /beclin- control of autophagy. recently, jnk was shown to be necessary for beclin- upregulation, and jnk-mediated phosphorylation of bcl- is associated with both, starvation-and ceramide-induced autophagy. the nfkappab pathway mediates critical survival signals during starvation, which have been linked to the inhibition of autophagy. we report here the novel findings that under conditions of starvation, pharmacological inhibition of nfkappab decreased the autophagic flux in mcf- cells, while jnk inhibition shows an enhancing effect on autophagy induction. ingenol -angelate (pep ), a novel activator of protein kinase c (pkc), has been shown to induce apoptosis in acute myeloid leukemia cells. we show here, that in contrast to leukemic cells, pep provides a strong survival signal to resting and activated t cells. this anti-apoptotic effect was dependent upon the activation of pkcv, a pkc isoform restricted to t cells and myocytes. expression of pkcv in the acute myeloid leukemia cell line nb turned their response to pep from an increased to decreased rate of apoptosis. furthermore, our data show that pep inhibited t cell apoptosis through the activation of nfxb downstream of pkcv, leading to increased expression of the anti-apoptotic proteins mcl- and bcl-xl. we conclude that pkcv expression determines whether pkc activation leads to an anti-or pro-apoptotic outcome in the cell types analyzed. this finding may be of considerable importance for the development of pkc -targeting antileukemic therapies. the neuronal growth factors, neurotrophins, and their receptors are widely expressed in a variety of non-neuronal tissues including the immune system. several reports indicate that survival and activation of normal b lymphocytes are regulated by nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf) autocrine circuits. however, the production and the role of neurotrophins were not evaluated in b lymphoma cells. diffuse large b-cell lymphoma (dlbcl) is a common and often fatal malignancy. despite major advance in the treatment (r-chop protocol) which improves the clinical outcome of patients, a subset of patients does not respond or relapses after the initial treatment; the exact mechanism of such resistance is not entirely clear. we hypothesized that autocrine neurotrophin survival circuits could contribute to the chemoresistance of dlbcl tumor cells. this hypothesis was investigated with dlbcl cell lines (su-dhl). thus, we evaluated the ability of su-dhl cells to produce neurotrophins (ngf, bdnf) and to express their receptors (p , trka and trkb) in different cell culture conditions. our preliminary data show for the first time the production of neurotrophins by dlbcl tumoral cells whose level decreased in apoptotic conditions, in association with bad dephosphorylation suggesting its pro-apoptotic role. furthermore our results suggest that up-regulation of autocrine circuits (expression of trka known to be involved in survival signaling pathways) may contribute to cell survival and thus drug resistances of tumoral b cells. objectives: ataxia telangiectasia (a-t) is a rare disorder caused by mutations in the ataxia telangiectasia mutated (atm) gene. this gene encodes atm, a protein kinase which has a major role in dna double strand break response. a-t patients suffer from a variety of immune system defects including lymphopenia, immunoglobulin deficiencies and impaired class switch recombination, they also have an increased incidence of cancer especially leukaemia and lymphoma. the susceptibility to lymphoid tumours and immunodeficiency could be partly due to failure of extrinsic apoptotic processes involved in regulation of the immune system. although atm is known to have a central role in the induction of apoptosis in response to unrepaired dna double strand breaks its role in extrinsic apoptotic pathways is unclear. this study aimed to investigate if atm has a role in fas induced apoptosis. a bank of lymphoblastoid cell lines (lcls) derived from a-t and normal individuals and tumour samples with wildtype or mutant atm were used in the study. apoptosis was induced by incubating cells with the fas activating antibody ch and analysed by flow cytometry. expression of the caspase inhibitor cflip which inhibits fas induced apoptosis was detected by western blot. results: there was no significant difference in the susceptibility to fas induced apoptosis or cflip protein expression between atm mutant and control groups. however cells expressing high levels of cflip protein do show greater resistance to fas induced apoptosis than those with lower expression. whilst the lcls expressed both long and short forms of cflip, the tumour cells expressed only the long form. conclusion: atm mutations do not affect susceptibility to fas induced apoptosis or alter cflip protein expression in lcls or tumour cells. cflip protein levels and fas susceptibility vary greatly between individuals but this is independent of atm status. high expression of cflip protein correlates with reduced apoptosis in response to ch treatment but there is no clear difference in cflip expression between atm wildtype and mutant cells. labdane diterpenoids have a broad spectrum of biological activities including antibacterial, antiviral, and anti-inflammatory properties. however, little is known about their possible role in the apoptotic cell death machinery. we report that labdane diterpenoids induce apoptosis in different tumor cell lines by activating caspase in the extrinsic death receptor pathway, with subsequent participation of mitochondrial signaling. activation of caspase by diterpenoids was followed by a decrease in mitochondrial membrane potential, the release of apoptotic factors from mitochondria to the cytosol, and subsequent activation of caspases and . diterpenoids also led to time-dependent cleavage of bid. inhibition of caspase- abrogated these processes, suggesting that the death receptor pathway plays a critical role in the apoptotic events induced by labdane diterpenoids. in addition, pretreating cells with neutralizing antibodies to fas ligand, tumor necrosis factor receptor (tnf-r ), and tumor necrosis factor (tnf)-a receptor (trail) inhibited diterpenoid-induced apoptosis, revealing it to be dependent on these death receptors. diterpenoid treatment also induced a significant increase in the generation of reactive oxygen species (ros). however, increased ros production was not directly involved in diterpenoid-triggered apoptosis. these results demonstrate that labdane diterpenoids induce apoptosis through activation of the death receptor pathway. conclusion: cell proliferation and differentiation are tightly regulated networks and it is believed that in cell differentiation, even in cancer form, cells precluded from proliferation. whether these changes affect the level of differentiation or the change of survivin expression can affect the proliferation and differentiation pathways are the hypotheses that need further investigation. synthetic alkyl-lysophospholipids (alps) are a group of unnatural lipids with promising anticancer capability. a prototypic member is the ether lipid -o-octadecyl- -o-methyl-rac-glycero- -phosphocholine (et- -och ; edelfosine), which induces selective apoptosis in tumor cells through activation of fas/cd independent of its ligand fasl/cd l. fas/cd is activated by edelfosine via its translocation in lipid rafts. in this study we showed that edelfosine promotes cell death in multiple myeloma and various solid tumor cell lines in a death receptor-independent manner. edelfosine-treated cells could not be protected against cell death after inhibition of caspases by zvad-fmk while fasl-stimulated cells stayed mostly alive. furthermore cells could not be rescued by addition of zvad-fmk in combination with necrostatin- , an inhibitor of death receptor-induced necrosis. fas resistant solid tumor cells overexpressing members of the anti-apoptotic bcl -family as well as cells overexpressing the cellular regulatory protein flip went in contrast to fas stimulation to apoptosis after treatment with edelfosine. therefore we suggest that edelfosine induces a death receptor-independent cell death pathway in a wide range of tumor cells. apoptosis represents a cellular "suicide" mechanism which allows the control of cell number from tissues and elimination of cells that present dna mutations or having an abberant cell cycle, those cells being predisposed to malignant transformation. thus, elucidating the mechanisms of programmed cell death process seems to be of great importance for malignant transformation, tumour evasion and therefore for anti-cancer therapy. many anti-cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction. the present study focused on the potential influence of oncolitical treatment ( -fluorouracyl) associated with natural compounds (curcumin, genistein, quercitin) on the dynamics of the cell cycle and levels of apoptosis in colon cancer cell lines (e. g. colo , sw , lovo, caco- , ht- ) . in addition, expression of antigens involved in tumour proliferation and apoptosis (ki- , pcna, p , bcl- ) was compared with gene expression in the presence or absence of stimuli treatment. percentages of apoptotic cells were detected by using annexin v/fitc and propidium iodide double staining, while progression through cell cycle phases was evaluated by using pi staining. correlation analyses between the individual profile of the stimuli modulated gene expression with the coded protein expression were performed by using data from rt-pcr with specific primers, and indirect immunofluorescence followed by flow cytometry, respectively. stimuli treatment of colon cancer cell lines differentially induced higher levels of apoptosis as compared to untreated tumour cells, while cell cycle distribution of dna changed. data obtained showed a various expression and functional behaviour of the markers under study associated to colon cancer cells, suggesting their possible involvement in regulating the interactions between tumour cells and host immune system. the results obtained might further lead to the establishment of an experimental pattern for the corroboration of cell and molecular mechanisms involved in the tumour progression and the treatment resistance of colon tumors using cell lines. the effect of modulatoy agents on proliferation and apoptosis could be used in clinical departments in order to elaborate new therapeutical approaches and act as useful instruments in elaboration of individualized treatment schemes. extensive tissue trauma and malnutrition results in disorders of programmed cell death influencing the patients susceptibility to infections. the purpose of our study was to assess the effect of pancreatic cancer surgery and immunonutrition on the apoptotic signaling pathways. the randomized studies were performed in patients after pancreatic cancer resection with preoperative standard or enteral immunonutrition. lymphocytes expressions of bcl- , bax, caspase , , , nfkb, parp / kda, tnfr /cd a and cd /fas were assessed by western-blot and flow cytometry. results: before and after surgery the expression of bcl- , bax, nfkb, parp was significantly lower and expression of caspases, tnfr as well as percentage of cd cells significantly higher as compared with control group. caspase expression was significantly higher as compared with nfkb, parp and tnfr . in comparison to the standard nutrition preoperative immunonutrition increased bcl- and nfkb expressions and decreased caspases and parp expressions. in addition, we found a significant down-regulation of bcl- expression after surgery, but insignificant in patients with preoperative immunonutrition. conclusion: preoperative enteral immunonutrition has an modulative effect on apoptotic signaling pathways after pancreas resection and possesses antiapoptotic properties. this modulatory effect of glutamine and omega- fatty acids has no influence on patients outcome. the capacity of medicinal herbs to modulate cellular and humoral immune response could have useful applications in some immune-mediated disorders, infections and cancers. in this study the immunomodulatory effects of salvia mirzayanii a native plant that is widely distributed to iran was investigated. s. mirzayanii is used for the treatment of infectious and inflammatory diseases and as a tonic in folk medicine. study of the effect of this plant on the activated human peripheral blood lymphocytes showed stimulatory effects at lower concentrations and inhibitory effects at higher ones (p x . ). in flow cytometry analysis, accumulation of apoptotic cells in the sub-g phase of cell cycle of the mitogen-treated lymphocytes exposed to the inhibitory doses of the extract was observed. dna fragmentation analysis of these cells showed a typical dna laddering. immunization of the extract-treated mice with the antigen decreased delayed hypersensitivity skin reaction as well as the antibody titer at higher concentrations (p x . ). these results indicated the presence of immunomodulatory compounds in the extract of s. mirzayanii and suggest that the induction of apoptosis in lymphocytes might be the mechanism responsible for the inhibitory effect of the extract observed at higher concentrations. a new randomized, double-blind, placebo-controlled clinical trial was conducted with healthy volunteers receiving either la ( cfu/day) or placebo, during days prior to uv ( × . med). blister roofs, liquid and skin biopsies were collected , and days after uv exposure from non-irradiated and irradiated skin areas and used for identification of cells involved in uv-induced immune response, quantification of inflammatory cytokines, a dna damage marker (p ). while a similar decrease of lc for both groups was observed on day after uv exposure compared to placebo, la- group presented a faster increase of a new subset of epidermal dendritic cells (dc), namely early lc precursors (cd a low cd -) associated with a minor recruitment of monocytes. concomitantly, inhibition of il- and a tendency to inhibit il- was observed in la- group compared to placebo. on day , la- group presented a greater recruitment of early lc precursors and a trend to increase cd a low cd + lc precursors compared to placebo. additionally, a faster reduction of inflammatory and immunosuppressive cytokines (il- , tnfa, il- , and il- ) was observed in la group compared to placebo. we show that la limits uv-induced immune-suppression and skin inflammation. this contributes to the recovery of the skin immune homeostasis, confirming the previously observed benefits of la supplementation for photoprotection at a lower dose ( log). the thymus is one of the primary lymphoid organs and plays a central role in the immune system. it provides the essential microenvironment for proper t cell development. in the thymus, the maturation of t cells depends on many interactions between t cells and different stromal cell types, mainly composed of epithelial cells (tecs). foxn is a winged-helix/forkhead transcription factor, which is crucially required for proper epithelial cell differentiation in the thymus. foxn appears to be expressed in all epithelial cells of the early thymic rudiment starting around e . . previously, we have used a lineage-tracing system to confirm the existence of a bi-potent epithelial progenitor cell. using the cre-loxp system, we showed that a single epithelial cell, when reverted to express foxn in a nude (foxn -deficient) background, can give rise to a functionally competent thymic microenvironment. hence, we hypothesize that the epithelial progenitor cell expresses foxn . if true, it should be possible to target this cell type by use of foxn -promoter driven transgenes. conditional targeted cell ablation is a powerful method to elucidate the physiological function of cell populations and their regenerative capabilities. currently, we are using three different strategies of conditional targeted cell ablation in order to examine functional characteristics of epithelial bi-potent progenitor cells within the thymus. intracellular accumulation of poly glutamines is known to cause neurodegenerative disorders, such as huntington's disease. considering this, we are expressing transgenic egfp variants containing either or glutamine residues under the control of foxn promoter, leading to different degrees of tec degeneration. furthermore, also under the foxn promoter, we are using the transgenic expression of human diphtheria toxin receptor and the transgenic expression of the bacterial nitroreductase enzyme that converts the pro-drug metronidatole (mtz) into a cytotoxic cross-linking agent for conditional cell ablation. preliminary results describing the phenotypes of these mice will be presented. t. kamei , , y. toriumi , k. kimura european university viadrina, frankfurt (oder), germany, shimane institute of health science, izumo, japan, shimane university faculty of medicine, department of pediatrics, izumo, japan, japan yoga niketan, yonago, japan as a method in relieving stress, yoga is popular today. many reports of physical changes describing how yoga improves respiratory, circulatory, endocrine, and metabolic functions by yogic practice have been reported until now. we examined changes of electroencephalograph (eeg) and cellular immunity before, during, and after yoga exercises, in an endeavor to detect the correlation between them. the subjects consisted of eight yoga instructors who had been practicing yoga for several years. a -minute-rest period, followed by a -minute yoga exercise called asana, a -minute respiratory exercise called pranayama (various specialized respiration methods continuously performed with the eyes closed), and a -minute meditation were performed. throughout rest and yoga, brain rhythms were continuously recorded via two disc electrodes placed on each forehead (fp ). blood samples were drawn before and after each exercise. nk activity and percentages of t-cell and b-cell subsets were measured. during the pranayama period, both a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in nk activity (r= . , p x . ), and a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in the number of t lymphocytes (r= . , p x . ) were observed. furthermore, a positive correlation was also observed between the change in amplitude of the activated alpha waves and the ratio of change in the number of cd (r= . , p= . ). these findings suggest that yoga creates a stress-free and mentally concentrative state which activates the functions of nk cells and t lymphocytes, mainly of cd , within a short period of time. we conclude from these results that yogic respiratory exercise may be able to activate cellular immunity and to help recover the mental and physical harmony of human. yoga is considered to have an effect of some re-activation of a latent ability of harmonization in which humans naturally possess. t regulatory cells play a central role in the suppression of immune responses thus serving to induce tolerance and to control persistent immune responses that can lead to autoimmunity. several recent studies suggest also that diverse populations of regulatory t cell play an important role in regulating t-helper response to allergens, maintaining functional tolerance and preventing allergy. here we demonstrate that cd + cd + t regulatory (t reg ) cells are critical in controlling the immediate hypersensitivity response of bone marrow mast cells (mcs) without affecting cytokine release. this effect is shown to require a cell-cell contact and depends on interaction between ox l expressed on mcs and the constitutive expression of ox (members of the tumor necrosis factor [tnf] and tnf receptor family, respectively) on t reg cells. this interaction does not alter the activation of plc-g, syk and lat in ige/ag stimulated mcs upon co-incubation with t reg cells, whereas it induces a decrease in the phosphorylation levels of akt. moreover, we find that upon co-incubation with t reg cells, mcs show increased levels of camp, which is known to inhibit mcs function, as a result of ox l signal. antagonism of camp in mcs reverses the inhibitory effects of t reg cells restoring normal ca + responses and degranulation. the cross-talk between t reg cells and mcs through ox -ox l interaction defines a previously unrecognized mechanism controlling mcs degranulation. loss of this interaction may contribute to the severity of allergic responses or inflammatory disease. active regulation has emerged as a very essential mechanism for both inducing and maintaining peripheral tolerance to non-pathogenic environmental antigens. a healthy immune system responds to antigens with a combination of polarized th or th effector cells and the induction of antigen specific foxp + regulatory t cells (treg). it is believed that the dominant subset determines the quality of the eventual immune response. in allergic asthma there is a clear dominance of th cell responses to non-pathogenic environmental antigens. recently it was shown that the specific transcription factors that characterize the th and treg subset, gata and foxp respectively, counter regulate each other (mantel y et al., ) . we hypothesize that children with allergic asthma will respond to allergens with low induction of foxp + tregs and high gata + th cells. in order to prove this hypothesis pbmc of children with allergic asthma and non-sensitized healthy controls are stimulated with allergens, tetanus toxoid, and lps. almost million allergic patients are sensitized to the major birch pollen allergen bet v , which cross-reacts with major allergens of fagales (e.g., alder, hazel, hornbeam, oak) pollen and plant food allergens. the epitopes of bet v recognized by allergic patients' ige antibodies belong to the conformational type and therefore have not been characterized in detail. here we used antibodies raised against peptides spanning the bet v molecule in ige competition experiments to search for sequences which are involved in ige recognition. the strongest inhibition (i.e., g %) of patients' ige binding to bet v was obtained with polyclonal and monoclonal antibodies specific for peptides comprising aa - (p ) and aa - (p ) of bet v . cross-reactive ige epitopes between bet v and related pollen allergens and plant food allergens involved primarily p . p and p are not adjacent peptides in the bet v sequence but define a surface-exposed patch on the three-dimensional structure of bet v . as determined by surface plasmon resonance, monoclonal antibody mab specific for p and mab specific for p showed high affinity, i. e., dissociation constants, k(d) = . e- m and k(d) = . e- m, respectively. interestingly, peptide-specific mabs inhibited allergic patients' ige antibodies equally well as peptide-specific polyclonal rabbit antibodies but only the latter inhibited strongly allergen-induced basophil degranulation. this finding indicates that the surface patch defined with anti-p and anti-p antibodies contains several ige epitopes. in summary, we have defined a surface-exposed patch on the bet v allergen which seems to harbor the majority of the ige epitopes and may be used for the rational design of active and passive immunotherapy strategies against birch pollen and related allergies. background: antigen-specific th cells as well as tc cells, induced by biolistic gene transfer using plasmid dna encoding the model allergen b-galactosidase (bgal) under control of the fascin promoter (pfascin-bgal), inhibited the elicitation of systemic th immune responses and suppressed ige production in an experimental mouse model. moreover, protective biolistic dna vaccination with pfascin-bgal prevented th -mediated lung pathology (eosinophilia) in sensitized mice locally challenged with bgal protein, but led to the recruitment of th /tc cells into the lung, associated with substantial neutrophilic infiltration and the induction of airway hyperresponsiveness (ahr). objective: to analyze the modalities of ahr induction in mice biolistically vaccinated with pfascin-bgal. methods: balb/c mice were immunized with pfascin-bgal using the gene gun. subsequently, mice were challenged by consecutive intranasal application of bgal protein. cd + and cd + t cells, respectively, were depleted before and during the provocation phase by intraperitoneal injection of anti-cd (gk . ) or anti-cd ( . . ) monoclonal antibodies. neutrophilic granulocytes were depleted by treatment of animals with either anti-gr- monoclonal antibody rb - c or monoclonal antibody nimp-r . one day after the last challenge airway reactivity was assessed by whole body plethysmography, bronchoalveolar lavage (bal) was performed and the frequency of ifn-g-producing cd + effector t cells in the lung was determined. results: whereas neutrophilia in the lung of immunized and challenged mice was considerably alleviated by depletion of cd + t cells, ahr was not significantly affected, implicating that the elicitation of ahr by cd + t cells is dissociated from the activity of neutrophils. this notion was verified by elimination of neutrophils during the provocation phase, likewise leading to unaltered ahr. in contrast to cd + t cells, cd + t cells induced strong neutrophilic infiltration and ahr. transfer experiments with cd + or cd + t cell, separated from the airways of vaccinated and challenged mice, will probably reveal details of the effector mechanisms of th and tc cells operative in the elicitation of airway inflammation. conclusions: robust type immune responses, although highly effective in the counter-regulation of local th -mediated pathology, might as well trigger inflammatory reactions in the lung and provoke the induction of ahr. respiratory epithelial cells function as physical barrier and have shown to be active participants within the process of defense against pathogens and recognition of allergens. upon activation they release inflammatory mediators thereby creating a micro environment in which recruited immunocompetent cells induce a local immune response. house dust mite (hdm) extract as a source of allergens has been shown to induce a broad panel of genes upon stimulation of epithelial cell line nci-h . the proteolytic activity of these hdm allergens has been proposed to be involved in the activation process. the aim of this study was to compare the influence of hdm extract on respiratory epithelial cells with grass pollen allergen-induced activation of these cells with regard to the mechanism of activation, gene expression level, and the level of induced cytokine and chemokine release. in contrast to the hdm major allergen der p , we were able to show that the major allergen of phleum pratense, phl p , although sharing molecular similarities with der p , does not display any enzymatic activity under physiological conditions. therefore, in this study respiratory epithelial cells were stimulated with grass pollen extract and purified phl p . chemokine and cytokine release was determined by multiplex enzyme-linked immunosorbent assay and mrna was used for cdna-microarray analysis. first data show that both, hdm extract and grass pollen allergens, induce the release of il- and il- from nci-h cells. furthermore, stimulation with hdm extract leads to the release of tnf-a, gm-csf and ifn-g. interestingly none of these mediators was induced after stimulation with grass pollen extract or purified phl p . in contrast to hdm extract grass pollen allergens induce the release of mcp- from respiratory epithelial cells, as well as moderate levels of il- . detailed characterization of the response on gene expression level might give new insights into the pathophysiology of grass pollen allergy and a comparison with hdm induced expression profiles will be helpful towards understanding the allergic response in general. (supported by dfg sfb tr ) results: collectively, responses to blg, but not to bsa, were observed in all groups analyzed, included healthy controls. nevertheless, distinct profiles of response were obtained: children with ige mediated cma had a significant increased level of proliferation (mean±sd of stimulation index(si): . ± . ) and of il- (mean±sd: ± pg/ml), and reduced il- (mean±sd of il- -spot forming units/ x cells (sfu): ± ), compared to healthy subjects ( . ± . si, p x . ; ± pg/ml, p x . ; ± sfu, for proliferation, il- and il- , respectively); children with non-ige mediated cma had a significant reduction of il- ( ± pg/ml), compared to ige patients (p x . ) and an increased, although not statistically significant, production of ifn-g ( . ± . sfu) compared to control ( . ± . sfu) and to ige-allergic patients ( . ± . sfu). finally, tolerant patients showed reduced il- ( ± pg/ ml, p x . ) and proliferation ( . ± . si), compared to acute ige-cma children. interestingly, the high level of il- observed in all groups might have a counter-regulatory effect, since its neutralization resulted in an increase of proliferation to blg; by contrast, il- was undetectable in all patients even blocking the il -receptor. conclusion: blg-specific, immune responses can be recalled in peripheral blood of cma patients, as well as of normal and tolerant children. a th -like response with il- and proliferation is dominant in ige-mediated cma patients; by contrast a th -skewed response with ifn-g is present in non-ige-mediated allergic and in those children who outgrew ige-allergy. y. f. tang , b. chua , f.c. lew , a. ho , k. wong , k.l. wong , d. m. kemeny national university of singapore, immunology programme, yong loo lin school of medicine, singapore, singapore allergic inflammation of the airways causes changes in the lung wall that can lead to chronic inflammatory disease such as asthma. using a mouse model, this response can be divided into an induction phase, in which cd th t cells specific for airborne allergens are produced, and an effector phase, during which they are recruited to the lung. in the lung, recruited th cells orchestrate the inflammatory response marked by eosinophilia, mucus hyper secretion and increased airway hyperresponsiveness (ahr). previously we, and others, have shown that transfer of cd t cells inhibits the induction of the th response. here we have investigated the effect of cd t cells on the effector phase of the inflammatory lung response. in vitro activated ot-i cd t cells were transferred to ovalbumin (ova)/ alum immunized mice one day before the first of airway challenges with ova. eosinophil infiltration was inhibited by transfer of cd + t cells ( . %± . % to . %± . %). when ifn-gamma -/-ot-i cd t cells were transferred, we found that the inhibitory effect on eosinophilia was reduced ( . %± . %), suggesting an important role for cd t cell ifn-gamma. cd c + cd + cd blung dcs from cd transferred mice secreted higher levels ( pg/ml) of il- p following ex-vivo stimulation as compared with animals that were not given cd t cells. these data show that, in addition to regulating the induction of the allergic immune response, cd t cells can subsequently divert the local lung environment to one that favors th immunity. the chain terminator drug abacavir triggers a serious hypersensitivity reaction in % of patients with hiv infection. this reaction is strongly associated with hla-b* and appears to be mediated by cd + t cells producing inflammatory cytokines. we show that cd +t cell responses can be primed in vitro, in normal blood donors who are hla-b* +, but not in non-b* + donors. cd t cells, but not cd t cells, are expanded by abacavir pulsed autologous apc over a -day culture, producing ifn-gamma and tnf-alpha upon re-stimulation with apc expressing hla-b* . similar responses were detected in abacavirhypersensitive hlab* + patients. responses were not detected using mutant apc deficient in tap or tapasin, or when normal apc were aldehyde fixed before loading with abacavir, indicating a reliance on the conventional mhc-i ag presentation. responses were exquisitely restricted to hla-b* since they were undetectable using apc expressing closely related hla-b or b allotypes. responses to apc expressing mutants of hla-b* demonstrated a crucial role for residue . isolation of peptide fractions from abacavir-loaded cells has led to the identification of specific fractions recognised by an abacavir-specific t cell line. our data suggests that abacavir forms a conjugate with an endogenous peptide that is presented by hla-b* triggering cd t cells. we speculate that this form of altered self is highly immunogenic, behaving like a form of allogeneic mhc-i, contributing to the responses observed in abacavir naï ve individuals. the molecular mechanisms underlying altered hla-b* may be relevant to the role of other disease-associated class i allotypes such as hla-b and b . a. jenckel , s. bulfone-paus , n. föger research center borstel, immunobiology, borstel, germany mast cells play a crucial role in acute inflammatory and allergic reactions. upon activation, mast cells secrete a vast array of preformed and newly synthesized inflammatory mediators. recent work has begun to appreciate an important role of the actin cytoskeleton in mast cell activation. the actin-associated protein coro-nin a (coro a), a coronin family protein preferentially expressed in hematopoietic cells, is critically involved in various actin-mediated cellular functions of leukocytes. recent data of our group also indicate a regulatory role of coro a in mast cell function. coronin proteins have been described to be differentially phosphorylated in vivo. however the molecular mechanisms by which coro a is regulated in response to physiological stimuli are still poorly characterized. here we investigated the modalities of coro a phosphorylation during the activation of mast cells. immunoprecipitation studies combined with phospho-specific western blotting techniques revealed a transient phosphorylation of coro a on serine residues upon antigen-specific engagement of fc-epsilon-receptors. as the phosphorylation status of coro a can influence its association with the actin cytoskeleton, we analyzed the subcellular localization of coro a during mast cell activation. cell fractionation experiments demonstrated that the association of coro a with the actin cytoskeleton significantly decreases in response to mast cell stimulation, concomitant with the increase in coro a phosphorylation. a functional correlation between coro a phosphorylation and its association with the actin cytoskeleton in mast cells was further indicated by structure function experiments employing specific phosphorylation mutants of coro a. thus, coro a is a downstream effector molecule of fc-epsilon-receptor signaling and likely is involved in the dynamic reorganization of the actin cytoskeleton during mast cell activation. allergen-specific t and b lymphocytes play an important role for the pathogenesis of asthma. t cells orchestrate the infiltration of the lung tissue with eosinophils and neutrophils and provide help for allergen-specific immunoglobulin production. recently, we have shown in a mouse model for allergic airway inflammation that b cells directly interact with t cells in the inflamed tissue and locally produce ige. to analyse t/b-interaction in the inflamed tissue in more detail, we developed a novel adoptive transfer system using ovalbumin-specific t cells and nitrophenolspecific b cells. recipient mice are then challenged intranasally with an np-ova conjugate. this system allows to track single allergen-specific t and b cells in all stages of the immune reaction using flow cytometry and immunohistology. in addition, cells can be re-isolated by flow-sorting for in-depth analysis. using this system we could define several phases of the inflammatory reaction. t and b cells first become activated in the lung-associated lymph nodes. granulocytes can be found very early in the lung tissue and also activated t cells very rapidly emigrate to the site of inflammation. however, clusters of allergen-specific t and b cells can only be found in later stages of the reaction. as another focus, we used our in vivo system to define the role of t cell costimulatory molecules for airway inflammation. costimulatory receptors are key regulators of t cell activation and differentiation and therefore promising targets for therapeutic intervention. of special interest is the t cell-specific icos molecule which is important for t/b cooperation as well as the regulation of chronic inflammatory reactions. using icos knock-out mice we were able to delineate the specific role of icos for the different stages of airway inflammation. in particular, we analysed the impact on t cell subset differentiation, cytokine production and allergen-specific immunoglobulin production. the integrity of the actin cytoskeletal network is critcal for a large variety of cellular functions. coronins constitute a family of evolutionary highly conserved wdrepeat containing proteins that have been implicated in the regulation of actin cytoskeletal dynamics. in mammalians seven coronin family members have been described. the high degree of sequence conservation amongst coronin family proteins suggests common features and functions. however, individual family members may also have developed additional selective and specific functions. our recent studies on coronin a (coro a) deficient mice have demonstrated that coro a exhibits an inhibitory function on the cellular steady-state f-actin content, is required for chemokine-mediated functions in t cells and is involved in the maintenance of t cell homeostasis. coronin b (coro b) is a closely related homolog of coro a and the two genes are co-expressed in hematopoietic cells. to address the question of functional redundancy in vivo, we have generated coro b deficient mice and crossed them with coro a deficient mice to obtain coro a/coro b double deficient mice. analysis of t lymphocytes from coro a/coro b double deficient mice revealed defective chemotactic responses and a severe peripheral t cell lymphopenia in double-deficient mice, which was significantly exacerbated as compared to the respective single knock-outs. an analysis of coronin deficient mast cells also revealed an involvement of coro a/coro b in the regulation of actin cytoskeletal dynamics and the function of mast cells. however, in contrast to the inhibitory effects of coro a/ b deficiency on t cell function, mast cell degranulation and migration was enhanced in coro a/ b double deficient mast cells. thus, depending on cell type specific requirements, coronin proteins can either exhibit positive or negative regulatory functions. additional studies will investigate molecular and regulatory mechanisms by which coronin proteins control actin cytoskeletal organization and function of immune cells. together, our studies here reinforce and expand our appreciation of the importance of actin-cytoskeleton regulatory proteins for immune cell function. initially found by serial analysis of gene expression, murine samsn (also known as hacs or sly ) is a putative adaptor and scaffold protein with a sterile-alphamotif (sam), a src homology (sh ) domain and a predicted bipartite nuclear localization signal. the samsn gene is located on mouse chromosome and encodes a well conserved protein with amino acids, which is predominantly expressed in hematopoietic tissues. initial overexpression studies suggest a contribution of samsn in b cell activation and differentiation, however its physiological function is yet unknown. to investigate samsn expression in lymphatic and myeloid cell types in greater detail we employed the sensitive method of quantitative real-time pcr. our data revealed an expression of samsn in all tested hematopoietic cell types. the highest expression level of samsn mrna was seen in mast cells compared to lower levels in macrophages, dendritic cells, cd + and cd + t cells and b cells. the other two members of the sly family of adaptor proteins -namely sly (hacs ) and sly (sash ) -were expressed only at a very low level in mast cells. the high level of samsn mrna expression in mast cells, together with minimal expression of other sly family proteins in these cells, implicates an important role of this adaptor protein for mast cells. to address the potential role of samsn in mast cell differentiation and function we are analyzing bone marrow derived mast cells from samsn deficient mice. initial in vitro experiments indicate normal proliferation and differentiation of samsn deficient mast cells. in additional studies we are now investigating the effects of samsn deficiency on mast cell activation processes, such as degranulation, cytokine production and the signal transduction cascade. analyzing the role of samsn in mast cells will help to define the biological function of this novel class of adaptor proteins. introduction: we showed previously that the ability of murine igg antibodies to mediate anaphylactic reaction is directly dependent on the amount of sialic acid residues attached to the carbohydrate chain n-linked to the antibody fc region (silva et al; j.immunol., ). then, we hypothesize that differences in the glycan composition mainly the sialylation grade observed between the anaphylactic and non-anaphylactic igg abs may be resultant of the differential expression of the glycosyltransferase, essentially sialyltransferase, coding genes during its synthesis in b cells. objective and methods: to prove this hypothesis it was analyzed the expression of st siai-v; st galnac i-iv, st gal ii -v genes quantitatively by real time-pcr in the hybridomas producer of these two types of igg abs. results: we observed that the expression of st gal i, iii and v coding genes was similar in both hybridomas, while the st gal ii and iv genes were less expressed in the hybridoma producer of non-anaphylactic igg . in addiction, the expression levels of st sia and st galnac genes in the hybridoma producer of anaphylactic igg were significantly higher when compared to those observed in the hybridoma producing of non-anaphylactic igg . conclusion: these data suggest a direct correlation between the sialylation grade observed in the carbohydrate chain attached to the igg abs and the expression of sialyltransferase enzymes in the hybridomas producer of these molecules. financial support: cnpq, capes, fapesp. basophils are innate immune cells endowed with important effector functions during allergic inflammation and parasite infection. their activation in terms of histamine and cytokine production is mediated through immunoglobulin-dependent and -independent mechanisms, raising the question whether stimulation of tolllike receptors (tlrs), which have been described in basophils, has a similar effect. we found that, in contrast to other tlr agonists tested, only the doublestranded rna poly(a:u) induced the typical t h cytokine and histamine production in vitro. this compound was also fully active when administered in vivo since it activated basophils and promoted their recruitment to the periphery. we took advantage of a murine model of allergic asthma to establish the pathophysiological relevance of this finding. using both adoptive transfer and depletion of basophils, we established not only that these cells contribute directly to the severity of asthma symptoms, but also that a mimic of viral infection can aggravate the disease through their activation. this is the first evidence for a mechanism of exacerbation of allergic asthma induced by a mimic of viral infections, mediated through basophils. ishes the airway hyperresponsiveness and airway inflammation in experiment murine asthma models. to investigate the effect of activation of nkt cells at different allergic asthma progression, we administered balb/c mice with a-galactosylceramide (a-galcer), a stimulator for nkt cell activation, before or after ova immunization and measured the airway inflammation of that mice after times of intranasal ova challenge. in our results, the total numbers of bronchoalveolar lavage (bal) cells were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. moreover, significant increased percentage and cell numbers of eosinophils in bal of mice administered with a-galcer before ova immunization was noted. il- and eotaxin are the most potent cytokine/chemokines for the recruitment of eosinophils. il- and eotaxin levels in the bal fluid were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. these data demonstrate that activation of nkt cells at different allergic asthma progression dictates the different outcome of asthma. in addition, the activation of nkt cells in naïve mice induces airway inflammatory responses. the potential risks of treatment with nkt cell activation on human diseases should be considered. objective: bronchial asthma is a complex disease of the lung and is characterized by a variety of symptoms such as airway hyperresponsiveness, reversible airway obstruction, high serum levels of ige and inflammation. histologically, there are infiltrations of eosinophils, degranulated mast cells and hyperplasia of airway globlet cells in addition to lymphocytes. the transcription factor irf mediates the differentiation into th cells by activating multiple genes which are independently crucial for the development of naive t cells into th cells. because irf is expressed in many different tissues, it can be considered as a master switch factor for th cell differentiation. methods: here, we tested mice deficient in irf in the murine acute asthma model to evaluate its importance in this th cell-mediated disease. the protocol setup was the following: sensitizations s. c. with ova, followed by challenges via ova inhalation and adoptively transferred wildtype cd + t cells prior to initial sensitization. in our experiments, we could demonstrate that only after priming of irf deficient mice with the help of adoptively transferred cd + t cells, asthma symptoms in these mice were more severe than in wildtype controls. as an example, eosinophil infiltration into the lung was increased by . fold. likewise, ovaspecific antibodies and numbers of goblet cells (fig. ) were also significantly higher in irf deficient mice. conclusion: interferon regulatory factor plays a role in the severity of the development of asthma. in its absence, proinflammatory parameters in the lung are increased significantly. this effect is only visible in the presence of wildtype cd + t cells. mechanistically, a potential counterregulation of asthma by th cells is not available in irf knockout mice. together with our previous report that irf represents a susceptibility gene for allergy in the human, our data highlight irf as key in regulating the severity of asthma. the sensory neuropeptide substance p (sp) acts as an important stress mediator with its own stress axis in the skin modulating mast cell as well as antigenpresenting cell (apc) activity. here we postulate that stress-dependent communication between nerve fibers and immune-competent cells can also occur in spleen and affects the course of inflammatory disease. to address this question, atopic dermatitis-like allergic dermatitis (ad) was induced in c bl/ mice by intraperitoneal sensitization and intradermal challenge using chicken egg ovalbumin. animals were additionally exposed to noise stress for hrs prior to challenge. in this model, stress lead to a relative hyperinnervation of the immune-competent areas of the spleen. at the same time, an increased number of apc could be observed in these areas and contacts between nerve fibers and apc were found. under the same conditions, we were able to show increased nk -receptor and ppt mrna levels. accordingly, sp had the capacity to raise the number of antigen presenting cells in spleen and altered the profile of cd c expressing apc substets characterized by cd and cd expression in vitro. in vivo we found a stress dependent shift of cytokine mrna levels towards a th- cytokine profile and increased levels of il- mrna. further the number of cd + t-regulatory cells was increased in vitro. additional analysis of the quality and function of neuro-immune interactions in the spleen will reveal the role of the observed stress-induced alterations in allergic inflammation. proton pump inhibitors (ppis) that are the cornerstone of gastroesophageal reflux disease therapy have been reported to improve asthma and eosinophilic esophagitis (ee) in patients with associated symptoms. the most accepted explanation for these findings is based on the belief that pathologic acidic reflux can act as a triggering factor for these diseases through proximal extent and laryngopharyngeal reflux in asthma and impairment of the epithelial barrier in ee. under these considerations, acid suppression is believed that could prevent these pathogenic mechanisms. nonetheless, a number of evidences suggested the possibility that ppis could have a direct effect in molecular pathways involved in asthma and ee: ) the inhibitory mechanism of ppis implies alkylation of cysteine residues in gastric atpase , ) asthma and ee are prototypic th diseases in which the cytokines il- and il- play a principal role through the activation of the transcription factor stat , and ) we have recently demonstrated that some chloromethyl ketones can downregulate stat by mechanisms involving cysteine alkylation. on the theoretical basis that cysteine reactivity of ppis may affect the regulation of stat , we analyzed its effect in the activation of stat by il- and il- . we found that treatment of cells with ppis inhibited the ability of il- and il- to signal stat activation in a dose-dependent manner in multiple cell types from different origin. given the important role of these mechanisms in asthma and related diseases, our findings show a novel mechanism to understand the effect of omeprazole in these diseases. in argentina more than three million people suffer from asthma, and the number is rising. asthma is defined as a disorder characterized by chronic airways inflammation that results in high mucus production and airways hyperresponsiveness. a th mediated immune response prevails in these patients. in the asthmatic exacerbation period, crisis (cr), triggered by viral infections or other factors, there is a high prevalence of bacterial overinfection. our objective is to compare immunological parameters and in vitro response of lymphocytes to bacterial antigens in the same patient, at that moment and at a time of stability between episodes (i). we studied asthmatic patients both at cr and i. we evaluated eosinophils, basophils and ige expressing b lymphocytes; as well as t regulatory cells (by expression of cd and cd high (treg)), that might inhibit the development of a th response, together with gdt cells, which function in asthma is not completely understood, but could have a role in the increased airway responsiveness. to evaluate the t cell response, mononuclear cells were cultured for hours in the presence of m. tuberculosis (m) or s. pneumoniae (spn), or absence of them (c). then the percentage of activated cells was determined (expression of cd or cd at hours). all the parameters were evaluated in peripheral blood by flow cytometry. discussion: even though the pathophysiological characteristics of asthmatic patients in periods of cr and i are different, no significant differences were observed in the parameters (cell populations and cell response to bacterial antigens) evaluated when compared for the same patient at cr and i. we might be able to detect differences if we studied cells from the lungs, the target organ. we demonstrate that murine and human lc expressed the h r. the level of intracellular ccl production in human lc was reduced after stimulation with h r agonists and basal production could be restored when h r was blocked with the specific antagonist jnj . moreover histamine and a h r specific agonist augmented the migration of lc from the epidermis as shown in ex-vivo migration experiments using human skin and in-vivo migration experiments in mice. in conclusion, the h r is expressed on murine and human lc and influences the immunomodulatory function and migration of these cells. these findings underline the relevance of the h r in allergic skin diseases and encourage further exploration of the h r as a therapeutic target in allergic skin diseases. expression data of the non-coding rna gene, prins (psoriasis susceptibility-related rna gene induced by stress) identified and characterized by our workgroup, suggests a role for prins in psoriasis susceptibility and in cellular stress response. in order to asses the function of prins, we aimed to identify genes regulated by prins and intracellular molecules interacting with this stress-induced non-coding rna. to identify prins regulated genes, we carried out a cdna microarray chip experiment on hela cells where the expression of prins was silenced. this experiment identified g p , an interferon-inducible anti-apoptotic gene that was down-regulated by prins silencing. g p was strongly expressed in proliferating keratinocytes and markedly upregulated in involved psoriatic epidermis compared to healthy epidermis. to detect prins interacting proteins we applied ribonucleoprotein (rnp) purification in hacat cells. with the help of matrix-assisted laser-desorption ionization time-of-flight (maldi-tof) method we identified nucleophosmin, a protein that physically interacts with prins rna. nucleophosmin is a ubiquitously expressed nucleolar phosphoprotein which shuttles continuously between the nucleus and the cytoplasm. immunohistochemical experiments revealed that the expression of nucleophosmin was significantly elevated in psoriatic involved epidermis, localized to the dividing cells of the basal layer. our data indicate that the non-coding prins rna forms a molecular complex with nucleophosmin that regulates stress-induced cellular processes. we suppose that the abnormal functioning of this complex may result in the altered regulation of genes among them the anti-apoptotic g p which can contribute to the pathogenesis of psoriasis. atopic dermatitis (ad) is a chronic inflammatory skin disorder based on a genetic predisposition and triggered by environmental factors characterized by eczematous skin lesions, pruritus, and typical histopathological features. rituximab is a monoclonal anti-cd antibody therapy that targets pre-b cells and mature b cells, but not plasma cells. ad is generally considered as a biphasic, with switch to initial th to chronic th -predominant disease, in which rituximab may have multiple effects. objectives: to report three patients with severe ad refractory to conventional treatments and to anti-ige monoclonal treatment (omalizumab). materials and methods: three patients with severe refractory ad with high levels of serum ige that received weekly intravenous infusions of rituximab at a dose mg/m body surface each. subsets of lymphocytes were analyzed with multiparametric-flow cytometry (facscalibur, bd) at baseline and at specific intervals after treatment. serum immunoglobulins levels were quantified by nephelometry. results: at baseline, all patients had highly elevated levels of total ige ( g , ; g , ; g , mg/dl, respectively). all patients underwent prior treatment with omalizumab for months, with only partial response. then, we started rituximab therapy, resulting in a clear and complete improvement of ad eczema area and severity of skin lesions in all patients. remission of pruritus was observed from the nd week after initiation of rituximab therapy up to year. whereas allergen-specific ige levels were not altered, we observed a large reduction in total serum ige concentrations after initiation of therapy with rituximab. in the first treated patient (follow-up year), ige levels decreased from , to , mg/dl. the other two patients are in the and -months of the follow-up period. importantly, during follow-up no other therapies were required for ad control. conclusions: treatment with an anti-cd antibody led to a dramatical improvement in our series of patients with severe refractory ad. this study support further evidence on the efficacy and safety of rituximab in severe ad. we have previously demonstrated that chronic topical exposure of mice to the contact allergen dncb or to the respiratory sensitiser trimellitic anhydride (tma) preferentially activates t helper (h) and th cells, respectively. in addition, a single application results in divergent cutaneous cytokine production and the migration of langerhans' cells (lc) with different tempos. to explore events occurring after allergen application, balb/c strain mice were exposed to a single topical dose of either %dncb, %tma or to vehicle alone for . - h. measurement of cytokine production from skin exposed to the allergens was performed by cytokine bead array. exposure to dncb provoked rapid production of il- (mean= pg/ml, n= , p x . ), il- (mean= pg/ml, n= , p x . ) and il- a ( pg/ml, n= , p x . ) in skin compared with tma-or aoo-treated mice. in subsequent experiments, mice received an intradermal injection of ng/ear of murine recombinant il- or of the known regulator of lc migration; il- b. interleukin- b induced a significant loss of epidermal lc numbers, measured as a function of reduced frequency of mhc class ii positive cells within epidermal sheets, after ( %) and h ( %) (n= , p x . ). in contrast, il- or control injections were without effect. however, il- administration caused an increase in cutaneous il- production ( pg/ml, n= , p x . ) compared with control injection and naï ve tissue ( and pg/ml, n= , ns). in addition, systemic treatment with anti-il- antibody failed to impact on lc migration provoked by dncb ( % reduction; n= , p x . ). in parallel experiments dncb-induced lc migration was blocked by treatment with anti-tumour necrosis factor (tnf)-a antibody, another cytokine known to regulate lc migration. however, dncb-induced cutaneous il- a ( pg/ml, n= , p x . ) and il- ( pg/ml, n= , p x . ) expression was reduced to baseline levels by anti-il- treatment. these data demonstrate that il- is not involved in the regulation of lc migration, unlike il- b and tnf-a. however, il- is involved in the regulation of the production of other cutaneous cytokines provoked by dncb. therefore it is hypothesised that il- may influence lc and dermal dc maturation, via the expression of il- a and il- . allergic contact dermatitis (acd) caused by nickel ions (ni) represents the most common form of human contact hypersensitivity. along with other allergies its incidence is increasing in the us and worldwide (nhanes iii survey ), but the majority of molecular events underlying this kind of t-cell mediated disease are still widely unknown. to elucidate initial molecular mechanisms (sensitization phase) taking place at the primary allergen contact site in human skin a differential proteomic approach was chosen. by applying dige technology (differential gel electrophoresis), software analysis and mass spectrometric protein identification to cell lysates of allergen stimulated human keratinocytes, seventeen proteins were identified that are specifically regulated by metal allergen ni. in the attempt to further characterize the role of a certain down regulated p -mapk-pathway related protein (p prp) in acd, we analysed its regulation, differential distribution of phosphorylated isoforms as well as its subcellular localization. our results strongly support an involvement of p mapk pathway in allergenspecific signaling responses. it is expected that identification of differentially allergen-regulated proteins and detailed analysis of acd-associated signaling events in primary keratinocytes will lead to a better biomolecular understanding of the initiation of human contact hypersensitivity. (work supported by eu-project novel testing strategies for in-vitro-assessment of allergens, lshb-ct- - , www.sens-it-iv.eu.) objectives: schnitzler's syndrome is a rare disease characterised by chronic urticaria, monoclonal gammopathy, fever, and arthralgia/arthritis with marked elevation of acute phase reactants. in the long term, % of patients develop a lymphoproliferative disorder. schnitzler's pathogenesis is unclear; immunosuppressive treatment is ineffective and high dose steroids are usually required. the recent finding that treatment with il- receptor antagonist (il- ra; kineret) is extremely effective has raised the issue of the role of the inflammatory cytokine il- b and of il- -like cytokines in the pathogenesis of the disease. methods: two patients with recently diagnosed schnitzler's syndrome were treated with kineret, obtaining rapid disappearance of fever and urticaria and normalisation of acute phase reactants in one month. blood samples were collected before and after initiation of therapy. serum cytokine levels were measured by elisa, and expression of il- -related genes by real-time pcr on mrna from blood cd + monocytes. results: compared to normal controls, schniztler's monocytes had similar expression of il- , il- bp, and caspase- , both before and after therapy with kineret. il- b expression was similar to controls before therapy, and was decreased five-fold after therapy. at the serum level, neither inflammatory (il- b, tnfa, il- ) nor anti-inflammatory cytokines (il- , tgfb) were detected. as expected, il- ra was only detectable after therapy. il- was detectable in schnitzler's sera at higher levels than in controls ( . vs. . pm) and decreased after therapy ( . pm). the circulating il- inhibitor il- bp was lower than in controls and not affected by therapy. thus, free il- levels were increased in schnitzler's patients as compared to controls ( . pm vs. . pm in controls) and decreased after therapy ( . pm). conclusions: schnitzler's syndrome is not associated to enhanced expression of il- -related cytokines (il- b, il- ), nor of the il- /il- -converting enzyme caspase- in blood monocytes. however, the high circulating levels of il- suggest an increased activity of caspase- , as in the case of autoinflammatory diseases. experiments are in progress to test this possibility. atopic dermatitis (ad) is a chronic relapsing allergic skin disease with a high and growing prevalence. currently around % of the children in industrialized countries are affected. in most cases patients exhibit increased systemic ige-levels (so-called extrinsic form) accompanied by sensitization to allergens. while ad is frequently cleared until adulthood, many patients develop allergic rhinitis and asthma. most ad-patients show topical colonization with staphylococcus aureus indicating a defective innate immune response. as a class of pattern recognition receptors, toll-like receptors (tlrs) are essential for pathogen recognition and critical for the induction of an effective adaptive immunity. all known tlrs except tlr signal via myd to induce nfxb-dependent gene transcription. tlrs are also known to be involved in the pathogenesis of autoimmune diseases. as chronic ad also has an autoimmune component, the study of myd signaling in ad might provide new insights into the function of tlrs. to investigate the role of tlrs in the immunopathology of allergic reactions and skin infections, we induced ad-like symptoms in c bl/ myd -deficient mice by repeatedly sensitizing the mice to ovalbumin (ova) after mechanical disruption of the skin barrier by tape stripping. first results show that myd -/-mice display reduced inflammation of the treated skin area compared to wildtype mice. immunostainings of skin biopsies reveal reduced acanthosis and infiltration of inflammatory cells into the dermis compared to wildtype. skin-draining lymph nodes are less enlarged in the ova-treated knockout mice compared to wildtype and differ in cellular composition. serum antibody levels determined by elisa show reduced systemic total and ova-specific igg -titers in ova-treated myd -/-mice compared to wildtype mice, although the nacl-treated myd -/-control group has higher total antibody titers than the wildtype nacl control group. total ige levels are increased in the knockout mice compared to wildtype mice under both conditions. to further investigate the role of staphylococcus aureus during ad development, we will include topical application of the superantigen staphylococcal enterotoxin b (seb) or living bacteria into our analyses. following this approach, we anticipate to obtain new insights into the role of the innate immune system in allergic reactions of the skin. introdution: the skin of vertebrates is the target for over , species of hematophagous arthropods. among these are ticks, which are long-term feeders and interact with host defenses for days to weeks. little is known about specialized strategies for eliminating ectoparasites, but ticks can induce immune responses in hosts. bovines present variable and heritable levels of resistance to the tick rhipicephalus microplus and are the only model in which distinct outcomes of infestation can be examined in the same species of host. in order to obtain some of the immune correlates of these outcomes, we examined expression of candidate genes and quantified populations of leukocytes and subpopulations of lymphocytes present in the inflammatory infiltrates elicited by tick bites in skin of genetically resistant and susceptible bovine breeds, respectively, nelore (bos taurus indicus) and holstein (b. t. taurus). methods: skin biopsies ( mm punch) were taken at the feeding sites of ticks from susceptible and resistant cattle (each phenotype n = ) or from non-infested contra-lateral sites. expression of mip- a, igf- , mcp- and ip- genes was quantified with realtime rt-pcr. sections of paraffin-embedded skin were stained with may grünwald-giemsa for differential cell counts. lymphocytes in sections of frozen skin were phenotyped with specific antibodies using immunoperoxidase technique; in infested skin, histological sections were limited to the area of the tick's cement cone. results: as expected, hosts recruit cutaneous inflammatory infiltrates around the tick's mouthparts. however the composition of infiltrates presented with significant differences that varied according to the phenotype of infestation. inflammation of nelores contained significantly more basophils, eosinophils and mononuclear cells expressing cd , cd , cd , cd , mhc class ii, and p than that of holsteins. lymphocytes expressing wc and cd antigens were significantly diminished in infested skin of holsteins when compared with control skin (p x . ). infested skin of nelores contained significantly more message for mip- a, igf- , mcp- and ip- than that of holsteins. conclusions: although ticks secrete molecules that inhibit cell adhesion and chemokines, resistance correlates with the capacity to recruit and maintain populations of leukocytes that generate effector immune responses. supported by cnpq, capes, fapesp, and icttd. in the last decade it has become clear that keratinocytes play an important role in the skin immune system. upon stimulation, keratinocytes produce high amounts of proinflammatory chemokines and cytokines and express receptors which are involved in immunoregulation. in a number of inflammatory skin diseases such as eczema or psoriasis infiltrating lymphocytes are found in close vicinity to keratinocytes, enabling interaction of these two cell types. it has been proposed (goodman et al.) that keratinocytes rather support a th response by interacting lymphocytes. we examined this hypothesis with autologous cultures of keratinocytes derived from the outer root sheet of the hair follicle co-cultured with cd + t cells from the same donor. during the coculture either seb or antigen were added. in all experimental approaches the addition of keratinocytes resulted in higher production of ifng by t cells. furthermore, we set up an experimental approach were autologous antigen-pulsed monocytes were also added. again, the induction of ifng by the presence of keratinocytes resulted in a marked and significant increase of ifng production by t cells. we were able to show that il- plays a crucial role in the induction of ifng in t cells keratinocyte interaction. in addition blocking of lfa- in the co-cultures resulted in significantly reduced ifng production by t-cells underlining that icam- /lfa- binding is also crucially important for ifng induction. we conclude from our study that keratinocytes rather support a th than a th local response pattern by virtue of il- secretion and icam- /lfa- interaction. this property of keratinocytes may account for the observed cytokine switch in allergic eczematous skin from a th like micromilieu in acute towards a th dominated milieu in chronic lesions. the genotyping of ccl l gene in patients with psoriasis could allow describing subcategories of patients based in clinical parameters and disease severity. therefore, it could be also used as a clinical diagnostic tool, potentially modulating the efficacy of new treatments, or even to be used as a therapeutical target of psoriasis. this work was supported by project grants of merck-serono and instituto de salud carlos iii (pi / ). psoriasis is an inflammatory dermatosis with % prevalence among caucasians. hla-cw allele is the gene that confers susceptibility to psoriasis and it is placed near to tnf loci with several snp in promoter region. the most common polymorphisms are two g to a transitions in - and - positions. strong association was found between polymorphisms in the - region with psoriasis. in several diseases, the association with hla and clinical manifestation is different between genders, for example in spondyloarthropathy and hla-b , and this is a question of increasing interest. the objective of this study was to identify clinical and molecular differences between male and female in brazilian psoriatic patients. sixty-nine individuals assisted at the dermatology outpatient clinic of the teaching hospital, university of campinas, with diagnosis of psoriasis of early-onset (up to years of age) were selected. hla-a -b -c -dr -dq alleles and tnf- and - snp were differentiated by pcr/ssp. analyzing the total group, patients ( . %) were male, ( . %) were female. in the male group, the mean age at disease onset was , years. severe forms were seen in this group (psoriatic arthritis in cases and erythroderma in ). seven patients ( , %) had a favorable evolution of the disease, but ( , %) developed extensive psoriasis, covering over % of body surface requiring systemic treatment. the main molecular risk factor for the disease, cw* allele was positive in cases ( , %), tnf g/a genotype was found in ( , %) and tnf g/a in ( , %). in the female group, the mean age at disease onset was , years, one case of psoriatic arthritis and one of erythroderma. twenty-nine ( , %) had a favorable evolution of the disease and ( , %) an unfavorable evolution. cw* allele was positive in cases ( , %), tnf g/a genotype was presented in ( , %) and tnf g/a in ( , %). severe disease was seen in male patients. there was no difference in frequency of cw* allele between male and female groups, but there was a tendency of significant difference in tnf g/a genotype. we found that c bl/ mice were more susceptible than balb/c and dba/ mice. higher susceptibility was reflected by higher footpad swelling and transient systemic dissemination. analysis of serum cytokine level revealed differences in production of proinflammatory cytokines, such as il- and mrp / , among different inbred strains of mice. furthermore, we identified the cells which are involved in this cytokine production. as expected, histopathological analysis showed that s. aureus infection induces an influx of monocytes and granulocytes. our study shows that not only bacteria-but also host-specific differences are associated with different courses of s. aureus skin infection. aims: to investigate the cause and to study the clinical symptoms and the laboratory findings of the anaphylactic reactions in the pediatric population of our country, considering that these are very often dangerous situations which demand direct treatment and increased alertness. methods: cases, which were studied retrospectively, included children ( boys and girls), aged - years, who had an anaphylactic reaction, out of the that were examined in total. the statistical analysis of the data was held with the spss program. the commonest causes were proved to be food ( %-particularly sea food and dried fruit), drugs ( %-usually antibiotics and non-steroidal antiinflammatory drugs), as well as insect bites ( %-mainly caused by hymenoptera). the symptoms included mainly the presence of pruritic pomphus with erythema ( %), and gastrointestinal symptoms ( %), while there were quite many cases with dyspnea, nasal congestion, but also angioedema. total ige g was found in out of the severest cases ( , %), in which the adequate control was held, while in their vast majority ( out of ) there was no previous anaphylactic reaction. on the other hand, it was proved in total, that in , % ( cases) there was a hereditary family history of atopy, while in children ( %) there was also a personal history of asthma. finally, at a great percentage ( %) eosinophilia was found, while a statistically significant seasonal distribution during spring and summer was registered. conclusions: it has, therefore, been shown that )the anaphylaxis is quite often in the pediatric population, with the commonest causes to be food and drugs, which are often thoughtlessly used. ) in particular, in many cases it is proved that there is a personal but also a family history of atopy. ) increased attention should be, thus, given in these cases -especially during spring and summer-for their early diagnosis as well as for their effective treatment (adrenaline, antihistamines and corticosteroids) particularly for the severest cases, where the hospitalization of the patient is also necessary. allergic contact dermatitis (acd) is an adaptive inflammatory response of the skin triggered upon exposures to certain chemicals or metal ions. as classical type iv delayed hypersensitivity reaction this response is mediated by t-cells. since many ingredients in consumer products might exert allergenic potency, there is a need for an appropriate screening and characterization of the chemicals used according to this toxicological endpoint. up to now the identification of potential allergens completely relies on animal testing, like buehler assay or guinea pig maximization test (gpmt). due to economical and ethical reasons, as well as driven by the enforcement of certain governmental regulations (i. e., cosmetics directive), the development of an in vitro test system for identification of potential sensitizers is mandatory. since dendritic cells (dcs) play a pivotal role in the initiation of contact dermatitis we chose dcs to characterize known sensitizers in their ability to activate these cells and subsequently examined the molecular interplay between dcs primed by allergens and t-cells in the test tube. the known allergens nickel, dinitrochlorobenzene (dncb) and cinnamic aldehyde were tested for their ability to alter the expression of several immunomodulating surface molecules on dcs derived from monocytes that display a langerhans cell (lc) type-similar phenotype. we used multicolour flow cytometry to detect differences in expression patterns of surface molecules that have been associated with maturation. in addition to the upregulation of cd we could observe dose dependent upregulation of programmed death ligand (pdl- ) and downregulation of the dendritic cell immunoreceptor (dcir). furthermore we observed enhanced t-cell proliferation in mixed leukocyte reactions (mlrs) applying lcs stimulated with allergens ex ante. since changes in the expression of only single cell molecules are unlikely of being sufficient for reliable identification of possible contact allergens, we are aimed at analyzing a wide pattern of various surface molecules by multicolour facs and propose that this might be a reasonable approach to screen for contact sensitizing properties of chemicals. our findings are of particular interest for further development of new in vitro assays, using immune cells, to detect the sensitizing potential and quantify the sensitizing potency of chemicals. we want to present the case of a year old iraqui patient with arabic ancestors who had been suffering from psoriatic arthritis since years. in march a treatment with fumaric acid esters in combination with ibuprofen was introduced. this led to the complete healing of the skin lesions. for this reason the dose could be reduced to one tablet fumaric acid esters ( mg) every second day. in april the patient presented himself in the consult with multiple livid papules with a diameter of mm in the area of the auricle. the histological examination showed an hhv- positive kaposi sarcoma. the differential blood cell count demonstrated a lymphocytopenia. the hiv-serology was negative. the staging examinations (chest x-ray, gastroscopy, coloscopy, abdominal and lymph node sonography) showed no signs of visceral involvement. after the diagnosis the treatment with fumaric acid esters was discontinued. over the course the livid papules showed a spontaneous complete regression. a spontaneous regression is known from the iatrogenic ks caused by immunosuppressive therapy when the immunosuppression is terminated. as our patient also showed a spontaneous regression of the kaposi sarcoma after stopping the treatment with fumaric acid esters we propose a causative relation. sarcoidosis is a multisystemic granulomatous disease with unknown etiology. although the immunopathogenesis of sarcoidosis remains unknown there are some supportive evidence for the significant role of th type immune response. recently, suppressor of cytokine signaling (socs) proteins have been identified as regulators of cytokine signaling pathways. in this study we aimed to evaluate the roles of socs , socs and foxp in the immünopathogenesis of sarcoidosis and their association with responsiveness to treatment. peripheral blood (pb) and broncholaveolar lavage (bal) mononuclear (m) cells from sarcoidosis patients in remission following treatment (responders, n: ), the patients who showed recurrence or progression after treatment (non-responders, n: ) and stage i/ii sarcoidosis cases which were followed up without any treatment (untreated, n: ) were evaluated for socs , socs ve foxp mrna expressions by taqman pcr, and also flow cytometric analysis was performed for lymphocyte markers including cd , cd , cd , foxp , cd + cd high , cd + foxp + . expression of socs and foxp- mrna in pbmcs and balmcs from responders were found to be significantly higher in comparison to other two groups . socs was found significantly elevated in pbmcs of responders when compared with other two groups. it was also elevated in balmcs of responders when compared with with those of untreated cases. the proportions of cd , foxp , cd +cd high , cd + foxp + cells in pbmcs and balmcs of responders were found to be increased in comparison to nonresponders and untreated cases. our data demonstrates that socs , socs and t regulatory cells may have potential roles in the control of sarcoidosis. we think that if the roles of socs and socs molecules and t regulatory cells are well characterized, new therapeutic approaches targetting cytokine signal supressors, which can strenghten the regulatory responses, may be beneficial for the sarcoidosis cases resistant to conventional therapy. the inorganic dust, containing free crystalline silicon dioxide (fcs) is critical for the development of silicosis. several studies supported the view that fibrotic responses mainly depends on the regulation of the immune response to the fcs in affected individuals. the role of fcs in induction of a local and systemic inflammation and pulmonary fibrosis are still debates.we studied the changes of neopterin, as a marker for ifn-g dependent macrophage activation and circulating immune complexes (cic), as a marker of humoral immune response, in patients with silicosis and workers exposed to dust containing fcs.we survey a group of silicosis patients, with mild ( ), moderate ( ) and severe ( ) silicosis, coal workers, exposed to inorganic dust containing fcs (exposed), and healthy workers without exposure to dust aerosol (controls).the serum quantity of neopterin and cic, containing iga(igacic), igg(iggcic) and igm(igmcic) was detected by elisa. differences between investigated groups were detected by student's t-test and a p-value less than . was considered significant.neopterin level was significantly elevated in exposed ( , ± , ng/ml) compared to controls ( , ± , ng/ml; p= , ). moreover, the neopterin level in exposed was similar to silicosis patients ( , ± , ng/ml).the levels of iggcic was significantly elevated in the exposed compared to controls ( , ± , au vs , ± , au p= , ) and to silicosis patients ( , ± , au p= , ). in contrast, igmcic was significantly elevated in silicosis than in exposed ( , ± , au vs , ± , au; p= , ).in comparison with exposed, significantly higher igmcic was found only in mild, but no in moderate and severe silicosis. in contrast, the level of iggcic in mild and moderate silicosis was significantly lower compared to the exposed (p= , and , respectively).the obtained results showed that activation of alveolar macrophages mainly depends on the presence of fcs in the respirable dust fraction and precedes the clinical data for pulmonary fibrosis. the dynamics of cic suggest the involvement of fc-receptors mediated regulation of the immune response in the progression of pulmonary fibrosis, and could be useful marker for exposure to inorganic dust containing fcs. described pathologic similarities between sarcoidosis (sa) and tuberculosis (tbc) suggest m. tuberculosis antigens as caustaive agentes. it seems that in the genetically different predisposed hosts, the same antigens may cause the development of sarcoid or tuberculous inmune response. so different hla haplotypes have been described as a predisposing factor to develop sarcoidosis (hla a* , b* , drb * (these of good prognosis) * /* /* /* ) or tbc (drb * /* /* /* and associations with dqa * /* /* ) we describe two cases of two female patients from the same geographic region with mantoux and zhiel-neelsen negative tests and high levels of tnf diagnosed of tbc and sa respectively. both of them debuted with the same clinical manifestations: fever, abdominal pain, and asthenia and shared similarities in the images from the tc study (pulmonary nodules and mesenteric adenopaties). we found the same results for the flow citometry analysis of the non-caseificant granulomes as well as the same anatomopathologic characteristics. after being treated with anti-tbc drugs, the first one presented a good clinical improvement, so she was diagnosed as tbc. the second one did not improved, so she was treated with corticosteroid, with good results. therefore, she was diagnosed as sarcoidosis. after hla analysis, we noticed that the tbc patient was hla a* , b* and drb * (sarcoidosis good prognosis haplotype) and the patient diagnosed as sarcoidosis was hla a* , drb * . as the results show, could there not be a direct relationship between the hla system and the development of sa or tbc, or in contrast, was the first patient missdiagnosed of tbc being a good prognosis sa? objectives: experimental mouse models for acute asthma are well established, yet models for chronic asthma have several shortcomings. for example, current chronic models show decreased inflammation over time and only marginal effects on airway remodelling. experimental models for chronic asthma are essential for development of new therapeutics and must include changes that closely resemble clinical conditions. ovalbumin (ova), house-dust-mite (hdm) and cockroach (cra) proteins are commonly used to trigger an asthma like response in mice and, for this reason, were used in the present study. the objectives of our work were to compare the most frequently used mouse models of chronic asthma and to develop a mouse model of chronic asthma that clearly displays pivotal features of severe human asthma. methods: for the induction of asthma, mice were initially sensitised by intraperitoneal injection of ova, hdm, cra or a combination of all three, followed by repeated challenge by intratracheal application of ova, hdm or cra. inflammation in lung was measured by analysis of cell influx into the bronchoalveolar lavage (bal) and by determination of chemokine and cytokine levels in bal and lung tissue using elisa and multiplex technology. additionally, serum levels of ige and igg antibodies were measured. airway remodelling was assessed by histological staining for mucus production, immune cell influx, smooth muscle thickening and fibrosis. results: significant differences were measured in cell influx, chemokine/cytokine and total ige levels. compared to hdm and cra, ova induced an higher cell influx in the bal, hdm showed an increase of chemokines in bal and increased ige levels in serum. using a combination of all three proteins resulted in the most severe form of asthma. conclusions: to our knowledge, this is the first study that directly compares the most commonly used mouse models in regard to their potential to display a pathology specific of severe asthma. the most sustained and severe form of asthma was induced by the combination model. this model offers particular advantages for evaluating existing and novel therapeutic agents. furthermore, this model could contribute to understanding of the mechanisms underlying chronic asthma. the present study focused on peri-smi connective tissue capsule formation, the most frequent post-operative local complication in patients receiving smi. we investigated the local immune processes via the phenotypic and functional characterization of lymphocytes within the fibrotic tissue. to this end, intracapsular lymphoid cells and peripheral blood mononuclear cells (pbmcs) from the same patients were isolated and analyzed via facs, concentrating on t-effector cells (teff) and t-regulatory cells (tregs: cd + , cd ++ , foxp + ), cytokine profiles, t-cell receptor (tcr) repertoire and reactivity against human heat shock protein (hhsp ). intracapsular tregs were visualized by immunohistochemistry and functionally tested in suppression assays. the cellular composition of intracapsular mononuclear cells showed a preponderance of cd + t-helper cells and a significant subset of tcrg/d + cells, exceeding that observed in peripheral blood. il- , il- , il- , tgf-b and ifn-g production prevailed, pointing to a th /th weighted immune response. furthermore, intracapsular t-cells displayed a restricted tcr a/b repertoire (monoclonal/oligoclonal) as well as a preferential reaction with hhsp . importantly, numbers of intracapsular tregs were inversely proportional to the degree of fibrosis and showed less suppressive capacity as compared to peripheral tregs. our results suggest that silicone triggers a specific local immune response via activated th /th cells, promoting fibrosis due to the production of profibrotic cytokines. clonal restriction of the tcr repertoire is a further indication for a specific antigen driven immune response preceding capsular fibrosis. in this context, hsp might be a prominent candidate. taking into consideration that it is ubiquitously expressed, it might be the "missing link" between local and systemic side effects of smi. the inverse correlation between the degree of capsular fibrosis and the number of intracapsular tregs suggest that tregs may initially be able to inhibit the progress of capsular fibrosis. however, as numbers of tregs, as well as their suppressive capacity decreases over time, fibrosis develops. supported by the competence center medicine tyrol (kmt) and the lore-and-udo-saldow donation. objectives: recent findings have proven that silicone induces a local inflammatory response with subsequent fibrotic reactions. the present project deals with the standardization and further development of a modified elisa test system (silisa ® ) for the identification of patients with a risk for fibrotic side effects to silicone mammary implants (smis) based on the protein signature adhering on the surfaces of such devices ( ) . the current silisa ® is a test system for the simple detection of the adhesion pattern of proteins from patients' sera to silicone. the optimization of the silisa ® comprised inter-and intra-assay standardization, robustness, specificity and sensitivity. the essay was further developed with antibodies against annotated proteins that were not yet tested in the past. all experiments were carried out in a well plate format for high-throughput analysis. statistical analysis has been performed using spss. the extended essay has been successfully established in the system with antibodies against seven already tested proteins, including c-reactive protein, collagen-i, collagen-iii, fibronectin, igg, c -complement, myeloid related protein and two new proteins, integrin-ß and fibrinogen. data from more than patients have been obtained and exploited so far. the intra-and inter-assay variability of the test was reduced to less than % and %, respectively. patients with fibrotic reactions to silicone were successfully identified using a pattern of protein deposition to silicone. conclusion: applying the silisa ® , sera from five different groups were tested: silicone patients with and without fibrotic reactions, female and male individuals without any contact to silicone and hospital's medical staff with potential silicone contact. the distribution pattern of eight proteins showed differences in patients developing strong fibrotic reactions to silicone compared to controls. muscular lesion is a frequent matter in sportive medicine and myodegenerative diseases. necrosis of the damaged tissue and activation of inflammatory response characterize the initial phase of muscle repair. this work aimed to analyze the tissue repair after induction of lesion in skeletal muscle from mouse lineages with distinct cytokine secretion patterns. it was included at least mice per group with distinct cytokine pattern: th (c bl/ , c bl/ ) and th (balb/c). muscular injury was performed by injection of bupivacaine. both th -dominant strains presented more areas with regenerating myofibers and macrophages at dpi. regional lymph nodes showed significant increase of cellularity and relative numbers of cd bupivacaine-inoculated balb/c mice compared to non-inoculated matched mice at dpi. balb/c mice showed increased collagen expression and decrease of mmp- activity associated with more mrna for tgf-b . this study shows that the immune background of the mouse may affect the remodelling processes in skeletal muscles that occur in response to bupivacaine injection promoting muscle regeneration (th cytokines) or myonecrosis and collagen deposition (th cytokines). the severe, life-threatening heart failure in some of the patients with dilated cardiomyopathy (dcm) is imputed to the stimulatory autoantibodies against the second extracellular loop (ec ii ) of the ß -adrenergic receptor (anti-ß ec ii ). to analyze their pathogenic impact as a single causal factor we used a human-analogous lewis rat model of dcm, where monthly subcutaneous immunization of the rats with the ß ec ii peptide as a glutathione-s-transferase (gst) fusion protein induced production of anti-ß ec ii and eventually dilated cardiomyopathy. in this model we isolated a ß ec ii -specific rat monoclonal antibody (clone f ), and showed by elisa that it binds to the linearized ß ec ii peptide. additionally, we confirmed with flow cytometry that f also binds the ß ec ii in its native conformation, i. e. directly labeled circular ß ec ii (dyl -ß ec ii ) peptide. moreover, we demonstrated activation of the ß -adrenoreceptor by f using a fluorescence resonance energy transfer (fret) assay system in vitro. these data further corroborate the pathogenic role of anti-ß ec ii antibodies in mediating dcm in this animal model, thus rendering them a potential therapeutic target. therefore, we investigated a novel anti-ß ec ii -specific peptide-based therapy, by intravenously applying a circular ß ec ii peptide in the dcm lewis rat model to neutralize the anti-ß ec ii antibodies. while the peptide therapy strongly reduced the anti-ß ec ii titers in the serum by up to % and consecutively lead to clinical remission, elispot assays for the detection of ß ec ii -specific antibody-secreting cells (asc) indicated no difference in the number of long-lived plasma cells in treated animals. in contrast, elispot and flow cytometrical analyses revealed a decrease in the number of ß ec ii -specific memory b cells in the treated animals, indicating that this cellular compartment is most likely also targeted by the peptide therapy. our newly developed anti-ß ec ii -specific therapy, thus, not only neutralized the pathogenic autoantibodies, but also depleted antigen-specific memory b cells involved in the generation of these autoantibodies. these results provide the rationale for further development of this therapeutic strategy for eventual application in patients with autoimmune dilated cardiomyopathy. cardiovascular diseases like myocarditis and subsequent dilated cardiomyopathy (dcm), are a frequent cause of mortality in humans with dcm being the most common reason for heart failure in young adults. infections with coxsackievirus b or cytomegalovirus can lead to an acute inflammation of the heart muscle that is followed by an autoimmune response directed autoantigens in the heart, such as the alpha isoform of cardiac myosin (myhca). immunization with the well-characterized myhca - epitope elicits autoreactive cd + t cell responses that have been shown to be the major mediators of autoimmune myocarditis in balb/c mice. it is known that professional antigen presenting cells (apcs) such as dendritic cells are crucial for initiating and maintaining t helper (th) cell responses affecting the heart muscle. however, the detailed analysis of the interaction between these cells in the context of autoimmune myocarditis has been hampered by the lack of appropriate analytical tools. we therefore generated a tcr transgenic mouse harboring t cells that specifically recognize the myhca - peptide. in a first step, hybridoma cells were generated by fusing bw tcra -cd lymphoma cells with myhca - -specific th cells. tcr expression and antigen specificity was assessed by facs analysis and elispot assay. following subcloning, the variable regions of the expressed tcr were characterized by pcr-sequencing. the rearranged v(d)j regions were subcloned into tcr cassette vectors and linearized constructs were injected into the pronuclei of fertilized oocytes. using this novel tcr tg mouse we plan to investigate in detail the activation of myhca - -specific t cells during the process of autoimmune myocarditis. furthermore, this new tool will help to generate a high resolution analysis of the contribution of different apcs in the activation and differentiation of autoreactive th cells during inflammatory heart disease. m. relle , a. schwarting , p.r. galle university medical center of the johannes gutenberg university mainz, medical clinic i, mainz, germany several mouse or rat models have been established to explore the role of proteinase (pr ), in anca-associated glomerulonephritis, vasculitis or pulmonary inflammation but these studies have demonstrated that anca alone are not sufficient to induce these diseases directly. therefore, we assessed the expression, mobilization and enzymatic activity of pr in mouse bone marrow, kidney, spleen and peripheral blood by immunohistochemistry and immunoblots, as well as the proportion of pr -positive neutrophils in the peripheral blood of frequently used mouse strains. neutrophils were mobilized from the bone marrow by an intraperitoneal injection with human il- . pr -mrna from the murine cancer cell line wehi- was amplified by race-pcr and subsequently sequenced. sequence comparisons were done with dnasis software package and the blast tool of the ncbi. promoter analyses were performed with the genomatix software matinspector. we could demonstrate, that mouse bone marrow is a reservoir for functional neutrophils, which are rapidly mobilized after injection of furthermore, we identified an alternative pr -promoter in the second intron of the mouse pr gene. this promoter is active in the bone marrow, in embyros and in cancer cell lines, indicating that its expression is not restricted to myeloid cells. fine structural analyses of this alternative promoter revealed differences not only between the rat and the mouse promoter but also between different mouse inbred strains. taken together, we have shown that the maturation processes of mouse neutrophils differ from those of human granulocytes. the identification of an alternative pr transcript and its promoter indicates that the murine pr may have additional, as yet not described, functions in hematopoiesis and cancerogenesis. objective: recent studies show that in vivo administration of och, a synthetic lipid that specifically activates natural killer t (nkt) cells, results in suppression of th mediated immune responses in autoimmune diseases. nkt cell activation depends on lipid presentation via the mhc-i like molecule cd d on antigen presenting cells such as mature dendritic cells (mdcs) and upon activation by och nkt cells rapidly produce large amounts of th cytokines. the goal of this study was to investigate the effect of och and och-primed dendritic cells on atherogenesis. methods & results: ldl receptor deficient (ldlr -/-) mice were fed a western type diet and atherosclerosis was induced via collar placement around both carotid arteries. subsequently the mice were treated i. p. with och (n= ) or pbs (n= ) twice a week for seven weeks. the injections with och did not affect atherosclerotic lesion size. to improve the presentation of och to nkt cells in vivo, bone marrow-dendritic cells were maturated via tlr activation, in the presence/ absence of och. subsequently we transferred . x mdcs (n= ) or och-primed mdcs (n= ) ( times) to ldlr -/mice. afterwards the mice were put on a western type diet to induce atherosclerosis. vaccination with och-primed dcs resulted in a . % reduction in plaque size compared to mice treated with mdcs (p x . ). during the experiment no effect on serum cholesterol levels was observed, but at the end of the experiment there was a significant . % (p x . ) reduction in cholesterol levels in the mice treated with och-primed dcs. the number of nkt cells in blood and liver was monitored and a to -fold increase in these cells was detected days after the last treatment with och-primed dcs (p x . ). additionally, the nkt cells in the liver of mice treated with och-primed dcs produced more il- . discussion: we conclude that immunotherapy using och-primed dendritic cells efficiently activates nkt cells, resulting in a th phenotype of the nkt cells and this leads to an efficient protection against atherosclerosis. these data indicate that immunotherapy based on ligand specific primed dcs may be a novel way to treat atherosclerosis. systemic lupus erythematosus (sle) is characterized by high serum titers of igg anti-nuclear antibodies secreted by plasma cells. however, the characteristics of the igg+ plasma cell antibody repertoire in sle has never been determined on a single cell level and little is known about the role of germinal center (gc) reactions for the development of sle autoantibodies. the igg inhibitory fcgriib knock-out mouse on the c bl/ background is a strain specific lupus autoimmune model that is characterized by the spontaneous development of autoantibodies to nuclear antigens such as dsdna and chromatin. to characterize the igg+ plasma cell compartment under normal circumstances and in autoimmunity we have cloned and expressed igg antibodies from single isolated gc b cells and plasma cells derived from spleen, bone marrow and lymph nodes of wild-type c bl/ and fcgriib deficient mice. igh and igl chain gene sequence analyses revealed no major differences in the ig gene usage between wild-type and autoimmune mice, but gc b cells of fcgriib were enriched for antibodies with positively charged igh cdr regions and anti-nuclear specificity. the overall frequency of autoantibodies was similiar between wild-type and fcgriib deficient mice. however, strongly autoreactive antibodies to dsdna and murine igg c were isolated only from fcgriib deficient mice, but not from c bl/ control mice and somatic mutations contributed to their generation. in summary, our data suggest that the gc reaction plays an important role for the development of self-reactive antibodies in fcgriib deficient mice. the finding that the frequency of autoreactive antibodies is higher in gc b cells than in spleen or bone marrow plasma cells may indicate that autoreactive gc b cells are partly regulated even in the absence of fcgriib. autoantibodies against double-stranded dna (dsdna) and nucleosomes (ncs) represent a hallmark of systemic lupus erythematosus (sle). however, the factors leading to the autoimmune response against these nuclear autoantigens are not fully identified. high mobility group box protein (hmgb ), a nuclear dnabinding protein and an extracellular proinflammatory mediator gets tightly bound to modified chromatin during apoptosis. it is not released, since apoptotic cells are immediately engulfed by phagocytes. conversely, in conditions of clearance deficiency, which is observed in a subset of patients with sle, non-ingested apoptotic cells, may undergo secondary necrosis, thereby releasing ncs containing the "endogenous adjuvant" hmgb . we investigated if hmgb -containing ncs contribute to the breakdown of immunological tolerance against dsdna and ncs. we found that hmgb remains associated with ncs released from late apoptotic cells in vitro. hmgb -ncs complexes were detected also in the blood of patients with sle. hmgb containing ncs from apoptotic cells induced secretion of il-b, il- , il- , and tnfa as well as expression of co-stimulatory molecules on human and murine macrophages and dendritic cells (dc), respectively. cytokine release from murine macrophages was dependent on myd and toll-like receptor . neither hmgb -free ncs from living cells nor from apoptotic hmgb -or hmgb / -deficient cells induced marked cytokine production or dc activation. specific inhibition of hmgb activity by the antagonistic a box domain significantly reduced capacity of "apoptotic "ncs to induce tnfa and il- release by macrophages. immunizations with hmgb -containing ncs from apoptotic cells induced anti-dsdna and anti-histone igg responses in non-autoimmune mice in tlr -dependent manner. in conclusion, hmgb in complex with ncs activate antigen presenting cells thereby contributing to the loss of immunological tolerance against ncs/dsdna and, hence, to the immunopathogenesis of sle. objective: apoptotic cells are considered to be a major source for autoantigens in autoimmune diseases such as systemic lupus erythematosus (sle). in agreement with this, defective clearance of apoptotic cells has been shown to increase disease susceptibility. still, little is known about how apoptotic cell-derived self-antigens activate autoreactive b cells and where this takes place. methods: injections of fluorescently labelled syngeneic apoptotic cells were traced using immunofluorescence microscopy. binding studies were performed using apoptotic cells and cho cells transfected with class a scavenger receptors (sr). repeated injections of syngeneic apoptotic cells in sr deficient and wild type mice were conducted and antibody production by autoreactive b cells was measured. autoreactivity against sr was followed in two sle prone mice strains over the development of disease and in a cohort of sle patients. an antibody against the sr was injected together with several antigens to directly evaluate the possible role of autoantibodies against the receptors. results: in this study, we find that apoptotic cells are taken up by specific scavenger receptors expressed on macrophages in the splenic marginal zone and that mice deficient in these receptors have a lower threshold for autoantibody responses. autoantibodies against sr are found before the onset of clinical symptoms in sle-prone mice, and they are also found in diagnosed sle patients. furthermore, injections of an antibody binding sr enhance the antibody production by b cells when co injected with either apoptotic cells or tnp-ficoll. conclusion: our findings describe a novel mechanism where autoantibodies toward scavenger receptors can alter the response to apoptotic cells, affect tolerance, and thus promote disease progression. because the autoantibodies can be detected before onset of disease in mice, they could have predictive value as early indicators of sle. e. glasmacher , k.p. hoefig , e. kremmer , v. heissmeyer stretches and helps in the selection of the correct splicing borders. a allele of (r h) creates a strong binding site for a splicing enhancer protein srp according to bioinformatics. our findings indicate that, the putative branch point, r h snp and the t stretch located downstream of exon two, plays a role in the alternative splicing of bank . finally, we believe that bank delta protein work as a dominant negative isoform in b cell activation and antobodies production, and may antagonize the effect of the full-length protein. these properties of the delta protein may contribute to the observed reduction in sle susceptibility. s. beermann , r. seifert , d. neumann hannover medical school, pharmacology, hannover, germany the biological function of histamine is mediated by four different receptors, namely histamine h receptor (h r), h r, h r, and h r. during an immune reaction histamine acts as a local proinflammatory mediator and contributes to the polarisation of the adaptive immune reaction by modulating the activity of dendritic cells and t cells. in these cells, histamine may modulate the synthesis of characteristic t cell cytokines such as ifng, which plays a central role in a number of autoimmune diseases. the present study was initiated to analyze the involvement of histamine on the induced production of ifng by immune cells. mouse spleen cells were stimulated in vitro by either immobilized a-cd antibodies or cpg-oligonucleotides (cpg-odn) in the presence or absence of histamine or -methylhistamine, a h r-selective agonist. ifng production was evaluated by analysis of cell culture supernatants by elisa. both, histamine and -methylhistamine concentration-dependently reduced ifng production in splenocytes obtained from control c bl/ mice induced by either a-cd antibodies or cpg-odn. this histamine effect was completely inhibited by the h r-specific antagonist famotidine, while h r-, h r-, and h /h r-selective antagonists had no or only moderate effects. interestingly, the h r-selective reagent jnj , which serves as an antagonist on human cells, did not inhibit the histamine-mediated reduction of a-cd induced ifng synthesis, but in contrast it slightly enhanced the histamine effect. thus, at the murine h r, jnj may be a partial agonist. we conclude that histamine modulates the induced production of ifng by t cells via mainly the h r and, to a much lesser extend, the h r. using this assay system, cells obtained from control c bl/ mice will be compared to those from sle-prone mrl lpr/lpr mice and the respective wild type strain mrl +/+ . i objectives: resolvins are products of omega- fatty acids and they exert potent anti-inflammatory properties. in this study we examined their effects on cytokine release in healthy subjects and autoimmune patients. to test the in vitro effects of ng/ml resolvin e (rve ) on the release of tgfb, il- and il- in the culture of peripheral mononuclear cells ( x /ml) stimulated by phorbol ester (pma) ( nm), and the combination of pma and ionomycine ( mg/ml) for hours. methods: mononuclear cells were prepared by ficoll-uromiro gradient centrifugation from healthy subjects and from patients each with sle and sjögren's syndrome (ss). level of cytokines was measured by elisa method. results: in the patients with sle (p = . ) and sjögren's (p= . ) mononuclear cell stimulation by pma resulted in a reduced release of tgf b compared with controls. rve significantly reduced tgfb release from control mononuclear cells stimulated by either pma (p= . ) or pma+ionomycin (p= . ), however rve was ineffective at reducing tgfb release in the sle and ss patients. rve caused a non-significant decrease in il- release from control mononuclear cells, but was again ineffective in sle and ss patients. the production of il- was not significantly modified by rve in any of the groups tested. the release of tgfb by ng/ml of rve can be significantly reduced in healthy control subjects but not in subjects with sle or ss. at the single dose of rve tested, il- and il- release were not significantly affected in healthy or autoimmune patients. omega- fatty acid derived rve may affect inflammation in healthy patients by reducing tgfb production but its effects on inflammation in sle and ss patients may be expected to be smaller or non-existent. in addition, the tgfb release in the pma activated mononuclear cells of sle and sjögren's patients is less than that of healthy subjects. g. e. fragoulis , a.k. tsirogianni , m. herrmann , h. m. moutsopoulos , m.n. manoussakis university of athens, dpt pathophysiology, athens, greece, university of erlangen-numberg, institute for clinical immunology, erlangen-numberg, germany objectives: altered phagocytic capacity has been shown to characterize systemic lupus erythematosus (sle) that is thought to lead to impaired clearance of apoptotic remnants. herein, we assessed comparatively the phagocytic capacity in the peripheral blood of ss and sle patients and investigated the phagocytosis of apoptotic/necrotic cells in the salivary glands of ss patients. methods: patients studied included with primary ss (american-european criteria ) and with sle (acr criteria ). age-and sex-matched healthy blood donors to the ss and sle groups ( donors each) were also studied in all assays. the phagocytosis capacity (phagocytosis index) was assessed by flow cytometry, as previously (gaipl et al, j autoimmunity, ) using heparinized whole blood from individuals studied mixed with a commercially available preparation of fluorescent microbeads (mb-phagocytosis) or a preparation of propidium iodide-stained necrotic cell-derived material obtained from heat-treated normal pbmc (snec-phagocytosis). salivary gland biopsies of patients with ss with and without malt lymphoma ( patients each) were also assessed by confocal microscopy for the presence of apoptotic/necrotic material (tunel assay) and the presence of macrophages (cd -staining). results: in agreement to previous studies, mb-phagocytosis was found significantly decreased in granulocytes and monocytes of sle patients (both for p= . ). in ss patients, defective mb-phagocytosis involved only monocytes (p x . ) and significantly correlated with the presence of extraglandular manifestations (p= . ). compared to controls, snec-phagocytosis was significantly increased in the granulocytes of sle (p x . ) and of ss (p= . ). in the salivary gland biopsies of ss patients, the lymphoepithelial lesions and germinal center-like structures manifested significantly increased infiltrations by macrophages. these lesions were also characterized by notable accumulation of apoptotic/necrotic material that resided both inside and outside the phagocytes. these phenomena were significantly more intense in the salivary gland lesions that manifested malignant in-situ b-cell lymphoma. conclusion: in a manner similar to sle, ss patients appear to manifest altered phagocytic capacity. this may be associated with the observed accumulation of apoptotic/necrotic cells in the salivary glands that in turn, may participate in the chronic autoimmune reactions and/or the lymphoma-generating processes that characterize the disorder. the autoantibodies to various enzymes are often found out in sera of systemic lupus erythematosus (sle) patients, but clinical value of such antibodies often is not understood. the purpose of work was to study the of antibodies generation to the basic enzyme of purine metabolism -adenozine deaminase (ada) in sle and to reveal the relationship of studied antibodies with clinical and laboratory features of pathological process. methods: healthy persons have been included in our study and sle patients ( women and men) with various clinical signs ( persons had st degree of disease activity, persons - nd degree of pathological process activity). women had habitual noncarrying of pregnancy (hnp) in anamnesis. antibodies of igg class to ada (anti-ada) determined by technique of indirect elisa developed by us with the use of immobilized form of ada as an antigenic matrix. b glicoprotein-i-dependent antiphospholipids (aphl) of igg classes were determined using commercial "anti-phospholipid screen igg/igm" test set (orgentec diagnostica). results: at admission an anti-ada was revealed in , %, aphl of igg class -in , % sle patients. it has been noted that igg-aphl were found out in anti-adapositive patients more often and in higher antibody titer, than in anti-ada-negative sle patients (x = , ; p x , ). development of cytopenic syndrome was noted reliable more often in sle patients with associated presence of igg-aphl and an anti-ada in comparison with patients who has not the combinations of these antibodies in blood (x = , ; p x , ). the increased levels of anti-ada were revealed in of women with hnp, and the combination of anti-ada and aphl ( / ) was found out more often than isolated anti-ada ( / , x = , ; p x , ) or isolated aphl ( / , x = , ; p x , ). conclusion: taking into account the imbalance of immunoregulatory functions in sle, the further studying of autoantibodies to ada generation seems to be very promising. presence of hnp in anamnesis is the evidence of necessity of careful biochemical monitoring of aphl and anti-ada in women for the prevention of abortus fetus and administration of adequate therapy. objectives: sjögren's syndrome (ss) is a chronic inflammatory and lymphoproliferative autoimmune disease, characterized by dryness of the mouth (xerostomia) and the eyes (keratoconjunctivitis sicca). dendritic cells (dc) are the most potent antigen-presenting cells that play a crucial role in initiating and maintaining primary immune responses. two main subsets of dc have so far been identified in human peripheral blood: myeloid dc (mdc), which can be further divided into mdc and mdc , and plasmacytoid dc (pdc), also known as ifn-a/b producing cells. the pivotal role of dc in inducing and maintaining tolerance could be critical in ss as alterations among dc populations might contribute to autoimmunity. purpose of this study was to quantify mdc , mdc and pdc in peripheral blood from primary ss patients by flow cytometry and compare the results with gender-and age-matched healthy controls. methods: blood samples from pss patients fulfilling the american european consensus group criteria (aecc) and gender-and age-matched healthy controls were collected in heparin tubes. dc populations were stained with the blood dc enumeration kit, miltenyi, according to the manufacturer's manual. cells were analyzed on a facs canto ii, bd, and data analysis was performed with flowjo software, tree star. for the statistical analysis, a two-tailed mann-whitney u test was performed using prism, graphpad. results: pss patients have significantly reduced amounts of pdc (p= , ) and mdc (p x , ) in peripheral blood. conclusion: alterations in dc populations have been considered to play a role in autoimmune diseases such as systemic lupus erythematosus (sle) or diabetes. in ss patients, up-regulation of interferon-regulated genes has been shown previously. therefore, decreased pdc numbers in peripheral blood from pss patients might explain the fact that an increased ifn signature is found in salivary glands of pss patients, but no elevated levels of ifn-a are measured in serum. recently we reported that malignant cd + b cells from patients with b chronic lymphocytic leukemia (b-cll) produce granzyme b (grb) and are rapidly undergoing apoptosis in a granzyme b-dependent manner following interleukin (il- ) stimulation. several autoimmune diseases have been linked to both elevated frequencies of cd + b cells and increased il- levels. we therefore hypothesized that il- may have similar biological effects on cd + b cells in autoimmune diseases. here we demonstrate that the amount of il- in the serum of systemic lupus erythematosus (sle) patients but not of healthy subjects highly correlated with serum levels of grb. in contrast to b cells from healthy individuals, where no baseline grb expression was found, we demonstrate that up to % of cd + b cells in sle individuals expressed grb. in-vitro experiments revealed that il- was able to induce expression of grb in b cells from individuals with sle and other autoimmune diseases including psoriasis and rheumatoid arthritis. this effect was direct and was strongly enhanced by engagement of the b cell receptor or toll-like receptor . importantly, il- significantly decreased the cd +/cd -b cell ratio in both sle peripheral blood and healthy cord blood samples, suggesting a preferential induction of cd + b cell death. these results suggest that il- -induced grb may play a regulatory role for cd + b cells similar to what we described earlier in b-cll cells. this is the first report uncovering an interrelation between il- and grb levels in sle and showing that il- reduces the cd +/ cd -b cell ratio in b cells from sle peripheral blood and healthy cord blood. endogenous il- may therefore play a disease-modifying role and may explain elevated grb serum levels in autoimmune diseases. further studies should evaluate the therapeutic potential of il- in sle and other autoimmune diseases. r. de palma , e. d'aiuto , s. vettori , g. abbate , g. valentini second university of naples, clinical & experimental medicine, napoli, italy ssc is considered an autoimmune puzzling disease whose pathogenesis is unknown. in the last years, there have been increasing evidences that an interplay between activated t cells and fibroblasts could play a pivotal role in promoting matrix accumulation in systemic sclerosis (ssc). we have previously shown that peripheral t cells from ssc patients with early diffuse disease co-cultured with autologous fibroblasts expand the same t cell clonotypes found in the affected skin. here, using the same experimental approach, we found that the t cell clonotypes expanded in co-cultures are ab positive, hla-dr positive, and promote apoptosis of autologous ssc fibroblasts. we also found that, in these co-cultures, ssc fibroblasts up-regulated fas and underwent apoptosis that paired with the expression of fas ligand (fasl) on cd + t cells. finally, when we added a blocking anti-fas antibody to the co-cultures, we observed a marked reduction of fibroblast apoptosis, suggesting that engagement of fas/fasl had a critical role in mediating apoptosis in co-cultured fibroblasts. it has to be reminded that the absence of fasmediated apoptosis in vivo could be due to several reasons, as the increase of soluble fas in sera of patients affected by ssc. moreover, in the co-culture supernatants we found tgf-beta, il- beta, il- and il- , cytokines known to have a role in promoting fibrosis in systemic sclerosis. taken together, these data suggest that t cell response in ssc may represent an attempt of the immune system to kill fibroblasts, cells that are likely to be altered and expressing (auto)antigens. indeed, fibroblasts of ssc patients have been shown to display a persistently activated phenotype characterized by excessive production of collagen and other extracellular matrix proteins. however, the overall outcome of the t cell response triggered by fibroblasts in ssc, while unable to control the activity and the growth of fibroblasts, contribute to sustain inflammatory loops leading to fibrosis. these findings may lead to change our view about the pathogenesis of this disease and other autoimmune diseases. systemic lupus erythematosus (sle) is a chronic inflammatory autoimmune disease that is associated with a major breakdown in b cell self-tolerance as reflected by elevated serum igg levels of predominantly antinuclear antibodies (anas). serum antibody titers are maintained by antibody-secreting plasmablasts and longlived plasma cells, which reside in survival niches of the bone marrow. however, the antibody repertoire of bone marrow plasma cells, which may include cells expressing autoreactive and potentially pathogenic antibodies, has not been characterized in sle. to determine the frequency, specificity and immunoglobulin gene characteristics of autoantibodies in the long-lived plasma cell compartment in sle, we cloned and expressed igg antibodies from single facs purified cd +cd +cd +cd + bone marrow plasma cells of patients with sle and tested the recombinant monoclonal antibodies for self-reactivity. our preliminary data on the ig gene repertoire and reactivity profile of human igg+ sle bone marrow plasma cells in comparison to healthy controls will be discussed. z. amirghofran , e. moazemi godarzi , e. kamali sarvestani , e. aflaki shiraz university of medical sciences, shiraz, iran, islamic republic of interleukin (il- ) has been shown to be related to the pathogenesis of systemic lupus erythematosus (sle). two polymorphisms in the promoter region of il- gene at positions - g/c and - g/c have been described that are key regulators of il- gene. in the present study the relationship between these two polymorphic sites and disease susceptibility in a group of iranian patients with sle was investigated using polymerase chain reaction-restriction fragment length polymorphism method. the genotype distribution and allele frequencies of il- gene polymorphism at - position showed no significant difference between sle patients and controls. at this position the frequency of gg genotype as well as g allele was higher than c allele in both patients and control groups. in contrary, both allelic and genotypic frequencies at the - position significantly differed in sle patients and controls. at this position gg genotype was observed in . % of patients compared to . % in the control group (p x . ). the frequency of - g allele in patients ( . %) was also higher than in controls ( . %) (p= . ). the haplotype study showed no significant difference between patients and healthy subjects. the relationship between these polymorphisms and clinical manifestations and laboratory parameters were investigated. - polymorphism was associated with the presence of antinuclear antibodies in all patients and rash and hematuria in male patients (p x . ). at - polymorphism, a significant difference with regard to photosensitivity in male patients (p= . ) was found. in conclusion, results of this study showed that - polymorphism plays an important role in susceptibility to sle and that - polymorphism could influence the presence of antinuclear antibodies in the patients. the eukaryotic constitutive proteasome is the main protease expressed in most tissues. recently we have elucidated a functional importance of the second proteasome form, inducible immunoproteasomes, in regulating nf-kb activity during the intestinal inflammation. in comparison with healthy controls and patients with ulcerative colitis (uc), there was increased expression of immunoproteasomes in the inflamed mucosa of patients with crohn's disease (cd) at both mrna and protein levels. in our very recent work we have shown that the proteasome subunit pattern might be suitable for diagnostic differentiation between cd and uc patients. since ifn-g has been shown to be the main inducer of immunoproteasomes in various murine and human cell lines and the ifn-g levels are highly elevated in inflamed intestine of cd patients, induction of immunoproteasomes in cd might be mediated by this cytokine. our data with human leukemic t cell lines and primary macrophages show a significant increase in the nf-kb controlled production of proinflammatory cytokines after the ifn-g-mediated induction of immunoproteasomes in these cells. in the dss-induced colitis model we have observed a diminished colonic inflammation in the absence of the proteasomal immunosubunits. therefore we here suppose that immunoprteasome are involved in the complex inflammatory response during the chronic intestinal inflammation by increasing nf-kb activity in the epithelial and immune cells. however, it remains to be determined whether these results have an important implication for the treatment of chronic gut inflamation in humans. objectives: inflammatory bowel diseases (ibd) including crohn's disease (cd) and ulcerative colitis (uc) are characterized by unknown etiology and chronic intestinal inflammation. noninvasive serological tests to differentiate cd from uc have been searched for a long time. testing for panca together with ascas has good predictive values to identify patients with ibd.the aim of this study was to find evidences for diversity of ascas and anti-mycobacterium paratuberculosis antibodies (anti-mpt) by elisa method. in addition, to examine whether combination of these elisas is useful for distinguishing cd from uc. methods: the study population contained patients with ibd ( with cd, with uc, with gluten sensitive enteropathy, gse) and healthy control subjects. serum asca igg, asca iga and anti-mpt antibody levels were measured by solid phase enzyme immunoassay. adsorption of asca positive sera was performed by baker's yeast suspension. results: elevated level of asca igg, iga and anti-mpt was shown in cd and gse but not in uc compared to healthy controls. serum levels of asca igg, iga showed a significant positive correlation with anti-mpt antibody levels in cd. repeated adsorptions with yeast removed asca igg and iga from sera of patients, but did not change levels of anti-mpt. these results indicate the diversity of asca igg, iga and anti-mpt (accordingly their antigens) and suggest that combination of these elisa can have a role in the differential diagnostics of ibd. it is now well recognised that the majority of lymphocytes may be located within tissues, not in blood, and yet these tissue-resident lymphocytes are relatively understudied, especially in humans. we have extracted cells from human gut biopsies (both normal and inflamed gut) in order to characterise the immune cell populations that exist therein and which molecules may be of paramount importance to their function. we show that distinct populations of t cells exist within the gut and that the ratio of these populations changes down the length of the gut, with the so-called 'unconventional' double negative t cell population (ie tcrab+ve, cd -ve cd b-ve) predominating in the healthy colon whereas these cells are overwhelmed by infiltrating cd (+) cells in inflammatory bowel disease (ibd). having previously shown in mice that gut-resident t cells express high levels of the regulator of g protein signalling- (rgs- ) protein, we have now found substantial over-expression ( - fold) of rgs- in human gut-derived t cells, particularly in this unconventional t cell population. furthermore, levels appear even higher in t cells derived from inflamed gut. transfection of rgs- decreases primary t cell responses to cxcl and ccl , strongly implying that it may regulate t cell localisation. thus, rgs- may be a novel target for modulating t cells in ibd, consistent with which snps in rgs- have been associated with both coeliac disease and type diabetes. mechanisms involved in the induction of oral tolerance (ot) or systemic immunization through the oral rout are still poorly understood. in our previous studies we have shown that when normal mice eat peanuts they become tolerant, with no gut alterations. conversely, if they are immunized with peanut proteins prior to a challenge diet (cd) containing peanuts they develop chronic inflammation of the gut. our aim is to evaluate the consequences of the introduction of a novel protein in the diet of animals presenting antigen specific gut inflammation. adult, female c bl/ j mice were divided in control (c) and experimental (e) groups. c -c received peanut protein immunization, animals of the control groups c were sham immunized, and control group c received ovalbumin (ova) immunization. the experimental group was immunized with peanut protein extract. before initial exposure to a day peanut containing cd, the experimental group was divided into groups (e -e ). ova feeding began days prior cd (e ) on day (e ), (e ), (e ) and (e ) during cd. our results show that oral exposure to a novel protein (ova) in the absence of gut inflammation (e ) leads to low levels of systemic antibody titers, comparable to tolerant animals. conversely, as off initial induction of inflammation, groups submitted to ova (ot) protocol develop increasingly higher systemic antibody (ab) titers similar to animals of the immune control group. in conclusion our protocol indicates that timing is more important than the antigenicity when a novel protein is offered, in the diet. nanoparticles of various types are increasingly used as constituents of food supplements and so called nanofood. since nanoparticles induce inflammatory reactions in the lung, there is an urgent need to also study the toxicological potential of nanoparticles in the intestine. therefore, we assessed the effect of particles on dendritic cells (dc) as key players in the manifestation of intestinal immunity.in in vitro studies we could show that ultrafine tio as well as ultrafine silica led to a mature phenotype of the cells when particles were added to cultures of immature bone-marrow-derived dc. this effect appears to result from enhanced cell death in immature dc but also from direct stimulation of the cells.to analyse the mechanisms underlying this effect we looked for apoptosis as well as for induction of the inflammasome since it has been shown that crystalline silica leads to activation of caspase and secretion of bioactive il -b.in our hands certain nanoparticles induced apoptosis of immature dc, as well as enhanced secretion of active il -b. we therefore hypothesize that particles can induce the inflammasome which leads to the activation of dc.to study the impact of nanoparticles on intestinal inflammatory processes in vivo, we induced colitis by applying % destrane sulphate sodium (dss) in the drinking water for days to wildtype mice. when ultrafine nanoparticles were administered on day and by gavage feeding, we observed an amelioration of disease symptoms when scoring the degree of epithelial disruption and inflammation. in future experiments we will also analyse the effect of different particles in the il -/model of colitis to assess the contribution of particles to the induction and pathogenesis of disease. m. schmohl , n. schneiderhan-marra , m. blum , g. stein , m. schmolz , t. joos nmi-natural and medical sciences institute at the university of tübingen, biochemistry, reutlingen, germany, edi gmbh, reutlingen, germany the human immune system represents a highly complex system that protects the organism against diseases. there is an impressive network of immunoregulatory signals within the immune system as well as between the different healthy and diseased organs. epithelial layers function as a barrier against pathogens. as the gastrointestinal tract, which is occupied with a large variety of microorganisms, represents the outside of the body, the immune system has to establish and maintain a strong presence at the mucosal boundary. the ability to discriminate between pathogens while remaining relatively unresponsive to food antigens and the commensal microflora is achieved by a plethora of largely unknown regulatory mechanisms. this ability appears to be breaking down with chronic inflammatory bowel diseases (ibd) like crohn's disease and ulcerative colitis [ ] . to date treatment options are restricted to controlling symptoms, putting and keeping the dis-eases in remission and preventing relapse. therefore, there is an urgent need for a more detailed understanding of the inflammatory events taking place during the disease. for this purpose a human organo-typic (hot) co-culture model is used, which allows analyzing the collaborative regulation between the immune system and the gut epithelial cells. the human caco- cell line, as a model for the gut-epithelium cells, are cultivated on the top side of special culture vessels, fitting as inserts into carrier wells of -well culture plates, containing whole-blood. this co-culture set up mimics the physiological barrier to perorally applied biologicals/drugs and allows measuring their effect on the immune system. as a read out miniaturized and parallelized sandwich immunoassays will be used to detect alterations in the intracellular mapk and rtk-signalling of the epithelial cells as well as in the extracellular communication via cytokines and chemokines at the interface of the two organs. this approach will provide new insight into the inter-and intracellular signalling of gut epithelium and the immune system, which will finally result in a better understanding of the etiology of inflammatory bowel diseases. inflammatory bowel disease (ibd), including crohn's disease (cd) and ulcerative colitis (uc), is characterized by an upregulation of pro-inflammatory cytokines that play an important role in pathogenesis. osteopontin (opn) is a cytokine implicated in several immunological diseases and, although expressed constitutively in normal intestine, is upregulated in intestinal mucosa and in the plasma of ibd patients. opn has been shown to be either pro-inflammatory or anti-inflammatory for experimental uc, indicating a controversy in this field, while its role in experimental cd remains unknown. in our study we investigated the role of opn in experimental colitis using two mouse models: trinitrobenzene sulphonic acid (tnbs) colitis, a t h -associated model that resembles cd, and dextran sulphate sodium (dss) colitis, a t h -like-associated model for uc. deficiency of opn (either by antibody-mediated neutralization or use of opn -/mice) resulted in suppression of disease phenotype in both colitis models, revealing that opn, and especially, the secreted isoform of opn (opn-s) is important for the initiation of acute intestinal inflammation. importantly, we discovered that opn drives il- production and t h polarization and decreases recruitment of cd + cd + foxp + t regulatory (treg) cells in mesenteric lymph nodes (mlns) of mice with colitis. also, there was an effect of opn on recruitment of cd c + dcs, which were significantly elevated in mlns of opn -/or anti-opn-treated, as compared to opn +/+ or ig-treated control mice. this finding implies that opn deficiency results in enhanced recruitment of regulatory cd c + dcs which may mediate treg induction and protect from colitis. overall, our findings indicate that opn is proinflammatory in both types of colitis, by promoting pathogenic t h and attenuating treg cell recruitment, implying also common mechanisms in the pathogenesis of cd and uc. c. shen , , g. van assche , p. rutgeerts , a. liston , j. l. ceuppens k.u. leuven, autoimmune & genetics lab, vib, leuven, belgium, k. u. leuven, experimental immunology lab, leuven, belgium, k. u. leuven, department of pathophysiology, gastroenterology section, leuven, belgium background: haptoglobin (hp) is one of the acute phase proteins synthesized during inflammation. hp- allele is associated with the disease behavior in crohn's disease but not in ulcerative colitis. however its role in inflammatory bowel disease has not been defined. aim: to determine whether hp modulates the immune responses in experimental colitis. methods: we induced types of colitis dss (th /th ), tnbs (th ) and oxazolone (th ) in hp ko mice. neutralizing anti-il- mab was injected into dss and tnbs hp ko mice. severity of colitis was evaluated by body weight, colon length and histology. th /th cells were analyzed by flow cytometry. cytokines were measured by elisa or rt-pcr. ) compared to the wt mice, hp ko mice developed much severer dss and oxa induced colitis. dss induced lethal colitis in hp ko but not in wt mice; ) in dss but not in oxa colitis mice, il- , ifn-g, tgf-b and il- were significantly increased (p x . , dss vs control) in lamina propria and mesenteric lymph nodes (mln), and this is much evident in hp ko mice compared to those in the wt (p x . , ko vs wt). in tnbs colitis, we found elevated il- and ifn-g (p x . , tnbs vs control). although not significant, il- was also somewhat upregulated; ) in dss colitis we observed that il- enhanced differentiated th cells in vitro, this effect could be abrogated by coculture with serum from wt but not hpko mice. furthermore, in vitro in the presence of tgf-b, il- and il- , more mln-t cells from hpko mice differentiated into th cells; ) anti-il- mab improved dss and tnbs colitis, and partially rescued hp ko mice from lethal dss colitis. in line with this, mice treated with anti-il- showed reduced il- , il- and ifn-g in both mln and lp (p x . , anti-il- vs control). our results reveal that hp has a protective role in the development of mucosal inflammation. in dss and tnbs colitis hp may exert its beneficial effect partially through inhibiting production of il- , supporting further pre-clinical and clinical application of hp for treatment of crohn's disease. p. engelmann , g. talabér , g. süt" o , p. németh , t. berki university of pécs, clinical center, department of immunology and biotechnology, pécs, hungary, university of pécs, clinical center, department of immunology and rheumatology, pécs, hungary objectives: inflammatory bowel disease (ibd) resembles as an autoimmune-like disease. ibd is most common in developed countries: it is calculated that . million people in europe suffer from ibd. several hypotheses are raised in the pathogenesis of inflammatory bowel disease. one of the most favored is the dysregulation of the immune response due to failure of regulatory t cells. the most well known regulatory t cells are the cd +cd hi+ t (treg) cells. furthermore, other immune-regulatory cells are known such as invariant natural killer t (inkt) cells producing both th and th cytokines rapidly upon antigen (lipid) stimulation. methods: based on this hypothesis we aimed to investigate the role of various immune-regulatory t cells in human ibd. we attempt to measure the proportions of inkt cells, treg cells in peripheral blood of patients with crohn's disease (cd) and ulcerative colitis (uc) compared to normal controls. blood samples were collected from normal controls and ibd patients; then lymphocytes were labeled for inkt and treg markers with specific monoclonal antibodies and measured with flow cytometry. results: according to our results a decline in the total inkt cells of ibd patients was observed, interestingly the proportions of cd + and double negative (dn) inkt subgroups showed a characteristic shift among the study groups. percentages of dn and cd + inkt subpopulations were assessed after gating of total inkt populations. in controls we observed high percentage of dn inkt cells ( . ± . %, mean ± sem), while cd + inkt cells ratio was moderate ( . ± . %). in uc and cd patients we found a reduced proportion of dn inkt cells (uc: . ± . %; cd: . ± . %, mean ± sem), while the percentage of cd + inkt cells was elevated (uc: . ± . %; cd: . ± . %, mean ± sem) in both disease groups. proportions of foxp + treg cells also showed a decline in ibd patients comparing to normal controls. conclusion: this study can provide useful data about the pathogenesis of ibd and can lead to identify and characterize new cellular and molecular targets with possible therapeutic use in human autoimmune disorders. objectives: the aim of this project is to explore whether exosomes from tgf-b gene modified bone marrow-derived immature dendritic cells (md-imdc) have the function of systemic immune inhibition and protective effect on the development of inflammatory bowel disease (ibd) in mice, the underlying mechanism was also investigated. methods: exosomes were isolated from supernatant of md-imdc transfected with tgf-b adenovirus (tgf-b -exo). the t cell inhibitory function of tgf-b -exo was determined by mixed lymphocyte reaction (mlr) in vitro. to evaluate the protective effect of tgf-b -exo in the development of ibd, dextran sulfate sodium(dss) induced murine ibd was established and mice were treated with tgf-b -exo. the main symptoms of ibd were observed. the inflammatory degree of colon was also evaluated by histological examination. the relative cd + foxp + treg cell numbers from spleens and mesentery lymph nodes (mlns) were analyzed by facs. results: it was demonstrated that tgf-b -exo could inhibit the proliferation of t cells in mlr in vitro. in murine ibd model, after treated with tgf-b -exo, the main symptoms of ibd such as weight loss, diarrhea and grume sanguinopurulent stool were all alleviated and the inflammatory degree of colon was also reduced. analysis of cd + foxp + regulatory t cells (treg) revealed that the relative numbers of cd + foxp + treg increased in lymphocytes from mesentery lymph nodes (mlns) of inflammatory site but not from spleens. conclusions: these results demonstrate that immunosuppressive exosomes obtained from tgf-b gene modified md-imdc can delay the development of ibd. this protective effect is mediated by the induction of cd + foxp + treg. tgf-b -exo might provide a novel strategy for the therapy of ibd. results: hcv-specific cytokine expression by cd + t-cells was similar in the four vaccinees as observed by ifng, il- production-profiles. however, the killing capacity of expanded cd + t-cells was distinct as observed by the competence to kill ns -peptide presenting transfectants in vitro. as depicted in figure , cd + t-cells cells from both vac (cleared ) and vac (chronic) produced il- and ifng after stimulation with ns -peptide . however, specific killing of the peptide loaded transfectants was only observed in vac , who was able to clear its hcv infection, and this was not observed not in any of the other chimpanzees, who became chronic carriers. [ figure ] killing of ns peptide presenting cells was restricted to the vaccinee that was able to clear hcv infection. these results suggest that controlling hcv replication as initiated by this dna-prime mva-boost vaccine-protocol was partly mediated by antigen specific cd + t-cells. hence, the effector mechanisms induced were distinct between the animals and clearance of the infection was correlated with induction of killing competent cd t-cells. objectives: infection by hepatitis c virus (hcv) is characterized by its high tendency to chronicity, which is usually associated with a low or absent t-cell response against viral antigens. immune response specific for non-structural protein ns from hcv was associated with viral clearance. we have demonstrated that fusion of an antigen to the extra domain a from fibronectin (eda) targets the antigen to tlr -expressing dendritic cells and improves its immunogenicity. thus, we tested if covalent linkage between eda and ns might constitute an alternative for vaccination against hcv infection. methods: recombinant plasmids expressing a secretable version of ns or eda-ns under the control of cmv promoter were prepared. recombinant ns and the fusion protein eda-ns were produced in e. coli. the recombinant proteins were tested in vitro on their capacity to activate maturation of bone marrow derived dendritic cells and to favour antigen presentation. hhd transgenic mice (expressing the human hla-a molecule) were immunized with the recombinant plasmids or with the recombinant proteins, in the absence or presence of poly(i:c) and anti-cd agonistic antibodies. elispot and chromium release assays were carried out to measure the immunogenicity of the different vaccination strategies. intrahepatic expression of hcv-ns rna was measured after a hydrodynamic injection with a plasmid encoding hcv ns . results: immunization of mice with the plasmids expressing eda-ns , but not ns alone, induced strong t cell responses against the main hla-a restricted cytotoxic t cell determinants from ns . the recombinant eda-ns fusion protein, but not ns , was able to activate in vitro maturation of bone marrow derived dendritic cells as well as the production of tnf-a by the thp- monocyte cell line. immunization of hhd mice with eda-ns fusion protein induced both cd + and cd + t cell responses against ns and, when immunized with poly(i:c) and anti-cd antibodies, was able to down-regulate the intrahepatic expression of hcv-ns rna. the recombinant eda-ns fusion protein may be considered for the development of prophylactic or therapeutic vaccines against hcv infection. vaccination is the most efficient strategy to prevent from microbial infections and to control epidemics but are still not available in the case of hiv infection even years after virus detection. therein we propose the intra-dermal inoculation of dna vaccine that present a plasmid vector exploiting the binding capacity of the bovine papillomavirus e protein encoding an artificial multi-component hiv antigen. this inoculation is followed by electroporation in order to increase dna uptake. we used skin as site for vaccination because, being the first line in host defence, it is populated with various cells of immune system. among them, langerhans cells (cd +cd a+), located in the epidermis, are dendritic cell subset capable to elucidate specific cd + responses. the present work emphasizes molecular and cellular biodistribution of the dna vaccine in the skin after intra-dermal vaccination in macaques, as one of the most relevant animal models in hiv studies. technical approach considers an intra-dermal injection of dna followed by topical electroporation of the injection sites. skin and draining ln biopsies were collected at different time points. these biopsies were used for ihc fluorescent staining in order to establish biodistribution dna-encoded antigens and co-localisation with different cell types. kinetic of antigen expression was studied by bioluminescence in vivo imaging. t cell responses were measured by ifn-g elispot assays up to years after dna vaccination. we show that a dna vaccine delivery method combining intra-dermal injection and electroporation dramatically increased the expression of the vaccine antigen selectively in the epidermis, increased the frequency of cd a+ cells in the draining ln in association with the antigen expression, and increased the cellular response persistence, at high levels, for more than two years after the last vaccine boost. our data suggest that electroporation after intradermal injection of dna vaccine involves langerhans cells from the epidermis that elucidate qualitative anti-hiv immune responses. this new approach that comprise new dna vaccine followed by non-invasive electroporation, induce long-lasting cellular response that could be crucial in prophylactic / therapeutic vaccine design. presenting cells was developed. murine coronavirus-based virus-like particles encoding epitopes from the lymphocytic choriomeningitis virus glycoprotein or human melan-a, in combination with the immunostimulatory cytokine gm-csf, selective targeted dcs in vitro and in vivo resulting in vector-mediated antigen expression, and efficient maturation of dcs. in mice, a single application of only low doses elicited strong and long-lasting cytotoxic t-cell responses which provided protective antiviral and antitumor immunity. furthermore, the efficient activation of human tumor-specific cd + t cells by mature dcs transduced with melan-a-recombinant human coronavirus e indicates that this novel vaccine platform mediates the delivery of antigens and immunostimulatory cytokines to those cellular components of the immune system that initiate and maintain protective immunity. as the application of gm-csf already enhanced immunogenicity, we are now trying to further modulate the coronavirus vector-induced immune response with the reverse genetic setup of recombinant coronavirus-based vectors expressing different immunostimulatory cytokines. thereby cytokines will be acting on t cell and dc level. to enhance t cell response interleukin (il ) and interleukin (il ) will be involved, and fms-like tyrosin kinase ligand (flt l) will be expressed to modulate dendritic cells. il is known to enhance early t cell expansion and limits t cell overshoot, whereaes il guarantees survival of high affinity t cells during memory phase. on the other hand flt l enhances dc proliferation and accumulation. with these approaches modulation of the immune response generated by this novel vaccine platform will be examined in viral and tumour models to get insight on the antigen specific ctl response, synergistic effects of the cytokines and protective as well as prophylactic vaccination approaches. f. demircik , ag waisman uniklinik mainz, . med, mainz, germany in murine cytomegalovirus (mcmv) infection, cytotoxic cd t cells and nk cells play a critical role. previously it was shown that mice deficient for b cells are more susceptible to mcmv-related disease, caused by virus reactivation. to better understand the role of b cells and antibodies in the response to mcmv, we made use of different mouse strains that lack b cells, secreted antibodies or il- production by the b cells. we found that for the initial t cell response to the virus b cells are important, but antibodies do not play an important role. this implicates b cells as potential important antigen presenting cells (apcs) in the activation of the virus-specific t cells. the reduced t cell response to the virus was observed whether the mice were b cell deficient from birth or if they were depleted later in life. six month after infection mice were tested for the memory cd t cell response. interestingly, we found that in mice that lack antibodies (mice that lack b cells all together and mice that have b cells but no secreted antibodies) maintain a rather high t cell response to viral peptides, in a level similar to the acute response days after infection. we conclude that antibodies probably remove residual viruses from the body and therefore prevent the continuous activation of t cells. finally, we tested the role of il- produced by b cells by conditional deletion of the il- gene in these cells. we found that b cell secreted il- has a suppressive effect on the t cell response to mcmv, as this response is elevated in these mice. we conclude that b cells are important for an efficient acute response to mcmv and that antibodies play a role in eliminating residual viral particles, thus implicating a dual role for b cells in the efficient acute and memory response to mcmv. this work is supported by the deutsche forschungsgemeinschaft grant sfb to aw. objectives: ebv infection leads to life-long viral persistence. although ebv infection can result in chronic disease and malignant transformation most carriers remain disease-free due to an effective control of the virus by t cells. ebv-specific ifng-producing t cells could be demonstrated in acute and chronic infection by many researchers. recent studies in hiv and leishmania provide, however, evidence that assessing ifng alone is insufficient to assess the quantity and quality of a memory t cell response and support the crucial role of multifunctional t cells in disease control. in this study we therefore analyzed ebv-specific t cell responses in peripheral blood (pb) and bone marrow (bm). methods: paired pb and bm samples were obtained from healthy virus carriers who underwent total hip arthroplasty. t cells were expanded for days in the presence of il- and il- with exposure to overlapping peptide pools of latent ebna- and lytic bzlf- antigens. ebv-specific immune responses were assessed exvivo and after expansion by multiparameter flow cytometry staining for live/dead discrimination marker, cd , cd , cd , ccr , cd ra, il- , tnfa, ifng and cd a. the majority of ex vivo ebv-reactive cd + t cells as well as ebna- -reactive cd + t cells were il- and tnfa-producing memory cells, the later being more frequent in bone marrow (cd +, median, ebna- : bm . %;pb . %; bzlf- : bm . %;pb . %, p= . ). after in vitro expansion a major subset of ebv-specific cd + and cd +t cells displayed a differentiated effector ifng/tnfa phenotype. a comparable number of ebv-specific cd + and cd + t cells retained, however, a tnfa single, tnfa/il- or triple producer phenotype resembling early differentiated or multifunctional memory t cells, respectively. interestingly, both cd + and cd + t cells generated from bm revealed significantly higher cytotoxic potential. sorting of ccr /cd ra differentiation subsets, revealed that ebv-specific t cells were predominantly expandable from the central memory compartment. conclusion: our data shows that multicolor assessment of ifng, tnfa and il- delineates various subsets of ebv-specific memory t cells, which reflect the profile of a protective immune response. human adenovirus (hadv) can cause serious morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation (allosct). reconstitution of hadv-specific t cells has been reported to be associated with sustained protection from hadv disease, but epitope specificity of these responses has not been further characterized. furthermore, the relative contribution of hadv-specific cd + and cd + t cells in the protection from hadv disease after allosct remains to be elucidated. in this study, we demonstrate, by sensitive measurement using intracellular cytokine staining combined with cd or peptide-mhc tetramer staining, that clearance of hadv was associated with a combined hadv hexon specific cd + and cd + t cell response in both pediatric and adult allosct recipients. based on this observation, we developed a clinical grade method for the rapid generation of t cell lines with high and defined specificity for hadv hexon epitopes for adoptive immunotherapy. activation of hadv hexon-specific cd + and cd + t cells in peripheral blood with a hexon protein-spanning pool of synthetic -mer peptides followed by ifng-based isolation allowed rapid expansion of highly specific t cell lines from healthy adults, including donors without detectable frequencies of hadv hexon-specific t cells. the frequency of hadv-specific t cells was increased to - % in the t cell lines and the absolute numbers of both hexon-specific cd + and cd + t cells were to log increased compared to the starting material. detailed analysis showed that hadv-specific t cell lines recognized multiple mhc class i and ii restricted epitopes, including known and novel epitopes, and showed specific and efficient lysis of hadv infected target cells. this strategy may be used for adoptive transfer of donor-derived hadv hexon-specific cd + and cd + t cells for treatment of disseminated hadv infection after allosct. several studies showed that hbv persistance correlates with a failure of an efficient virus-specific t-cell response. induction of hbv-specific t cells by vaccination may be an innovative approach to overcome virus persistance. dna prime-recombinant adenovirus serotype (ad ) boost strategy proved to be effective in stimulating t cell responses and control of viral infections. woodchuck hepatitis virus (whv) and its host the woodchuck are a useful peclinical model for investigating the new therapeutic approaches. the efficacy of plasmid dna and ad vaccine vectors expressing whv core protein was first examinated in c bl mice. groups of mice were immunized with a dna prime-ad boost regimen or with dna and ad alone. ad was injected i. m. or s. c. t cell response was evaluated by intracellular ifng staining of splenocytes stimulated in vitro with whc-derived peptide pools. anti-whc antibodies were detected by elisa. we detected cd + t cell responses against peptide pools and in spleens of dna and dna-ad immunized mice. however, in prime-boost group the percentage of of detected ifng+ cd + t cells was lower in comparison to dna group. in splenocytes of animals vaccinated with ad very weak cd + t cell response was observed. in dna vaccinated animals we determined high level of anti-whc already after second immunization. after boosting with ad level of antibodies did not change. those antibodies were only igg a subclass what indicates th t helper type of response. ad -immunized mice had over -fold lower level of anti-whc: both igg a and igg subtypes were detected. the weak response induced by ad may be due to the low expression of whcag. in ongoing expreriments we improved the protein expression level by insertion of an intron. we currenly investigate the new construct in mice. the new peptide construct containing four m e-peptide sequences coupled to t helper epitopes from the plasmodium falciparum cs protein and the hepatitis b virus antigen was administered together with adjuvants intranasally and subcutaneously as described (mozdzanowska et al., virology journal ) into various mouse strains. in contrast to its predecessor peptide, we found that vaccination induced much higher anti-m e serum ab titers against peptide and native m e. this correlated with a large number of m -specific ab-secreting cells in lungs and bone marrow. moreover, the serum of vaccinated mice was also crossreactive against the influenza virus subtype a/fm (h n ), which contains a variant m e-sequence different in amino acid positions. importantly, this new peptide vaccine regimen showed significant protection against viral challenge with influenza a strains x (h n ) and the highly pathogenic pr/ (h n ) with remarkably reduced viral titers in lungs and noses of mice. in conclusion, our studies show promising results towards the further development of vaccination with m e as a potential "universal" influenza vaccine. this research is supported by a nih t fellowship ca - , the nih grant ai and a grant from the commonwealth of pa. l. yu zhejiang university, zhangzhou, china interleukin- (il- ) is a cytokine produced by stimulated mononuclear macrophage system. in this report, -day-old chicken embryos were vaccined with the plasmid dna (pci-chil- ) encoding chicken interleukin- and the copy numbers of chil- in peripheral blood, spleen and bursa of fabricius at different time points post-embryonic-vaccination were detected by real-time fluorescent quantitative pcr. the polyprotein of infectious bursal disease virus (ibdv) was prepared into dna vaccine, and the dna vaccine was co-administrated with pci-chil- in -day-old chicken embryos, then boosted after two weeks, and challenged with virulent ibdv four weeks later. the results indicated that allantoic cavity vaccinated with pci-chil- could accelerate high concentrations of chil- in nonage peripheral blood, accelerate high expression of chil- in nonage spleen and bursa of fabricius and promote the body early immune response capacity. embryo vaccination with chil- could significantly enhance the nonage proliferation responses of t lymphocytes from spleen and b lymphocytes from bursa. meanwhile, it could raise the nonage neutralization antibody level and inhance the protection against virulent ibdv induced by dna vaccine. the results indicated that the nonage immune responsing to ibdv dna vaccine was highly enhanced by embryonic coadministration with chil- (p x . ). due to the unique role of the hair follicle in percutaneous penetration, drug delivery systems, which target active compounds to the hair follicle, may result in a better penetration and a higher efficiency of hair and skin therapy ("follicular targeting"). applications in immunotherapy, e. g. transcutaneous vaccination, are of particular interest, because skin antigen-presenting cells (apcs) can be found at particularly high densities in hair follicle-bearing skin, where they are concentrated around the upper portion of the hair follicles. in in vitro studies on human skin explants, we demonstrated that nanoparticles, due to their ability to aggregate in the hair follicle openings and to penetrate along the follicular duct, are promising carrier systems for transfollicular drug delivery. transcutaneously applied nanoparticles in the size range of nm, were capable of penetrating the epithelium and entered into human epidermal lcs, suggesting that such particles may be used to transcutaneously deliver active vaccine compounds, via the hair follicle. the use of the skin as target organ for vaccine has been spurred by recent implication of epithelial dendritic cells (dc) in cd cell cross-priming and suggests that vaccination via the transcutaneous (tc) route may be relevant in the induction of cellular immune responses. advanced studies in vivo using functional vaccines are, however, essential to further assess the potential of particle-based vaccines in transcutaneous vaccination. for this purpose, we developed a standard operating procedure (sop) for transcutaneous vaccine delivery on human skin based on our current knowledge on follicular penetration. in a pilot study on volunteers and a phase i study on volunteers vaccinated with an influenza vaccine, we found that this newly developed sop is safe and efficient at inducing a significant increase in cellular immune responses mostly composed of antigen-specific cd cells. induction of t cell responses has become one of the major goals in therapeutic vaccination against viral diseases and cancer. this study proposes new perspectives for the development of vaccination strategies that triggers t cell immune responses in humans. objectives: all anti-hiv- neutralizing antibodies are directed toward the viral envelope glycoproteins (gp) and the transmembrane protein gp . two sites on gp and gp are attractive targets for vaccine design: the epitope in the third hypervariable region (v ) is recognized by the human monoclonal antibody - d and the epitopes in membrane proximal external region (mper) were recognized by the human monoclonal antibodies e and f . in order to elicit anti-hiv- neutralizing antibodies we have designed virus like particles (vlps) displaying either the gp -v region or the gp -mper. the vlps are based on the acyltransferase component (e chain) of the pyruvate dehydrogenase complex of geobacillus stearothermophilus. the e chain self-assembles into a nm protein scaffold resembling a vlp and that contains copies of e . efficient display and refolding of the v and mper regions in e vlps are obtained by using engineered plasmid which allows insertion of exogenous oligonucleotides at the ' of the gene coding for e . the priming and boosting with a combination of vlps and specific hiv- envelope dna were used to immunize mice and rabbits. results: the v -e and mper-e vlps were purified as stable mers from e. coli cells after refolding in vitro from inclusion bodies followed by gel filtration chromatography. binding of - d, e and f antibodies to hiv-e monomers was confirmed by western blot. we obtained high titers of hiv- gp -specific antibodies in mice immunized with a combination of vlps plus dna (hiv- sf gp ). these antibodies generated a low ( %- %) level of neutralization. moreover immunizations were also performed in rabbits, a better model for induction of neutralizing antibodies. three doses of e vlps plus dna elicited a low titer of hiv- gp specific antibodies. additional rounds of immunizations in rabbits will be performed, in combination with gp plasmid dna, to enahance the responses to envelope and to induce neutralizing activity against these key epitopes. our results demonstrate that e vlps are able to display antigenic determinants of hiv and to induce high titers of hiv- -specific antibodies. the e vlps represent a promising tool for a vaccine design. now a day we paid for vaccination of previous generations. as a result morbidity sharply increases, but we haven't well-tried scheme of immunity renewal yet. every clinic, every center do it in there own way, while vaccination is continued, even when it's not necessary, for example, grip, nobody know strain exactly. the most unpleasantly think is that most of physicians don't know what immunity mean specifically, general they think about vitamins, that isn't fit for forming immunity because of many reasons. we offer a way of immunity according to the world scientific theory and practice. the method is based on biochemical, electrophysiology, and biology way of correction physical status. at first we normalize and activate current settings that are going to the diseased organ, vascular system, gastrointestinal tract, spleen. all of it attends indemnity necessary microelements that were extracted from wild officinal herbals. we don't concentrate only on the one or two types of immunity, fist of all we take into account structure and dynamic of immunogeneration system. in our clinic we use this method; immunity is restored very quickly and kept during long time even if organism gets any complications, which can worsen the situation. that's why when we secure new physical statement in the cns program we forming new nearest and distant men health. we tell local state mechanism of disturbances from disturbances, that develop in blood, lymphatic system, tissues and hypothalamus, when pathological process exist long time. it's completely different disturbances of physician state, which should have different therapeutic approach. the threat of an influenza pandemic has become evident in recent years, emphasizing the requirement for influenza vaccines that are broadly cross-reactive against different subtypes with pandemic potential. we have previously shown that baxter's vero cell-derived h n whole virus candidate vaccines are highly immunogenic both in animal models and in human clinical studies, and cross-protective in mice and ferrets. more recently, it was reported that cross-reactive heterosubtype immune responses against highly pathogenic h n influenza virus could also be achieved by immunizing subjects with a trivalent seasonal influenza vaccine; however the induction of cross-subtype protection could not be addressed in this study with human subjects [ ] . the study reported here evaluated whether the seasonal influenza vaccine, when used either as a monotherapy or in combination with a h n whole virus wild-type vaccine, could induce an immune response and protect mice against h n influenza virus infection. a trivalent seasonal influenza vaccine was shown to elicit anti-h n antibody and t cell responses and partially protected mice against a lethal challenge with wild-type h n virus. the protective efficacy of the trivalent vaccine derived mainly from the h n component. moreover, passively transferred serum of mice immunised with seasonal influenza vaccine protected naïve mice from infection with h n virus, suggesting that antibodies are the main contributor to protection. h n specific serum did not inhibit neuraminidase activity of h n virus suggesting that protection was not mediated by neuraminidase n -specific antibodies. next, we investigated the combination of the trivalent seasonal influenza vaccine and the h n whole virus wild-type vaccine. a prime with the seasonal influenza vaccine followed by immunisation with the h n vaccine enhanced anti-h n antibody response, cellular immunity and protection compared to a single immunization with an equivalent sub-optimal dose of the h n vaccine. hence, hetero-subtype immunity can be achieved by immunization with a trivalent seasonal influenza vaccine, which can be further boosted with a h n candidate vaccine. [ ] gioia c et al. aims: to register the compliance of the population to the old and new vaccines of the national vaccination program for the children up to years old, and to investigate the possible causes of the potential shortages, in order to approach even more successfully the further goal of this whole attempt, which undoubtedly is the future control of important generalized infections. methods: in the study we checked the vaccination history of children in the first grade of primary school in the area of central and west macedonia. there were greek and foreign children. as fully vaccinated were considered those who had already undergone at least one dose of hib, meningococcus and pneumococcus, two doses of hav, as well as four doses of dtp-sabin, while in the cases of a lack of vaccination, the causes were investigated and the adequate recommendations and information were given. in all the cases, except for the nationality, the sex, and the educational and social level of the parents were registered. results: the percentages of the compliance found, are presented in the following ) it should be underlined that, as shown in the table, the percentages of the obligatory-free of charge vaccines were close to %. ) high percentages were noted also for meningococcus, either because it is an old vaccine (it has been available for seven years), or because the bacteria is considered quite dangerous (it has been emphasized through the media). ) on the contrary, as far as the hepatitis a and the pneumococcus vaccines are concerned, low percentages were found, either because of the lack of adequate information-fact that was also shown in our study-or even because of their cost. ) finally, a statistically significant difference was found relating the response to the vaccination coverage, between greeks and foreigners, but also between the greeks themselves, in relation to their educational and socioeconomic level. objective: over the past three decades, the incidence of type diabetes has dramatically increased in europe and north america, inversely correlated to the decrease of infections. according to the hygiene hypothesis, pathogens may prevent the onset of the disease. om- , a bacterial extract of both gram positive and gram negative bacteria already used as an immunomodulatory treatment in children, has been shown to protect non obese diabetic (nod) mice from diabetes development. we aimed here at understanding the mechanism underlying this protection. methods: nod mice and nod-cd -/mice, which are devoid of natural regulatory t cells (tregs), were treated with om- . cytokine secretion, activation and proliferation of b cells and foxp + tregs were monitored. as toll-like receptors (tlr) recognise microbial molecules and trigger innate and adaptive immunological response, cells from mice deficient for tlr , tlr or the myd adaptor protein were used to further address the mechanisms driving the immunomodulatory activity of om- . two synthetic tlr agonists used as adjuvant in human (om- -dp and om- -mp-ac) were also tested for their capacity to protect nod mice from diabetes. the om- -induced protection of diabetes required natural tregs, as nod-cd -/mice were not protected. remarkably, om- activated b cells and not t cells, promoting their proliferation and il- secretion, two phenomena that were tlr -and myd -dependent. om- -dp and om- -mp-ac two synthetic murine tlr agonists effectively prevented diabetes onset in nod mice, promoted the expansion of cd + cd + foxp + t cells and the proliferation of il- secreting b cells in a dose-dependent manner. conclusion: our results argue for the involvement of tlr signaling in the protective effect of om- on development of diabetes and show that two other tlr agonists induce proliferation of b cells and their secretion of il- as well as stimulation of regulatory cd + cd + foxp + t cells. activation of the innate immunity by tlr-stimulation using those products already used in clinics, may prevent the onset of diabetes in those at risk of developing the disease. d. de wit , a. legat , s. thomas , m. van mechelen , p. hermand , m. goldman institute for medical immunology/université libre de bruxelles, gosselies, belgium, glaxosmithkline biologicals, rixensart, belgium aminoalkyl glucosaminide -phosphates (agp) are lipid a mimetics which are considered as interesting candidates for the development of synthetic vaccine adjuvants targeting toll-like receptor (tlr ). since natural lipid a from bacterial lipopolysaccharide (lps) depends on membrane-bound or soluble cd (scd ) for its tlr ligand activity, we investigated the involvement of both forms of cd in the responses elicited by crx- , a prototypical agp. first, we found that crx- efficiently induces nf-kb and irf activation in hek cells transfected with tlr and md- genes, whereas the responses to lps required co-transfection of the gene encoding membrane-bound cd . likewise, crx- efficiently induces the synthesis of nf-kb and irf- dependent cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which a defect in membrane-bound cd prevents lps responses. we then observed that monocyte-derived dendritic cells (dc) which are devoid of membrane-bound cd respond to crx- but not to lps in serum-free medium. the addition of the soluble form of cd did not modify the levels of il and tnf produced by crx- stimulated dc but increased the levels of interferon-b (ifn-b). when scd was added to hek cells expressing tlr /md- , nf-kb activity was not modified but irf activity was increased in a dose-dependent manner in response to crx- . we will further compare the responses induced by crx- in wild-type and cd deficient mice. we previously showed that the transcriptional transactivator (tat) of human immunodeficiency virus possesses the unusual ability to raise a humoral immune response in the absence of adjuvant. these observations prompted us to examine whether such a property can be used to boost the immune response raised against poorly immunogenic peptides. as we previously observed that the autoadjuvant property is controlled by a determinant located within the core-and cysteine-rich regions of the protein, we decided to investigate whether the grafting or the co-injection of a peptide partially containing this determinant (ptat) can raise a humoral immune response against two model peptides. these two peptides, which originate from diphtheria toxin (pdt) and from toxin alpha (pt), both contain an i-ad restricted t-cell epitope but are nonetheless non-immunogenic in balb/c mice of the h- d haplotype when injected with alum. the ptat, pdt, pt, ptatpt and ptatpdt constructs were prepared by chemical synthesis, purified by reverse phase hplc and characterized by mass-spectrometry. pdt+ptat, pt+ptat, ptatpt and ptatpdt were respectively injected twice at two weeks interval in balb/c mice and animals were bled and days after the second immunisation. the sera were subsequently incubated in microtiter elisa plates previously coated with pt and pdt peptides respectively in order to assess the humoral immune response. we observed a lack of antibody response for the immunizations made with the mixture of peptides (pdt+ptat and pt+ptat) but an anti-pdt and anti-pt response for the immunizations made with the two hybrid constructs (ptatpt and ptatpdt). our results indicate that a humoral immune response can be raised towards non-immunogenic peptides using a determinant involved in the autoadjuvant property of tat, that the phenomenon requires the covalent coupling to the peptide antigen and that it is therefore not related to a bystander effect. interleukin- gene polymorph isms (c t, g a, c a and a t) and susceptibility to brucellosis in iranian patients russian federation some epidemiological and observational data suggest that farm and pets exposure [ ] in early childhood may be conducive to reduced atopy. currently, there is a lack of consensus regarding underlying immunological mechanisms, especially in prenatal period. as we previously reported the decreasing of intracellular ifn-g production by cbmc statistical analysis was performed using the kruskal-wallis and mann-whitney tests. results: we revealed that newborns from rural mothers (n= ) have higher amount of both nonactivated (subtype infg+/cd -, p= . ) and activated (subtype infg+/cd +, p= . ) cbmc, producing ifn-g, as compared with newborns from urban mothers (n= ) exposure to pets and the risk of allergic symptoms during the first years of life intracellular interferon-g production by cord blood mononuclear cells as predictor of atopic dermatitis forming in infants: a one-year prospective birth cohort study pc / to what extent t-spot.tb could be used in the diagnosis of tuberculosis in children exposed to tb infection? s. a tb) in children, especially in bcg-vaccinated is difficult for diagnosis because of the low percentage of smear positivity ( - %) and clinical futures only in severe forms of disease. the purpose of the present study was to evaluate the diagnostic value of t-spot.tb (oxford immunotec, oxford, uk) compared to tuberculin skin test (tst) in children exposed to tb contact in the family. forty three children with a history for bcg vaccination/revaccination, treated in the university clinic for lung diseases in children sofia, bulgaria were enrolled in the study. the patients were divided according to age in the following groups: months - years (n= ), - years (n= ) and - (n= ) tb has the highest diagnostic value in children n years of age in early childhood the diagnostic value of t-spot.tb and tst does not differ cfp- antigen is more sensitive for detection of tb-specific t cells compared to esat- antigen. . in children with tst - mm t-spot.tb has a high diagnostic value objectives: the goal of this study is to determine the role of tlr and tlr in the development of spontaneous lupus disease by creating tlr or tlr deficient c bl/ lpr/lpr mice. methods: tlr and tlr deficient lupus prone mice have been generated by crossing c /bl -tlr -/-or c /bl -tlr -/-mice with c /bl lpr/lpr mice which develop a moderate type of lupus related to fas deficiency. we analysed the phenotype of the disease, autoantibody production and renal injury. statistical comparisons were performed using the mann-whitney u-test. results: these mice developed a less severe disease and few immunological alterations. indeed, in tlr or tlr deficient lpr mice, glomerular igg deposits and mesangial cell proliferation were dramatically decreased and anti-nuclear, anti-dsdna and anti-cardiolipin autoantibody titers were significantly reduced. however, the response against nucleosome remained unaffected, indicating a role of tlr or tlr in the production of autoantibodies directed against certain slerelated autoantigens. analysis of b cell phenotype showed a significant reduction of mz b cells, particularly in tlr deficient mice suggesting an important role of tlr in the sustained activation of these cells likely involved in autoantibody production. interestingly, the lack of tlr also affected the production of cytokines involved in the development of lupus disease. conclusion: our data show that deficiency in tlr pc / expression of full length mcl- and its splice variant in juvenile systemic lupus erythematosus (jsle) neutrophils: differential modulation by gm-csf granulocytemacrophage colony-stimulating factor (gm-csf) can prolong neutrophil survival by increasing mcl- , an anti-apoptotic protein. a splice variant of mcl- arises by removal of exon and induces cell death rather than preventing it. here we investigate the expression of both the full length mcl- (mcl- l) and its splice variant (mcl- s) in jsle neutrophils compared to controls and investigate whether the addition of gm-csf changes the expression of both isoforms of mcl- . method: neutrophils were isolated from children (diagnosed x years) with jsle (n= ) and non-inflammatory conditions (control, n= ) and incubated with control serum, jsle serum alone or with jsle serum plus pg/ml gm-csf. quantitative real time pcr was used to assess mcl- l and mcl- s mrna expression (mean ± sem) following incubation in the above conditions and immediately following neutrophil isolation the ratio of mcl- s to mcl- l was also higher in jsle patients compared to controls (p x . ). the addition of gm-csf to jsle serum was associated with an increase in mcl- l ( . ± . ) and a decrease in mcl- s ( . ± . ) mrna expression the addition of gm-csf to jsle serum can abrogate the increased neutrophil apoptosis. alternative splicing is recognised to play a significant role in the regulation of proteins involved in cell death. our results suggest that jsle neutrophils may be more apoptotic due to differential expression of mcl- compared to controls, with jsle neutrophils having greater expression of the pro-apoptotic isoform mcl- s, and less anti-apoptotic full length mcl- cyld is a tumor suppressor gene known to play an important role in the nf-kb pathway. to analyze the function of cyld in vivo we used the cyld ex / mouse strain, which is characterized by loss of the full-length transcript and overexpression of a short splice variant of the cyld gene (scyld) to further investigate the connection between scyld overexpression in t cells and colonic inflammation, we used an adoptive transfer model of colitis. therefore naive cd + cyld ex / t cells were transferred into rag -/-mice which were analysed by mini-endoscopy weekly after cell transfer. here we could demonstrate that cyld ex / cd + t cells exhibit less capacity to induce colitis compared to control cells. consequently we investigated if regulatory t cells (t regs ) of cyld ex / mice are capable to control inflammatory responses. for this purpose cd + cd + cells were co-transferred with naïve wt cd + t cells into rag -/-recipients. interestingly, rag -/-recipients of cyld ex / t regs displayed strong features of colitis compared to control recipients showing that these cells were unable to inhibit inflammatory responses. our findings demonstrate that overexpression of scyld leads to a hyperresponsive t cell phenotype and higher production of inflammatory cytokines by t cells pc / the role of hla complex in inflammatory bowel disease: crohn's disease and ulcerative colitis de investigación biomédica en red de enfermedades hepáticas y digestivas (ciberehd). university hospital virgen arrixaca the allele frequencies of hla class i in cd and uc patients were not different to those observed in controls, although we found an increased frequency of a* in cd vs uc. haplotype frequencies of hla class i and ii in cd and uc were also not different to those observed in controls. however, we found increased frequencies of drb * , * and * alleles, and a decreased allele frequency of drb * in cd vs uc patients and controls. these data are in concordance with other previous studies suggesting that, in patients with isolated colonic cd, drb * is associated with the development of severe disease and positive association of cd with drb * and drb * . indeed, drb * was negatively associated with cd. this allele appears to confer protection against all subgroups of cd, in all ethnic groups including japanese. however, hla-drb * frequency allele, associated in unselected patients with cd in other studies, was not different in our cd and uc patients, and controls. additionally, an increased frequency in hla-drb * in cd was not found in our patients in a different manner to other reported studies. on the other hand, in our uc patients, allele frequencies of drb * were strongly increased with respect to cd and controls. however, the frequency of drb * was decreased in uc with respect to cd and controls. in this sense, our data are agreed with other reports showing that hla-drb * is associated with uc in european, north american, japanese and korean populations methods: a total of children were studied, ( boys and girls), up to years of age, with symptoms suspicious for epstein-barr virus infection. the elisa method was used to look for specific antibodies against the capsid of the virus vcaigg and against the nuclear antigen ebv-igm, while taking into consideration the possible increase of the vcaigg title between two serum samples. results: totally, positive cases of children were found ( %) with active infection : boys ( x years of age, - years of age and g years of age) and girls ( x years of age, - years of age and g years of age). pharyngitis was present in children ( , %), had fever ( , %) and had lymphadenitis ( %). the lab tests revealed leukocytosis up to . leukocytes in cases ( , %) and leukocytosis g . in cases ( , %). the most frequent complication documented was streptococcal superinfection in children ( , %) and thrombocytopenia in children ( , %). a past infection (negative ebv-igm values and positive vcaigg values) was virus infection is common among children and teenagers serum negative are mainly the children of little age and ) there is no statistically important difference between the two sexes, while on the contrary there is a seasonal distribution of the infection, with winter and summer outbreaks general hospital of rethymno, rethymno, greece shows that the il- ra , previously believed to be a decoy for il- only, is able to transmit a signal via il- . our results support this and may suggest that il- / il- ra signalling causes disease in oxazolone-induced colitis. currently we are dissecting the role of single cell populations expressing il- ra to establish which cells play a role in regulating the immune response to oxazolone-induced colitis. together this data can define a role for il- or il- ra and identify specific cell populations methods: splenic apcs exposed to enteroantigen (eag) +/-probiotics were used to stimulate cultured cd + cd -t cells to which titrated numbers of tregs were added. neutralizing antibodies against il- and il- b and elisa-based cytokine analyses were used to monitor the effect of cytokines secreted in the t cell cultures. results: exposure of apcs to eag and probiotics did not influence eag-specific cd + cd -t cell proliferation. however, exposure to three of the six probiotics tested (b. bifidum bi- , l. acidophilus ncfm tm and b. bifidum bi- ) consistently reduced regulatory activity of tregs in a cell-dose dependent manner. the tregreducing activity of probiotics was analyzed using fractionated components of the b. bifidum bi- strain. data indicated that bacterial cell-wall components were responsible for reducing treg activity and not components of nucleus or cytoplasm. the probiotic-induced down-regulation of treg activity was not mediated by increased intra-culture secretion of inflammatory cytokines such as il- or il- b. conclusion: we conclude that certain probiotic strains can modify apcs to cause reduced treg activity in an eag-specific t cell proliferation assay. this effect apparently depends on a direct apc-to-treg cell contact and not secreted cytokines. the apc/probiotics-mediated inhibitory effect on tregs may oppose antiinflammatory activities desired from probiotic therapy palmieri 'la sapienza dysregulated innate and adaptive immune responses against commensal flora lead to crohn disease (cd) and ulcerative colitis (uc), two different forms of inflammatory bowel disease (ibd), a lifelong inflammatory condition of the gastrointestinal tract methods: we analyzed pediatric cd patients ( active, remission), pediatric uc patients ( active, remission), and age-matched non-ibd controls. nkg d/ligand expression was evaluated by immunostaining and multiparametric facs analysis (on pbmc subsets), and by immunohistochemistry and twocolour immunofluorescence (on intestinal biopsies). differences between groups were analyzed with non-parametric and parametric tests; a level of p x . was considered significant. results: nkg d expression is selectively upregulated on circulating "innate-like" t cell populations (g/d and cd +cd + nkt cells), in active, but not in quiescent ibd patients; receptor upregulation correlates with disease type (observed in uc, but not in cd patients). in the same patient groups, the appearance of nkg d ligands on circulating monocytes is also observed. the dramatic increase of nkg d+ lymphocytes, and the strong upregulation of nkg d ligands on both epithelial and immune components, are observed in active ibd lesions. conclusions: our observations document the dysregulated expression pattern of nkg d/ligands on selected innate immunity populations in pediatric ibd patients, both at mucosal and systemic level pc / peripheral and intestinal regulatory t cell dynamics in pediatric ibd patients is a chronic inflammatory condition of the gastrointestinal tract characterized by dysregulated innate and adaptive responses against commensal flora. regulatory t cells (t reg) represent an important mechanism to suppress uncontrolled immune responses to bacterial flora. aims: to evaluate the frequency of regulatory t cells in the peripheral blood, and in inflamed and non inflamed mucosae of pediatric ibd and non mucosal regulatory t cells were identified by immunohistochemistry; circulating regulatory t cells were analysed by immunofluorescence and facs analysis. differences were analyzed with parametric and non-parametric testsconsidered significant. results: foxp + t reg were significantly increased in the intestinal lesions of active ibd patients (cd or uc), and returned to normal levels in post-therapy remission phase. at variance, circulating cd + t reg frequency was elevated in patients affected by both forms of ibd, independently of disease activity, as it persisted in the remission phase. a selective imbalance in the frequency of t and nk subsets characterized the abundant inflammatory infiltrate present in active intestinal lesions, and the normal immunological profile was only partially restored in mucosal samples of quiescent ibd patients. conclusions: regulatory t cells dynamics are differently regulated in mucosal tissues and at the systemic level, during the distinct phases of disease; t reg dynamics in pediatric ibd patients only partially matches previous data obtained in the adults; quiescent ibd is characterized by the imbalance of selected lymphocyte subsets, both in the mucosa and systemically the increased expression of immunoproteasomes in the inflamed mucosa of ibd patients was shown to contribute to this pathology by enhancing nf-kb activation. due to the relation between nf-kb and the immunoproteasome we have investigated whether specific inhibition of immunoproteasomes is suitable for therapeutic intervention in ibd. lmp knock-out mice are deficient in the essential catalytic immunoproteasome-subunit ß i and therefore are devoid of immunoproteasomes. to test our hypothesis, we employed the dss colitis model. in contrast to wild-type mice, colitis was attenuated in lmp knock-out mice characterized by reduced weight loss and less infiltration of lymphocytes in the mucosa confirmed by histology. in addition, lmp knock-out mice had lower levels of proinflammatory cytokines and chemokines compared to wild-type mice validated by rt-pcr and elisa. especially nf-kb regulated genes show enhanced induction in wild-type mice unlike lmp knock-out mice synaptic systems gmbh, braunschweig, germany objectives: although more than million people worldwide are chronically infected with hepatitis c virus (hcv) no prophylactic or therapeutic vaccines do exist to prevent or cure hcv infections. our major objective is to develop a dendritic cell (dc)-based immunotherapy enhancing virus-specific cellular immune response for treatment of hcv infections based on this approach we aim at generating adec- antibodies conjugated with immunodominant hcv proteins to induce hcv-specific protective immunity. methods: recombinant hcv proteins are expressed using "expression-ready-clones" containing n-terminally his-tagged hcv-core (aa - ) or hcv-ns (aa - ) sequences. protein purification is performed by metal-affinity chromatography on ni-nta-agarose hcv-specific t cell responses are monitored at different time points after immunization by facs and in vitro t cell proliferation assays. results: to obtain high amounts of recombinant ns and core we successfully optimized culture and protein purification conditions. briefly, ns was purified natively using pbs-based buffers with ph-gradient. in contrast, purification of core was performed under denaturing conditions in presence of guhcl and urea and a ph-gradient elution. moreover, optimized conditions allowing conjugation of adec- to recombinant hcv proteins were established with respect to duration of conjugation and buffer requirements needed to avoid protein precipitation. efficient conjugation was verified by western blot analysis. after successful generation of adec- / hcv-protein conjugates we are currently establishing optimized vaccination conditions to induce hcv-specific immune responses pd / mva-nef vaccination induces polyfunctional cd t-cells and increases the proliferative capacity of cd t-cells in hiv- infected individuals under haart several vaccination trials have made use of the modified vaccinia virus ankara (mva) as delivery vector. in a therapeutic vaccination trial, we demonstrated that mva expressing the hiv- protein nef (mva-nef) was safe in hiv- infected individuals under haart and immunogenic in regard to the elicitation of ifn-g mediated cd t-cell responses. recent advancements in polychromatic flow-cytometry technology revealed that the sole evaluation of ifn-g provides limited information on the quality of antigen-specific t-cell responses. the evaluation of several functions is essential, as simultaneous production of multiple cytokines by t-cells is associated with superior control of viral replication. methods: in a retrospective setting, we simultaneously assessed the production of ifn-g, il- and mip- b, the expression of the activation marker cd and the differentiation marker cd ra in nef-specific cd and cd t-cell populations during the course of the vaccination trial. furthermore we applied a multi-colour cfse based proliferation assay investigating the proliferative capacity and the simultaneous expression of ifn-g, il- and mip- b. results: following mva-nef vaccination, we observed a significant increase of the total nef-specific cd t-cell response and a significant increase of polyfunctional nef-specific cd t-cells, simultaneously expressing ifn-g, il- and cd . using the standard ics no increase of nef-specific cd t-cell responses was observed. however, by the cfse based proliferation assay, we could show a clear expansion and a generally enhanced proliferative capacity of nef-specific cd t-cells following mva-nef vaccination. notebly, we observed a correlation between the increase of ifn-g, il- and cd expressing cd t-cells and the increase of proliferating cd t-cells suggesting the possibility of a causal link between the two functions. conclusions: the mva-nef vaccine is able to change the quality and quantity of the nef-specific cd t-cell immune response and has the potential to increase the proliferative capacity of nef-specific cd t-cells in hiv- infected subjects under haart this preferential binding to the complex was evident in classical immunochemistry assays, as well as in surface plasmon resonance (spr) tests. this ab inhibited hiv- mediated membrane fusion and p -detected replication. db was found to nicely recapitulate the characteristics of the unconventional, protective immune response, which is taking place in naturally resistant esn individuals. further characterization of the antibody and of its binding epitope is ongoing following intradermal vaccination with mg dna and electroporation of balb/c mice, splenocytes have been incubated with peptides representing class i and ii epitopes, and specific t cell-responses were examined by elispot-assays. the specific antibody responses have been measured by sandwich eli-sas, and neutralizing antibodies have been investigated by hi-assays. results: the vaccibody constructs have been found to be expressed and correctly folded in vitro. the in vivo experiments further demonstrate the presence of neutralizing antibodies as well as the strong induction of antigen specific cd + and cd + t cells. conclusion: antibody and cellular immune responses against influenza hemagglutinin are enhanced when targeted to apcs. methods: the hcv recombinant proteins rns ( - aa) and rns a ( - aa) were conjugated with immunomax using the heterobifunctional reagent sulfo-smcc. balb/c and dba/ j mice were immunized intraperitoneally times at a month interval with different doses ( . - mg/mouse) of the proteins without adjuvants, as conjugates with immunomax, or with complete freund's adjuvant (cfa) the other combinations were not immunogenic at given doses. it should be noted that only conjugates stimulated production of antibodies that bound not only to recombinant protein but also to peptides imitating epitopes of ns protein. immunization with rns a-immunomax conjugate and rns in cfa ( . mg/mouse) induced a similar antibody activity, but a different t-cell responses. the conjugate induced splenic accumulation of t cells specifically reacting in vitro with ns a recombinant proteins of various genotypes, with peptides and with phages by cell proliferation and/or cytokine secretion. immunization with rns a in cfa induced cells proliferating in vitro after stimulation only with peptides; none of the antigens stimulated cytokine secretion. conclusion: covalent conjugates of hcv nonstructural proteins with immunomax effectively induce humoral and cell immune responses pd / degree of cross-genotype reactivity of hcv-specific cd t cells directed against ns the existence of multiple hcv genotypes characterized by marked sequence differences is a challenge for immune control. the aim of this study was to compare the antiviral cd t cell response targeting hcv genotype (gt ) and genotype (gt ) as the most predominant genotypes in germany and to determine the extent of cross-genotype reactivity of specific t cells. we analyzed a cohort of patients with past or ongoing intravenous drug use (ivdu) hypothesizing that multiple exposures to different genotypes may occur. methods: subjects ( with gt , with gt and anti-hcv-pos/hcv-rna-neg) were analyzed. hcv-specific t cells were expanded from pbmc in the presence of peptide pools covering ns from gt or gt . individual reactive peptides and the degree of cross-reactivity between the gt and gt variants were determined by ics. complete ns is sequenced from all viremic patients pd / anti-retroviral effects of type i interferon subtypes in vivo ifna subtypes , , or suppressed fvreplication in vitro, but differed greatly in their antiviral efficacy in vivo. treatment of fv-infected mice with the ifna subtypes , or , but not led to a significant reduction in viral loads. decreased splenic viral load after ifna treatment correlated with an expansion of activated fv-specific cd + t cells and nk cells in the spleen, whereas in ifna -and ifna -treated mice it exclusively correlated with the activation of nk cells. other ifna subtypes like ifna , and are under investigation pd / elimination of immunodominant epitopes from multispecific dna-based vaccines allows induction of cd t cells that have a striking anti-viral potential immunodominance limits the tcr diversity of specific, anti-viral cd t cell responses elicited by vaccination or infection. to prime multispecific t cell responses, we constructed dna vaccines that coexpress chimeric, multidomain antigens (with cd t cell-defined epitopes of the hepatitis b virus (hbv) surface (s), core (c) and polymerase (pol) proteins, and/or the ovalbumin (ova) antigen as stress protein-capturing fusion proteins. priming of mono-or multispecific, hla-a* -or k b -restricted cd t cell responses by these dna vaccines differed. k b /ova - -and k b /s - -specific cd t cell responses did although chronic infections remain asymptomatic in most cases, immunocompromised patients can suffer from severe and life-threatening ebv-associated diseases, such as posttransplant lymphoproliferative disorders (ptld). thus, immunotherapeutic strategies using adoptively transferred ebv-specific t cells are promising. one option is the generation and expansion of cd + and cd + t lymphocytes by using ebv-specific synthetic peptides for the stimulation of pre-existing memory t cells. aim of our study was to identify a set of mhc class-ii peptides for each antigen promiscuitive peptides with high syfpeithi scores were tested for immunogenicity using an ifn-g-elispot. pbmcs of at least healthy, randomly chosen blood donors were cultured for days in the presence of each candidate peptide. functional and phenotypic analysis of t cells of several donors was performed by multicolor flow cytometry. out of tested peptides could be identified as t-cell epitopes. two of them were defined as immunodominant, as more than % of tested blood donors showed peptide-specific t cell responses. so far, eight of the tested peptides could be identified as mhc class-ii epitopes. furthermore, a highly immunodominant class ii peptide mix consisting of peptides was selected. in conclusion, we could identify several new ebv-specific mhc class-ii epitopes which can be used for united kingdom, hospital de clínicas during persistent hbv infections, patients usually develop poor or no protective immune responses against viral antigens, which not only leads to the chronicity but also the unresponsiveness to conventional treatments.in order to overcome the unresponsiveness and to generate an effective therapeutic strategy for treatment for chronic hbv infections a chimeric tcr against hbsag, which aims to increase the percentage and quality of antigen-specific cd + t cells, was developed moreover, we pre-conditioned the liver microenvironment by injection of cpg oligodeoxynucleotides (odn) to optimize the recruitment of transferred cd + t cells to the liver and to overcome the tolerogenic microenvironment of the liver. we found that the il- -exposed cd + t cells showed at least five-fold increase of survival rate in vivo than il- -exposed cd + t cells did treatment of the recipients with cpg-odn could increase the percentage and also the total amount of transferred cd + t cells mainly in the liver. by in vivo brdu incorporation, we demonstrated that the higher in vivo survival rate of il- -exposed cd + t cells and the effect of cpg-odn were due to the up-regulation of the proliferation of those cells. to sum up, the cocktail therapeutic strategy could not only increase the survival rate of transferred cells but also direct the antigen-specific cd + t cells to the liver to exhibit their effector functions. the detailed mechanisms responsible for the il- and cpg-odn effects on the regulation hyper igm (him) and wiskott-aldrich syndrome (was) than those of corresponding controls (p x . ) . there was a significant elevation of t ada and ada activities in iga deficient patients as compared to healthy individuals (p x . ) . our results hypothesized that altered ada activity may be associated with altered immunity. therefore, serum ada level could be used as an indicator along with other parameters pd / hiv- sequence evolution after dendritic cell-based immune therapy in a phase i/ii clinical trial hiv rna was extracted from plasma samples collected before the startof haart and early after vaccination when haart was terminated. rna was amplified by rt-pcr and sequenced using standard protocols. sequences of the vaccine genes tat, rev and nef as well as control genes vif, vpr, vpu and parts of env were analyzed for variation between pre-and post vaccination time points. hiv sequences spanning known and predicted epitopes of the relevant hla alleles from each participant were analyzed in detail. results and conclusion: immune therapy was well-tolerated and no severe adverse effects occurred. after haart termination, plasma viral load became detectable in all patients after - weeks. follwing the viral rebound a set point was reached, that was lower than the viral load before start of haart. using various methods we evidenced newly induced or enhanced immunity after immune therapy (see abstract b. de keersmaecker et al.). for studying sequence evolution, complete sets of both pre-haart and post-vaccination sequences were obtained in out of patients. with one exception, variation in sequences of vaccine and control genes of pre-haart samples compared to samples taken early after vaccination was limited. this indicates that there was no significant impact of the immune response on virus evolution at this stage. more focussed analysis on viral sequences spanning specific hla islamic republic of newcastle disease (nd) is regarded throughout the world as one of the most important diseases of poultry, not only due to the serious and high flock mortality, but also through the economic impacts. the purpose of this study was to be informed from the possible influence of infectious bronchitis virus on immune response of chickens to nd live vaccine. one hundred and twenty, -day-old ross broiler chickens divided randomly into groups, experimental and a group as control one. the first experimental group vaccinated by a monovalent nd live vaccine with cl/ strain, and the second experimental group vaccinated by a bivalent newcastle disease and infectious bronchitis live vaccine with cl/ and h strains, via the drinking water at days of age at the same time, and the control group received no nd vaccine. the antibody response to vaccination was assessed using the hemagglutination inhibition (hi) test by taking blood samples three times, first the day before and the next, & days post vaccination. results indicated that, although the strain of studied nd live vaccines were the same united kingdom t cell-based ifng release assays from blood are an important advance for diagnosing tuberculosis infection but do not permit reliable treatment monitoring or distinction of active tb from successfully treated disease or latent infection. t-cell cytokine profiles vary with in vivo antigen load in viral infections cd t cells from patients with active tb and patients with successfully treated tb were analysed for simultaneous expression of ifng and il at the single cell level using multi-colour flow-cytometry after hours stimulation with ppd. moreover, cells were stimulated with esat- and cfp- receiver operator characteristics analysis revealed that a percentage of ifng /il dual positive cells x % served as an accurate marker for active tb patients (specificity %, sensitivity %), while frequencies g % were observed in treated as well as active tb patients. in conclusion, quantitation of antigen-specific t cells based on the analysis of ifng only does not allow distinction of patients with active and successfully treated disease pd / necessity of postpone bcg vaccination -lesson from primary immunodeficiencies v. thon methods and results: the czech national database of primary immunodeficiencies (pid) was established in and is connected with the european database of primary immunodeficiencies (esid). the prevalence of pid in the czech republic (approximately inhabitants) is . to . among these patients there are children diagnosed with severe combined immunodeficiency (scid) and chronic granulomatous disease (cgd) too. according to the czech national database of pid, out of children with later proved scid were immunised with bcg vaccine in the first days of life. nine of them developed disseminated and generalized bcg infections. five children with scid died. moreover, reactivation of bcg was also seen in healthy children after admission of combined vaccines with hepatitis b given at the age of twelve weeks. on the other hand, this was not the case in thousands of children of hbsag positive mothers who were vaccinated against hepatitis b after delivery in the first place and later immunized with bcg vaccine. systematic vigilance against tuberculosis (tb) and vaccination significantly lower the prevalence and risks of tb. in the czech republic, the prevalence of tb is currently . to inhabitants. unfortunately, temporary interruption of bcg vaccination in three large districts in the period of to led into higher incidence of tb and appearance of new cases of aviary mycobacteriosis. these complications were not observed in vaccinated children. conclusion: we recommend a change of current practice of bcg vaccination considering new immunization schedule with hexavalent vaccine pd / novel analogues of thalidomide inhibit cd expression and production of tnf-a, il- , ifn-g, cxcl- this work describes the synthesis and characterization novel thalidomide analogues, prepared in good yields using simple methodology. our results suggest that anti-inflammatory and immunomodulatory activity of these diamine compounds is potentially applicable in treating enl and other diseases. supported by: cnpq, fapemig and capes, brazil. of the b cell follicle. cta -dd augmented gc-formations, specific antibody responses and cell-mediated immunity to the t cell-dependent antigen np-cgg, but failed to do so when used together with t cell independent antigens, such as np-ficoll or np-dextran. this effect required adp-ribosyltransferase activity, as mutant cta r k-dd failed to exert an adjuvant effect. the adjuvant function appeared to correlate with the fdc-localization and turned out to require complement and/or complement receptors (cr) chitosan formulations varying in molecular weight, counterion and structure (i. e. soluble v/s particulate) were used in assays to examine expression of maturation markers via flow cytometry and cytokine production by elisa. we found that, in contrast to alum, plg and ps particles, chitosan induced bmdc maturation on its own, as determined by the expression of cd and cd . these effects were most prevalent with soluble chitosan chloride formulations but were also notable with soluble chitosan glutamate chitosan. the effect of chitosan on cytokine production was investigated using a panel of different tlr agonists in combination with chitosan particles. results show an increase in the secretion levels of il- a and il- b, while il- levels were not affected. finally we studied the role of inflammasome activation in the enhancement of il- b production. using bmdc from nlrp -/-mice we examined il- b production in response to different tlr and chitosan combinations. results show that the ability of chitosan to enhance il- b production is dependent on nlrp . collectively our data indicate that upregulation of maturation markers and enhancement in proinflammatory cytokine secretion mediated by chitosan severe sepsis, induced in mice by cecal ligation and puncture (clp), led to ho- expression in infiltrating peritoneal leukocytes, kidney and liver. mortality rate of clp increased from % in wild type (hmox +/+ ) mice to % in ho deficient (hmox -/-) mice. hmox -/-but not hmox +/+ mice developed end-stage multiorgan failure. mortality of hmox -/-mice was associated with increased peritoneal leukocyte infiltration, but not with increased pro-inflammatory cytokine secretion or bacterial load in peritoneum, blood or organs. clp induced a significant increase in cell-free hemoglobin free heme was found to sensitize primary hepatocytes to tnf, anti-fas antibody, h o or peroxynitrite mediated apoptosis. this cell death was associated with outward nuclear translocation and extra-cellular accumulation of the late-stage pro-inflammatory cytokine hmgb . similarly, circulating and cytoplasmic hmgb was increased in hmox -/-relative to hmox +/+ mice following clp. in conclusion, these data suggest that free hemoglobin and heme, released during severe sepsis, are important factors in the organ failure and death associated with severe and b- , -linked mannose residues elicit inhibition effect. it was found that inhibition activity of oligosaccharides increases with chain length. immunization with mannan-hsa conjugate allowed for the maturation of immune response generating specific antibodies with high avidity/affinity, whereas immunization with mannan alone elicited only low-affinity antibodies. in the future, an effective antifungal subcellular vaccine would be constructed using selected mannooligosaccharidic epitope and the appropriate carrier protein as inductor of immunological memory. acknowledgements: this work was supported by the grant agency of slovak academy of sciences all subjects received dtwp vaccine at - years of age (booster vaccination), following the national vaccination schedule of iran. blood samples were collected before and - weeks after the vaccination. immunogenicity of the vaccines was assessed by elisa using commercial kits. results: the geometric mean titers (gmt) of the antibodies induced against diphtheria and tetanus by dtwp-local were . and . iu/ml and those of dtwp-pasteur were . and . iu/ml, respectively. there was no significant difference between the immunogenicity of the two vaccines against diphtheria and tetanus. the gmts of antibodies produced against pertussis were . eu/ml for dtwp-local and . eu/ml for dtwp-pasteur vaccines, respectively (p x . ). no significant differences were observed in the antibody titers against diphtheria, tetanus and pertussis between the two vaccines before vaccination. conclusion: immunogenicity against diphtheria and tetanus was similar for the two vaccines pd / united kingdom haemorrhagic septicaemia (hs) is an acute disease of cattle and buffaloes in tropical countries, caused by pasteurella multocida serotype b: , a gram-negative coccobacillus. jrmt , an aroa mutant of pasteurella multocida, constructed previously in our laboratory, attenuated for virulence in the mouse and protects mice from challenge with the virulent strain. in this work, the immune response of calves was tested after intramuscular vaccination with single dose of cfu of jrmt . a possible contributory role of cellular immunity against hs was investigated in vaccinated and in control calves after challenge. a lymphocyte stimulation assay was used to assess the effects of a cell-free extract (cfe) of p. multocida on peripheral blood mononuclear cells (pbmcs) isolated from calves at different times after challenge. the results were indicative of a possible immunosuppressive effect of challenge with p. multocida b: on calf pbmcs. the suppressive effect was further investigated by in vitro experiments. calf pbmcs obtained from normal calves were treated with cfe for h before adding concanavalin-a (cona) pd -vaccination and immunotherapy against parasitic diseases pd / evaluation of simian adenoviral vector adch expressing msp- as a candidate blood-stage malaria vaccine this successful regime incorporated a human adenovirus serotype (adhu ) prime, boosted eight weeks later with a modified vaccinia virus ankara (mva) vector. adenoviral vectors have generated great scientific interest in recent years and appear to be superior viral vectors with great potential in vaccine regimes. their potential use in humans, however, is limited by natural anti-vector immunity to human adenoviruses, but this problem could be largely circumvented by the use of simian adenoviral vaccine vectors. recent clinical trials have suggested that the simian adenoviral vector adch is a promising clinical candidate. we have developed vectors (of human and simian origin) and mva encoding a novel construct based on p. falciparum msp- and have undertaken comparative immunogenicity studies in mice. the antigen, termed 'pfm while asymptomatic per se, the heterozygous sickle cell trait confers a survival advantage against malaria, the disease caused by plasmointo carbon monoxide (co), iron and biliverdin. when infected by plasmodium hb sad mice are protected against experimental cerebral malaria (ecm), a lethal neuroinflammatory syndrome that in many aspects recapitulates human cerebral malaria. ho- expression and activity are strictly required to suppress ecm in hb sad mice, as demonstrated by functional deletion of the hmox locus or pharmacologic inhibition of its enzymatic activity. the protective effect of ho- is mediated by co, which inhibits the accumulation of protein-free heme in plasma following plasmodium infection conclusion: topical treatment of cutaneous leishmaniasis with gsno accelerated healing and reduced local parasitism in the mouse suggesting that it may be ben gp expression was confirmed by sds-page and elisa using monoclonal antibody against gp . discussion: today researchers attempt to find a suitable vaccine for leishmaniasis. although some researchers have reported proper vaccines of interest, a - recp is recognized only by sera collected from resistant bovines infested with all stages of r. microplus, but not by sera from similarly infested, susceptible hosts. furthermore, this recognition was specific since sera from resistant non-infested bovines (naï ve animals) did not react with a - recp. our results show that reverse immunogenomics can be useful for discovery of new antigens for development of an anti-tick vaccine. supported by cnpq and fapesp. for the maintenance of ab-mediated vaccine-induced protection after re-challenge with the pathogen or the vaccine antigen. memory b-cell elispot together with ab titres might therefore prove useful as independent marker for duration of protection. objective: this study focused on establishing experimental conditions and optimizing the performance of the memory b-cell elispot assay by detection of specific memory b-cells against anti-tetanus vaccine and naturally acquired toxoplasma gondii infection as a model. methodology: twelve healthy subjects who had received the tetanus vaccine at least month previously were enrolled. peripheral blood mononuclear cells (pbmcs) were isolated from each donor using cell preparation tubes (cpt). plasma was obtained after centrifugation of cpt and stored at - °c until used for elisa. specific igg-secreting b-cells were determined by elispot assay, using tetanus toxoid (tt) and t. gondii surface antigen as model antigens. results: to optimize our assay, conditions were changed and compared to the previously established protocol. we detected low frequencies of total igg memory b-cells and tt-specific memory b-cells in all donors four seropositive and seronegative donors had positive responses in elispot. no correlations were found with serum antibody titers and frequencies of memory b cells (r= . , p= . ) or with t. gondii-specific b-cells conclusions: following optimization of several assay parameters, we demonstrated that the memory b cell elispot could be reliably used to determine low numbers of antigen-specific memory b-cells in individuals naturally exposed to infection or following vaccination our previous work demonstrated that il- also affects the cells of erythroid lineage, by stimulating development of early erythroid progenitors, bfu-e, but inhibiting the growth of late stage erythroid progenitors, cfu-e, from normal murine bone marrow. we also provided in vitro evidence that at least part of its effect on cfu-e is mediated by nitric oxide (no) generation. in the present study we demonstrated that the in vivo reducing effect of il- on bone marrow cfu-e was prevented by co-treatment with the no synthase (nos) inhibitor, l-name, implying that this effect is mediated through nos activation. the data obtained in cultured bone marrow cells showed the ability of il- to upregulate the expression of mrna for both the inducible (i)nos and the constitutive, endothelial (e)nos isoform. both the nos-inducing effect of il- and il- -related inhibition of cfu-e growth were dependent on p mapk activity, since the p mapk inhibitor, sb , markedly downregulated il- -induced activation of nos and reversed the growth inhibitory effects of il- on cfu-e. the in vivo stimulating effect of il- on bfu-e colony growth in the bone marrow was not affected by co-treatment with the nos inhibitor, pointing to different mechanisms for il- effects on bfu-e and cfu-e. however, the in vivo exposure of the mice to l-name, increased the number of various hematopoietic progenitor cells in the bone marrow, indicating that no itself is important regulator of hematopoietic progenitor cell activity. overall, the data presented gave an insight into the mechanisms by which il- acts on bone marrow cells and also revealed a link between the il- , no and hematopoiesis. further studies on il- -mediated induction of both inos and enos methods: a total of blood donor samples were tested for hbsag and anti-hbc with the immunoenzymic method elisa, while simultaneously, molecular blood test (nat) was applied. the positive samples for anti-hbc were also tested for anti-hbs and anti-hbc igm. results: a total of samples ( , %) were found anti-hbc positive conclusions: it is proven, therefore, that in some cases the levels of hbsag, following an infection from the hepatitis b virus, are probable to remain low, so that it is not possible to detect them using elisa method. in these cases anti-hbc can be the only serological marker of the infection. consequently, patients with positive anti-hbc and levels of anti-hbs x iu/l are possibly not immune enough, so that they can become blood donors. that was the reason why some blood donation centers in our country, until recent years when there was no capability for nat testing of blood donors, had iu/l as a limit for anti-hbs levels. however, in present days that nat testing of blood donors is used in our country, it has offered great safety and it is possible that anti-hbc testing will not be necessary, despite the fact that many blood donor centers have preserved the safety limit of iu/l anti-hbs in all the blood units, which also goes for our study pd / neonatal allo immune thrombocytopenia and igg glycosylation patterns michaelsen , national institute of public health in milder cases it can cause petechia and in more severe cases it can cause intracranial hemorrhage and death. the reason behind the variation in clinical symptoms is not fully understood, but is probably not due to differences in immunoglobulin isotypes or antibody affinity. recently influence of glycosylation patterns of igg on the biological activity has been realized. variation in carbohydrate structures attached to asparagine can cause differences in the interaction with fc-receptors, and hence a difference in thrombocyte elimination capacity of the igg molecule. patient sera from norway and the netherlands with different levels of antibody titres and severity of symptoms have been used to affinity isolate igg antibodies against the hpa- a alloantigen and analyze the glycopeptides using mass spectrometry. the glycosylation patterns have been analyzed for a possible link between severity of symptoms and variation in the glycosylation patterns. so far patients with serious symptoms seem to have increased galactosylation and sialylation and a high level of non core-fucosylated n-glycans on their anti-hpa- a iggs we monitored children ( boys and girls) in ages from . to . years with average age of . years. in of them all was diagnosed for the first time. subject had the second relapse of all. one patient received maintenance chemotherapy, all the rest ( subjects) induction chemotherapy. methods: leukocyte count and hemiluminescent analysis of whole blood were performed for all the patients during infectious complications twice: on neutropenia background and after the recovery of neutrophil number. hemiluminescent analysis for whole blood allows to estimate the functional activity of phagocytes, namely their bactericidal power and phagocytosis completeness. we valuated spontaneous and zymosan induced hemiluminescence. we used onsonised zymosan as the inductor of "respiratory paroxysm mice with a homozygous mutation in the rc h gene, that encodes the zinc finger and ring finger containing protein roquin, develop severe autoimmune disease. the observed lupus-like phenotype involves follicular helper t cells, which express higher levels of icos. these cells provide inappropriate t cell help to b cells, leading to the production of autoantibodies (vinuesa et al. , nature , - ). it has been shown that the half-life of icos mrna is shortened when roquin is over-expressed. such repression requires the 'utr of icos, in which a bp sequence, containing a possible mir- binding site, was sufficient (yu et al. , nature, , - ). mnab is the paralogue of roquin, and has been shown to bind to nucleic acids (siess et al. , j biol chem , - ). we demonstrate that in primary mouse t cells and embryonic fibroblasts roquin, but not mnab, inhibits translation of icos. we map critical domains in the roquin protein to icos repression using deletion-and point-mutants of roquin, as well as chimaeras that swap sequences from roquin to mnab and vice versa. addressing the mechanism of roquin mediated icos repression; we demonstrate binding of roquin to icos mrna in primary mouse t cells and in cotransfection experiments. our current work dissects the requirement of cellular rnai, the stress response pathway or p-body function by testing roquin repression of icos mrna in dicer-, tia- -and ago -deficient mef cells and in knockdown approaches. acknowledments: j m m-v is a recipient of a harvard real colegio complutense (rcch) grant. work in dr tsokos' lab is supported by grant phs nih r ai . we have recently reported that -hydroxyl- -methylindole- -acetonitrile ( -hma) isolated from brassica rappa inhibit nuclear factor-kappa b (nf-xb) activity in raw . macrophages. in this report, we investigated the effect of -hma on dextran sulfate sodium (dss)-induced colitis model in mice. methods: we induced colitis with dss in mice and evaluated disease activity index (dai), including body weight, stool consistency and gross bleeding, and tissue myeloperoxidase (mpo) accumulation. through h&e staining, histological change was observed. the expression of inducible nitric oxide synthase (inos), inhibitory kappa b-a (ixba) and nf-xb were detected by western blot and immunohistochemical staining. in-vitro system, the expressions of interleukin- (il- ), monocyte chemotactic protein- (mcp- ) in ht- human colon epithelial cells were measured by rt-pcr. results: in dss colitis model, the dai score and detection of mpo accumulation brevealed -hma significantly inhibited loss of body weight, suppression of diarrhea and bleeding, and infiltration of macrophages, leukocytes. moreover, h&e staining also indicated -hma suppressed the thickness of muscle layer, edema, mucosal damages by dss. these results were related to the regulation of nf-xb activation. -hma attenuated the dss-induced phosphorylation and translocation of nf-xb subunit p . in addition, this effect was accompanied with parallel blocking degradation of ixba. moreover, pretreatment of -hma significantly reduced the mrna levels of il- and mcp- stimulated by tumor necrosis factor-a (tnf-a) in the ht- cells. pretreatment of -hma also significantly blocked the ixba degradation and nf-xb p nuclear translocation stimulated by tnf-a in the ht- cells. these results were concurred with the effect on nf-xb activation in dssinduced colitis model. conclusions: these results for the first time demonstrated that alleviation of -hma mediated by regulation of nf-xb activation and suppression of chemokines in vitro and in vivo. therefore, -hma could be new potential therapeutic agent for inflammatory bowel disease.cd serves as receptor of hiv and is a self-antigen. we have previously characterized the anti-cd igg immune response in hiv- -exposed, seronegative (esn) subjects and we know that there is a peculiar specificity of these antibodies for epitopes induced by gp -binding and that there is an epitope specificity distinct from that seen in hiv-infected patients (second cd domain preferred). to generate antibodies able to inhibit the infection of hiv virus trying to learn from what happen in nature in esn we used a particular immunization procedure. we immunized mice with autologous cells expressing gp , reacted with the external domains of soluble human cd , in the absence of the target cells expressing the co-receptor ccr . the latter is the membrane molecule, which allows the complete reshuffling of the epitopic make-up of the cd -gp complex and trigger the membrane fusion between effector (gp expressing) cells and target (ccr expressing) cells. thus, in the absence of ccr we specifically enriched our immunogens with "frozen" conformational intermediates, that are presumably transiently exposed on the cell membrane during hiv- infections. a conventional protocol for the generation of monoclonal antibodies was used. db- (igg , x), one of the anti-cd antibodies obtained, recognized preferentially cd complexed to gp , as compared to cd alone, not competed for the gp binding site on cd and was specific for the second extracellular domain of cd . g. röder , l. geironson , a. darabi , m. harndahl , c. schafer-nielsen , k. skjödt , s. buus , k. paulsson copenhagen university, institute of international health, immunology and microbiology, department of experimental immunology, copenhagen, denmark, lund university, immunology bmc d , lund, sweden, lund university, rausing laboratory, division of neurosurgery, department of clinical sciences, lund, sweden, schafer-n, copenhagen, denmark, department of immunology & microbiology, university of southern denmark, odense, denmarkcytotoxic t-lymphocytes become activated by binding to mhc-i molecules presenting antigenic peptides. the loading of peptides onto mhc-i takes place in the er and involves different chaperones and enzymes. tapasin binds mhc-i molecules, integrates them into peptide-loading complexes, and assures that only 'optimal peptides' are bound to surface exported mhc-i molecules. how tapasin exerts this quality control, and the criteria for being an optimal peptide, are still unknown. here, we have generated the first n-terminal amino acids of human tapasin, tpn , and shown that this fragment of tapasin facilitates peptide dependent folding of hla-a* . to further investigate the properties of tpn and tapasin, we generated multiple mouse monoclonal antibodies towards tpn and wildtype human tapasin. one clone, atpn - / , was found to be specific for natural human tapasin and stained cellular er localized tapasin. using peptide chip technology, the epitope of atpn / was demonstrated to be located on tapasin [ ] [ ] [ ] [ ] [ ] , which recently was shown to be a surface-exposed loop of the tapasin structure. together, these results demonstrate that, the first n-terminal amino acids of tapasin are able to facilitate peptide-binding to mhc-i, and as well, this fragment can be recombinantly expressed in e.coli and fold into a structure, which at least partially, resembles that of wild-type human tapasin. we speculate that this region of tapasin might support empty, open and receptive mhc-i peptide-binding clefts effectively allowing an otherwise inherently unstable molecule to exchange peptide; i. e. this tapasin region might be essential for enabling peptide editing. a objectives: antigen processing and presentation through hla class i molecules is critical for an effective destruction of infected or transformed cells by cd + t lymphocytes. different intracellular routes governing the processing of endogenous and exogenous antigens have been described. we show here a strategy to introduce epitopes inside the cells for a productive cross-presentation to ctls. methods: to produce genetic in-frame tat fusion proteins, dna sequence encoding for the amino acid region - of the influenza a virus nucleoprotein (np) was inserted into the expression vector ptat-ha. starting from tatnpflu recombinant protein we produce hybrid proteins, in which the hla-b* -restricted np-flu epitope (aa - ) was replaced by hla-b or hla-a -restricted epitopes of ebv and hcv, respectively. cross-presentation was evaluated according to the standard cr release assay and through the ifn-g production. results: using hla-b or hla-a restricted viral epitopes we show that the two molecules cross-present the epitopes following two different pathways of processing: the hla-b molecules follow a proteasome-independent pathway which is active in different cell types, whereas the hla-a molecules present the epitopes in a classical proteasome-dependent pathway performed by dcs. furthermore, different hla-a restricted epitopes can be inserted in tandem and presented to the specific ctls without interfering each other. the data reported here offer new insights on how a same construct containing multiple epitopes from different viral or oncogenic proteins could be designed for vaccinal strategies. these findings also enlighten hla-b as a remarkable hla-class i molecule that, differently from hla-a , can present peptides through additional, unconventional antigen presenting routes. this could concur to an imbalance of the immunological properties of the hla-b molecules leading to a more effective response towards viral as well as self -antigens. objectives: although cytotoxic t cells (ctl) in human immunodeficiency virus (hiv- )-infected individuals can potentially target multiple virus epitopes, the same few are repeatedly recognized. ctl play a key role in limiting viral replication in infections caused by e. g. epstein-barr virus, cytomegalovirus, hepatitis c virus and hiv . consistent patterns of immunodominant and subdominant ctl-responses have been found between individuals with the same hla-alleles in both acute and chronic infection. as the ctl-response frequency in a population closely correlates with its relative magnitude in an infected individual, the terms immunodominance/subdominance have been used in both contexts. however, the factors determining these ctl-response hierarchies are largely unknown. while structural differences between peptide-hla class i complexes may be important for tcr-repertoire selection and clonal expansion, it is less obvious how they impact ctl-response hierarchy formation and timing. other factors may also contribute, e. g. epitope abundance at the cell surface. methods: antigen processing efficiency of ctl epitopes from the p -gag and p region was determined in vitro. mer peptides were digested with i s and c proteasomes and the fragments identified by mass spectrometry. for epitope precursor peptides generated by the proteasome, we then determined tap affinity, trimming by eraap and hla-binding affinities and analyzed patient responses by elispot. results: we show that ctl-immunodominance in regions of hiv- p -and p -gag correlates with epitope abundance, which is influenced strongly by proteasomal digestion profiles, transporter-associated-with-antigen (tap) affinity and endoplasmatic reticulum aminopeptidase (eraap)-mediated trimming, and moderately by hla affinity. proteasomal cleavage-preferences were affected by flanking and intra-epitope ctl-escape mutations and could modulate the number and length of peptide-epitopes, thereby affecting t cell response avidity and clonality. conclusion: our analyses reveal that antigen processing plays a pivotal role in determining ctl-response hierarchies, that viral evolution may modify cleavage patterns, and suggest strategies for in vitro optimization of ctl-epitope-based vaccines. t. f. gregers , g. koster , o. landsverk , f. skjeldal , o. bakke university of oslo, molecular biosciences, oslo, norway mhc ii is synthesized and assembles in the er together with invariant chain (ii). ii facilitates mhc ii assembly followed by transport to the mhc ii loading compartment (miic) where peptide loading occurs. miic is multivesicular late endosomal compartments resembling conventional multivesicular bodies (mvbs) found in all cells. it is not known whether the biogenesis of miics is regulated by the same mechanisms as formation of mvbs. expression of ii induces the formation of enlarged endosomes and we have previously shown that ii modulates antigen processing and presentation. we have suggested that ii itself can act as a tethering factor involved in fusion of ii containing endosomes, and our main question is whether ii can regulate the formation of an endosomal pathway dedicated for antigen processing and mhc ii loading.in order to investigate this we use cell lines expressing ii controlled by an inducible promoter, thus being able to control the ii expression level and thereby the endosomal size. live imaging and high through put microscopy of ii expressing cells treated with inhibitors and/or specific sirnas have revealed that ii induced endosomal fusion is independent on type iii pi kinases and thus ptdins( )p. this is in contrast to conventional endosomal fusion and mvb formation. thus other factors might be important for miic biogenesis. by using small rnai libraries targeting proteins known to be involved in endosomal pathways and microscope based screening we aim to identify factors that are able to knock out the formation of enlarged endosomes in ii expressing cells, and thus potentially identify molecules defining an antigen presenting cell. m. bouvier , l. visvabharathy , j. fu university of illinois at chicago, microbiology and immunology, chicago, united statesobjectives: adenoviruses (ads) cause persistent infections. the e - k protein from ad targets class i mhc molecules for retention in the endoplasmic reticulum (er), thereby preventing the cell-surface presentation of viral peptides. this escape from immune surveillance allows ads to freely replicate in host cells. the molecular mechanism of e - k-mediated class i retention is mostly undefined. it is clear that further characterization of this mechanism is important to understand the susceptibility of the class i antigen presentation pathway to immunomodulatory proteins and to elucidate the molecular basis of ad pathogenicity. we used biophysical and cell-based approaches to examine interaction between ad type e - k and class i molecules.results: we showed that e - k associates with immature (peptide-deficient) and mature (peptide-filled) hla-a molecules, with the mature form being more tightly associated. we also provided evidence that e - k does not compete with the class i assembly proteins for binding onto class i molecules. importantly, immature class i molecules sequestered by e - k can still bind peptides. together, these results suggest that ads have evolved to interfere with the early and late stages of the class i antigen presentation pathway. evidence was also provided that e - k displays an allele-and locus-specificity towards class i molecules with high-density lipoprotein (hdl) reduces the risk for atherosclerotic cardiovascular disease by promotion of cholesterol efflux from macrophage foam cells and by antioxidative as well as anti-inflammatory properties. recent data indicate that qualitative changes of hdl including oxidative modifications and alterations of the protein cargo of hdl may alter its biological activity. here we analyzed the anti-inflammatory potential of hdl and compared it with hdl obtained from patients with end-stage renal disease (esrd), which are characterized by a proinflammatory state and an associated significantly increased cardiovascular mortality. we demonstrate that freshly isolated, but not oxidized hdl from healthy individuals exerts profound anti-inflammatory properties on professional antigenpresenting cells (apc) such as monocytes and dendritic cells, which are regarded as the most potent apc. production of typical proinflammatory cytokines (il- , il- , tnf-a) were significantly suppressed by hdl after stimulation of monocytes or dendritic cells with toll-receptor ligands and , but also with the t-celldependent stimulus cd l (cd ) indicating an immunomodulatory effect independent of agonist neutralization by hdl. moreover, surface expression of crucial activation and costimulatory molecules like cd , cd , and cd was inhibited by freshly isolated, but not oxidized hdl. the negative regulatory effect of hdl on cytokines and surface receptors occurred at the transcription level, while hdl did not modulate the activity of the major inflammatory transcription factor nf-kb or the map kinases p and erk- / . strikingly, hdl from esrd patients not only failed to block, but rather promoted proinflammatory cytokine production and apc activation. these data identify hdl as a novel potent anti-inflammatory regulator of professional apc, which may help to dampen excessive inflammatory responses of the innate immune system. conversely, qualitative changes of hdl leading to a loss of its anti-inflammatory function might contribute to a proinflammatory state that is linked with excessive cardiovascular mortality in esrd patients. objectives: cd + t cell abnormalities may play a role in the autoimmune pathogenesis of churg strauss syndrome (css). on one side, th (il- +) cells may sustain autoantibody formation and eosinophilia, which are hallmarks of css. on the other, th (ifn-g+) cells could participate in vessel wall damage and granuloma formation. in order to define this th / th balance and to identify potential t cell target antigens (ags), we analyzed circulating cd + t cell responses to polyclonal stimuli and to myeloperoxidase (mpo) in css and healthy subjects. methods: ifn-g and il- expression in peripheral blood cd +cd + lymphocytes were measured in ccs patients and healthy subjects (hs) upon polyclonal stimulation, both by intracellular staining and by elisa. mpo-driven il- /ifn-g production was assessed by elispot on t cells co-coltured with autologous dendritic cells, stimulated either with heat-inactivated mpo, negative control protein or hexavalent vaccine (positive control recall ags). results: upon polyclonal stimulation, higher il- and lower ifn-g intracellular expression were detected in cd + t cells from css patients, as compared to hs (il- : . ± . % vs. . ± . %, p x . ; ifn-g: . ± . % vs. . ± . %, p x . ). similar results were obtained by elisa (il- : . ± . vs. . ± . pg/ml, p x . ; ifn-g: . ± . vs. . ± . pg/ml, p x . ). elispot counts of hexavalent vaccine-stimulated cd + cells were positive for il- in / ( %) css patients and in / ( %) hs, and for ifn-g in / ( %) css patients and / ( %) hs. mpo stimulation determined significant ifn-g release in / ( %) css patients, but not in hs ( / ) no il- response to mpo in both groups was observed. conclusion: polyclonally or recall ag-stimulated cd + cells from css patients show a th -polarized cytokine profile. mpo is here first identified as a css-related ag targeted by cd + t cells, and responses towards it are instead th -polarized. these data unfold one molecular target and possible pathogenic mechanisms of cd + t cells in css. a. voigt , e. opitz , k. savvatis , k. klingel , k. stangl , u. kuckelkorn , p.-m. kloetzel charité -universitätsmedizin berlin campus mitte, berlin, germany, charite -campus benjamin franklin, berlin, germany, universität tübingen, tübingen, germanymurine models of coxsackievirus b (cvb )-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection. immunoproteasomes (ip) are crucial in the modulation of adaptive immune responses, in the maintenance of protein homeostasis and in the preservation of cell viability under stress conditions. our previous work has established that ip expression in the infected myocardium is linked to a strong enhancement of viral epitope generation.here, we investigated the impact of ip function in enterovirus myocarditis. mice, which are deficient in immunosubunit lmp of the stress-induced ip, were infected with x e pfu cvb nancy strain. in concurrence to wt littermates, we observed a pronounced up-regulation of cardiac ip subunit lmp as early as day p. i. in lmp -deficient mice. however, lmp -deficiency was linked to less severe myocarditis at day p. i. (he stain of cardiac tissue sections: wt . ± . vs. lmp -deficiency . ± . (grade of myocarditis; scale - ; p x . ). whereas the cardiac output (co) was reduced in wt littermates in enterovirusmyocarditis (p x . ), there was no difference in lmp -deficient mice in comparison to sham-treated mice. maximal left ventricular pressure and dpdt max were impaired in acute myocarditis in wt littermates. in contrast, systolic function was not affected by cvb infection in lmp -deficient mice. likewise, diastolic function was preserved in lmp -deficient mice upon enterovirus infection. our findings of less severe myocarditis in lmp -deficient mice were associated with tremendously reduced viral load in the myocardium of this strain.in conclusion, this study suggests an impact of lmp -immunosubunit function in regulatory processes of viral replication. absence of lmp confers host protection in enterovirus myocarditis. h. w. liao , j. xu , j.q. huang sun yat-sen university, guangzhou, chinathe characteristic of the dengue hemorrhagic fever/dengue shock syndrome (dhf/dss) is hematologic abnormality, which results from multiple factors including thrombocytopenia, coagulopathy and vasculopathy. the pathogenesis of endothelial dysfunction associted with vascular leakage syndrome however remains unknown. in this work, we showed that dengue virus serotype (den- ) strain induced apoptosis in human umbilical vein endothelial cells (huvecs). additionally, fas expression was increased on infected huvecs. trailr and tnfr - were constantly very low whereas trailr - decreased after den- infection. fasl was expressed at similar levels on huvecs throughout den- infection. the apoptotic rates in huvecs were decreased upon addition of caspase family inhibitors and activated caspase , caspase were also observed by western blot after by den- infection. there were no significant changes of no in our study. we thus proposed that the fas/fasl pathway might be involved in apoptosis induced by dengue virus in vascular endothelial cells in vitro. dermatolymphangioadenitis is a common complication of interruption of afferent lymphatics by cancer surgery combined with partial lymphadenectomy. it seems that skin microbes normally penetrating epidermis during hand work or walking are retained in the skin and subcutis because of lack of lymph drainage and evoke host reaction. aim. to study lymph node cellular reaction to bacterial antigens before and after ligation of afferent lymphatics. materials & methods. group i. s. epidermidis was injected daily for days into wis rat paw web tissue in saline containing . x cells. group ii. s.epidermis was injected as in group after ligation of lymphatics below the popliteal lymph node. nodes were isolated on day .they were weighed, the cell number was counted and cells were stained with mabs for immunohistochemical analysis. immunohistochemical pictures were analyzed by microimage program. results. group i. skin contained some mhcii cells. the popliteal lymph nodes became enlarged on the bacteria injected side. there was an increase in lymph node weight and cell concentration per g of tissue, compared to controls by factors , and , respectively (p x . ). immunohistochemical pictures showed increase in percentage of ox (migrating dendritic cell), mhc ii, his (granulocytes), ox (stem cells) and cd (icam i) subsets in the subcapsular , follicle, paracortex and medullary areas. group ii. after ligation of afferent lymphatics the weight of nodes was not significantly increased. skin showed presence of multiple mhc ii, ed (macrophages) and ox cells. popliteal lymph nodes contained evidently less of ox , his and mhcii cells than in group i (p x . ). summary & conclusions. afferent lymphatics transport microbial cells and/or microbes phagocytized by dendritic cells and macrophages to the regional node. local skin reaction is limited, whereas lymph nodes reveal acute reaction with mobilization of granulocytes from blood perfusing nodes. interruption of lymphatics saves nodes but skin reaction is strong and long-lasting. these observations seem to explain why damage to lymphatics during mastectomy or groin dissection is followed by recurrent attacks of skin inflammation. omega- fatty acids, and in particular docosapentaenoic acid (dha) and eicosapentaenoic acid (epa) from fish origin, have recently emerged as nutrients capable of modulating the expression of genes involved in inflammation and atherosclerosis and thus reduce the risk for cardiovascular events. our presentation focuses on the role of omega- fatty acids in the prevention and treatment of cardiovascular disease. it is based on reviewing and processing data obtained by search of scientific and medical databases. search terms used were: atheroma, atherosclerosis, cardiovascular disease (cad) ,coronary disease, antiinflammatory drugs, omega- fatty acids, epa, dha. we also searched epidemiological research web sites and screened the results of numerous controlled clinical trials which monitored the effects of omega - fatty acids consumption. the results indicate that omega- fatty acids supplementation is associated with a significant cardioprotection effect on both healthy individuals and patients with an established cardiovascular disease. omega- fatty acids appear to work by decreasing endothelial responsiveness to pro-inflammatory and pro-atherogenic stimuli, affecting molecular events not targeted by other drugs thus allowing their use as complementary treatments for the already implemented pharmacological treatments in inflammatory diseases. combined therapy with omega- fatty acids and statins shows a synergistic effect. methods: on ultracentrifugation of serum at density . and , g, top % layer contained lipoproteins only and - % layer contained lipoproteins as well as immunoglobulins. the bottom layer was shown to contain immune complexes (ic) by binding to coated anti apo(a) and detection with peroxidase labelled anti human immunoglobulins.both these forms of lp(a) were western blotted and probed with jacalin-hrp, anti-gal-hrp and anti apo(a)-hrp. anti-gal was prepared by affinity chromatography on guar galactomannan and complexed with lp(a) in vitro. ic formation by lp(a) was measured in terms of reduction in response in a new elisa for lp(a) involving addition of lp(a) sample to plate-coated jacalin, followed by anti-apo(a)-hrp detection. ic formation was also shown by migration of lp(a) from free lipid layer to ic layer below in ultracentrifugation. results: anti-gal and lp(a) could be liberated from precipitated ic using specific sugar. immune complexed lp(a) in serum was found to be more o-glycosylated, larger in size and binding more anti-gal than lp(a) in free form in western blots. while ic formations within homologous free anti-gal-free lp(a) pairs were few, those within heterologous pairs were more rampant. conclusions: lp(a) is a risk factor in vascular disorders including atherosclerosis, aneurysm, stroke and peripheral vascular diseases and is a component of atherosclerotic plaques, though mechanism of its uptake remains unclear. anti-gal comprising % of serum igg is rich in igg capable of complement fixation and macrophage mobilisation. present results offer a viable mechanism of lp(a)-mediated immune injury to vessel walls leading to vascular damage. even though no receptors have been detected for lp(a), unlike for ldl, the present results may explain the internalization of lp(a) in the form of lp(a) immune complexes by macrophages since the latter can phagocytose ic. extended specificity of the a-galactoside-specific anti-gal for t-antigen in lp(a) is akin to that observed in jacalin, pea nut agglutinin and galectin- . objective: the aim of this study was to determine if the combination of two genetic alterations, one affecting cell cycle regulation, such as the e f mutation, and other affecting b cell apoptosis control, such as bcl- over-expression, can induce the development of ais. methods: mice: mice with both genetic abnormalities were generated in a non-susceptible c bl/ (b ) genetic background. e f -/-bcl- tg were obtained backcrossing e f -/-and e f +/+hbcl- tg mice. e f -/-bcl- tg, e f -/-, e f +/+hbcl- tg and control mice (e f +/+) were followed up to mo-old. serologic studies: serum samples obtained at , and month of age were test for igg and iga ana and anti-dsdna by elisa. histopathologic studies: kidney paraffin sections of , and mo-old mice were stained with hematoxylin-eosin (h&e) and masson's trichrome to identify histological changes. immunecomplex deposits were studied by direct immunofluorescence on kidney using fluoresceinated goat anti-mouse igg, igm and iga. to evaluate b cell homeostasis, absolute number of b cell in blood, primary and secondary lymph organs were assessed by flow cytometry. in vitro proliferation was measured with [h ]-thymidine and brdu was used to assess in vivo proliferation capacity of immature b cells. results: overexpression of hbcl- tg in b lymphocytes of e f -/-mice induced the production of high titres of igg and iga ana and anti-dsdna, together with the development of a glomerulonephritis characterized by a moderated mesangial proliferation, mesangial immunecomplex deposits, mainly of the iga isotype, and the presence of tubular casts and lymphoid infiltrates with the presence of glomerular deposits. e f -/-bcl- tg mice showed an altered b cell homeostasis as demonstrated in proliferation and apoptosis studies. e f -/-mice showed neither autoantibodies nor nephropathy. this study demonstrates that the isolated deficiency of e f or the overexpression of a bcl- tg in the b genetic background do not induce an ais. when combined both genetic alterations, involving deregulation of cellular proliferation and survival affect lymphocyte homeostasis, induce a mild ais with overproduction of iga autoantibodies. an alteration in the b cell compartment, but not in the t cell compartment, seems to be underlying the syndrome described in the present work. in different mouse models of the autoimmune disease systemic lupus erythematosus (sle) loss of toll-like receptor (tlr ) abolishes the generation of antinucleosome igg a and igg b autoantibodies but exacerbates lupus disease. however, the tlr -dependent tolerance mechanism is unknown. here we show that loss of tlr in b cells of lupus prone mice prevents the generation of protective t cell-dependent self-reactive igm and thereby enhances the development of th and th t cells. transfer of a synthesized monoclonal polyreactive igm to tlr deficient lupus prone mice inhibits t cell activation and abolishes development of lupus disease. thus, these results document a protective tlr -dependent tolerance mechanism in b cells that induces the generation of self-reactive igm to prevent autoimmunity. cloning and production of polyreactive or antigen-specific igm might therefore be a powerful tool to treat autoimmunity. objectives: to investigate cytokine and autoantibody levels in serum from patients with primary sjögren's syndrome (pss), and to determine possible associations with focal mononuclear cell infiltrates, lymphoid organization, and age at the time of biopsy. methods: minor salivary gland tissue was obtained from a group of patients fulfilling the revised eu-us criteria for pss (n= ) (vitali et al. ) . ninety-seven of ( %) patients had focal mononuclear cell infiltrates corresponding to focus score (fs) g (fs+), while biopsies from / ( %) patients lacked characteristic focal mononuclear cell infiltrates (fs-). germinal center (gc)-like lesions were determined in / ( %) minor salivary gland biopsies. serum samples were used for cytokine and autoantibody evaluations. the mean level of unstimulated whole saliva was significantly lower in the fs+ patients compared with the fs-patients, and in the gc+ patients compared with the gc-patients (p x . ). interleukin (il) , il- ra, il- beta, il- p , il- , macrophage inflammatory protein (mip) alpha, mip- beta, eotaxin, interferon (ifn) alpha, and il- levels were significantly increased in the gc+ patients (n= ) compared with the gc-patients (n= ). in addition, minor differences in cytokine levels were found when comparing age groups. degenerative changes such as atrophy/fibrosis and fatty cell infiltration observed in the minor salivary glands of patients with pss may represent "burned out" inflammation. no significant differences were found in autoantibody levels in either of the groups, nor when comparing cytokine levels in the fs-and fs+ subgroups. the reduced salivary flow observed in gc+ patients may be influenced by the elevated levels of il- found in these patients (gao et al. ) . increased titers of th -associated cytokines, il- , il- beta and the il- subunit il- p , may indicate a higher activity of these cells in gc+ patients (nguyen et al. ) . differences in cytokine levels may be utilized when sub-grouping the ss patients into disease phases and may consequently have implications for treatment. objectives: c-reactive protein (crp) is an acute phase protein, produced by hepatocytes in response to the pro-inflammatory cytokine il- . the rapid increase of crp during inflammation makes it an excellent inflammatory marker, but for unknown reasons, blood levels of crp typically remain low in disease flares of systemic lupus erythematosus (sle), a systemic autoimmune disease. another feature of sle is the so called 'interferon (ifn) signature' which implies high levels of ifn-alpha and/or up-regulation of ifn-alpha related genes. ifn-alpha has a wide spectrum of immunomodulatory functions but is mainly known for its antiviral and anti-tumour effects. since high levels of ifn-alpha coexist with a muted crp response in sle disease flares and in viral infections, we hypothesized that ifnalpha inhibits crp synthesis. methods: crp promoter activity was studied in a crp-promoter and luciferase reporter transfected human hepatoma cell-line, hepg . production of the acute phase protein serum amyloid a (saa) and the negative acute phase protein transferrin were analysed by elisa as reference. results: the crp-promoter activity was inhibited by all ifn-alpha subtypes. mixes of type i ifns that were induced by sle-like immune complexes or virus also inhibited the crp-promoter activity. virus-induced purified leukocyte ifn-alpha had the most prominent inhibitory effect ( g %) on crp promoter activity. saa synthesis was inhibited by ifn-alpha in a similar fashion as for crp promoter activity, whereas transferrin was unaffected. conclusion: our data indicates that ifn-alpha is an inhibitor of crp-promoter activity. we suggest that this could explain the muted crp response seen in sle disease exacerbations. further, i may contribute to differences in crp response between viral and bacterial infections. background: b cell activating factor of the tnf family (baff) is an essential b cell survival and maturation cytokine. mice overexpressing baff (baff tg mice) develop lupuslike autoimmunity, b cell hyperplasia, and lymphomas. autoimmunity in these mice involves proinflammatory autoantibodies driving nephritis and sialadenitis, and was previously found to be t cell-independent (ti) and myd -dependent. this suggested the involvement of transmembrane activator and caml interactor (taci), which is a receptor for baff that is essential for ti immune responses and is upregulated by myd -dependent tlr activation. we assessed the role of taci in baff-driven ti autoimmunity. methods: we tested the importance of taci in ti autoimmunity by generating baff tg bone marrow (bm) chimeras reconstituted with taci -/or taci +/+ bm then comparing their disease severity by flow cytometry, autoantibody elisa, immunofluorescence microscopy for ig deposition. results: as expected, baff tg chimeras reconstituted with taci +/+ bm produced high levels of circulating proinflammatory autoantibody isotypes and rheumatoid factors (rhf), and ig deposition in the kidneys and salivary glands was observed. by contrast, baff tg chimeras reconstituted with taci -/-bm had greatly ameliorated levels of circulating proinflammatory autoantibodies, rhf, and ig deposition. b cell hyperplasia was greater in taci -/- baff tg chimeras. defects in the regulation of apoptosis contribute to the pathogenesis of human systemic lupus erythematodes (sle). autoantigens not being properly removed and thus exposed to the immune systeme might lead to the emergence of autoantibodies. physiologically apoptotic cells are removed without initiation of an inflammatory immune response and myeloid dendritic cells are believed to actively tolerize t-cells after phagocytosis of apoptotic material. these processes of silent apoptotic cell clearance seem to be disturbed in sle patients. a characteristic of apoptotic cell death is the shedding of membrane coated vesicles from the cellular surface (apoptotic cell blebbing). these microparticles have been recognized as mediators of intercellular communication. therefore, we were interested whether apoptotic cell derived microparticles can influence the function of monocyte-derived dendritic cells and whether those interactions might play a role in the pathogenesis of human sle. we observed an engulfment of microparticles by monocyte-derived dendritic cells. further, apoptotic cell-derived microparticles stimulated differentiation of immature dendritic towards a mature phenotype. however, microparticles caused a remarkable downregulation of mhc class ii molecules. further, we observed only a minor release of proinflammatory cytokines from monocyte-derived dendritic cells pulsed by membrane microparticles when compared to lps stimulated dendritic cells. finally, these dendritic cells pulsed by membrane microparticles did not cause a significant t-cell expansion. interestingly, dendritic cells obtained from sle patients showed significant variations in phenotype and cytokine secretion compared to normal healthy donor cells with absence of the mhc class ii downregulation and a higher constitutive secretion of il- . objectives: increased levels of il- , an innate and inflammatory cytokine of the il- family, can be detected in serum and organs of human autoimmune pathologies, as well as in autoimmune animal models. here, expression of il- and other genes of the il- /il- r families was examined in human systemic lupus erythematosus (sle) and in mrl lpr/lpr mice, which develop a chronic progressive lupus-like syndrome. methods: serum, urine, and monocytes were collected from patients and healthy controls. lymphoid (lymph-nodes, spleen, thymus, peyer's patches) and non-lymphoid organs (kidney, lung, liver, salivary and lacrimal glands) were collected from mrl +/+ and lpr/lpr mice of different ages. il- and il- bp were measured by elisa. gene expression was assessed by real-time pcr and expressed relative to b-actin. results: in sle, serum and urine levels of total and free il- are higher than in controls. serum il- correlates with disease activity and decreases upon remission. monocyte expression of the receptor il- rb is increased and correlates with disease severity, while expression of tir /sigirr (a down-regulatory receptor of the il- r/il- r family) is reduced. in mrl lpr/lpr mice, expression of il- , caspase- and il- rb genes precedes disease onset in lymph-nodes. in other organs, changes in il- -related genes (il- and tigirr- up-regulation, tir /sigirr down-regulation) occur after disease onset. free il- levels are abnormally high in lpr/lpr lymph-nodes before disease onset, while in other organs the increase occurs with disease. conclusions: free il- levels correlate with autoimmune lupus both in mice and humans. free il- may be pathogenic in murine lymphadenopathy, while is a disease correlate in lpr/lpr and a severity correlate in sle. both in human and mouse syndromes, upregulation of il- rb is a marker of pathology, suggesting increased il- -dependent activation. both in mouse organs and human monocytes, tir /sigirr expression decreases with disease, suggesting impaired control of il- r activation. thus, il- may be involved in autoimmune lupus pathology, and il- -related molecules can be both original diagnostic markers and novel therapeutic targets in autoimmunity. in this study we compared the epitope specificity of anti-topo i autoantibodies present in sera of dcssc, lcssc and sle patients. we have constructed an antigen fragment library displayed on bacteriophage lambda and screened this library with igg purified from patients' sera. regions of topo i selected from the library were expressed as recombinant fusion proteins and were tested with elisa and western blot. we unexpectedly found that antibodies against a fragment of topo i (fragment f (amino acid (aa) - ) could be detected in sera of healthy individuals and patients with inflammatory rheumatic diseases other than ssc and sle. using sera of dcssc, lcssc and sle patients we showed that the pattern of recognized epitopes is different between these patient groups. fragment f was recognized by all patients. fragment f (aa - ) was recognized by of dcssc patients. fragment f (aa - ) was recognized by of sle patients. analysis of clinical data revealed a significant difference between the f negative and f positive groups of ssc patients in age and in the duration of the disease. according to our results the newly identified fragments f and f could represent characteristic epitopes for dcssc and sle, respectively. background: previous studies demonstrated that depletion of regulatory t cells (tregs) results in autoimmunity in mice while their adoptive transfer prevents autoimmune diseases. studies performed by us and others showed that in human connective-tissue diseases a reduced number of tregs exists and this abnormality seems to be correlated with autoantibodies production and disease activity. objectives: based on these observations and the fact that rapamycin (rapa) has the ability to expand tregs and to induce anergy, we proposed to study the possibility to restore peripheral tolerance of cd + t cells isolated from systemic lupus erythemaosus (sle) patients by ex vivo expansion of tregs. methods: pbmcs or peripheral cd + t cells from sle patients were cultured in the presence of specific stimulation with or without rapa and ril- . by facs the initial percent of tregs and after expansion protocol were determined. in order to verify the suppressive capacity of expanded tregs, cd + t cells enriched in tregs were co-cultured with activated cd + effector t cells (teff) stained with cfse, after one week teff cells proliferation was measured by facs. additionally, cytokine and igg release in cell culture media were analyzed by multiplex and elisa, respectively. expanded cd + t cells anergy was also evaluated based on cbl-b, grail and foxp mrna by realtime rt-pcr. results: in vitro expansion of tregs was more efficient when the starting cells were cd + t cells. the presence of rapa during expansion protocol significantly increased the number of tregs. sle tregs cells expanded in vitro in the presence of rapa had the capacity to suppress proliferation of both sle and hd teff cells. rapa inhibits igg secretion in the pbmcs culture, inhibition dependent on tregs level. rapa during tregs expansion protocol stimulated some type of cytokines while suppressed others. rapa had the capacity to re-establish sle cd + t cells anergy by induction of anergy genes, grail and cbl-b. conclusions: our data show that the above described protocol permits ex vivo tregs expansion and that suppressive capacity of the expanded tregs depends on the source of both tregs and teff cells. in this study, we look for a more specific approach to remove b- cells through targeting p d by shrnas strategy. methods: we used the drugs, ly and wortmannin, pan-specific inhibitors against pi ks. then we designed shrnas carried by the lentiviral system and validated that several segments of them can sufficiently knock down the expression of p d. we then introduced either pan-specific inhibitors against all pi ks or p d-targeting shrnas into an sle-prone animal model, nzb/w f mice, for therapeutic purposes. the results suggested that pi ks are not only important for the development of b- cells but also remain essential to maintain their population after birth. shrnas carried on lentiviral systems were designed to knock down the expression of p d. either pan-specific inhibitors against pi ks or p d-targeting shrnas were introduced into the sle-prone animal model, nzb/w f mice. one inhibitor, ly , and shrnas delivered by low dose of lentivirus exhibited certain potential to retard the rising of anti-dna auto-antibodies and prolonged the life span. conclusions: our findings are promising for developing treatments for sle. moreover, knowing pi ks are critical for the maintenance of b- cell populations might shed light on future treating other diseases associated with b- cells, such as certain melanoma, lymphoma, or leukemia. a. m. zaghlool , m. alarcón-riquelme , s. kozyrev institution of genetic and pathology, uppsala university, uppsala, swedenrecently, we discovered that the bank gene, which plays a role in b cells activation pathway, is associated with systemic lupus erythematosus through a nonsynonymous substitution g/a (rs , r h). we identified that bank gene expresses two alternatively spliced isoforms, a full-length, and a shorter isoform that lacks exon (delta ). the two isoforms were detected differently in susceptible lupus patients depending on the presence of a risk haplotype. to address the question of how bank is spliced and what are the signals governing the expression of each isoform, minigenes with different genetic variants were constructed and the expression of the bank isoforms were tested in vitro. qpcr analysis revealed that, another t/c snp (rs ), which is in complete ld with r h snp and located in the putative branch point, has a strong affect on the isoforms expression levels. deletion of a polypyrimidine (py) stretch downstream of the skipped exon produced a dramatic decrease in the full-length expression levels, probably due to the loss of the binding site for protein tia , which bind to t objectives: cerebral ischemia is the most common presentation of antiphospholipid syndrome (aps), but several other neuropsychiatric features, including chronic headache, dementia, cognitive dysfunction, psychosis, depression, transverse myelitis, multiple sclerosis-like disease, chorea, and seizures have been associated with the presence of antiphospholipid antibodies (apl). we report the case of a subject with atypical movement disorder related to aps successfully treated with oral anticoagulation agents. case report: a -year-old woman with a previous history of recurrent foetal losses was admitted to our hospital due to cognitive dysfunction and headache. she presented involuntary movements that were characterized as mioclonic seizures and tonic spasms lasting from few minutes to several hours, followed by bilateral arrhythmic rapid purposeless jerks of the legs. mild executive dysfunction was observed. her deep tendon reflexes were symmetric and normal. pathological reflexes were absent. biochemical analysis, renal, hepatic and thyroid functions were preserved, prothrombin time and partial thromboplastin time were all normal. the immunoglobulin g (igg) isotope of anticardiolipin antibody (acl) was elevated, whereas igm isotype and anti- gpi antibodies were undetectable. the lupus anticoagulant (la) was negative such as antinuclear antibodies (ana). no evidence of epilepsy was revealed from electroencephalogram or signs of denervation from electromyographic studies. brain magnetic resonance imaging (mri) showed multifocal encephalomalacia probably linked to previous cerebrovascular accidents. she was diagnosed as having an atypical neurologic manifestation probably linked to aps. she was thus discharged with a low-molecular-weight heparin therapy subsequently changed to mild oral anticoagulation . the therapy leads to a late, gradual improvement of symptoms that persisted at the last year follow-up evaluation. conclusions: antiphospholipid syndrome may constitute a rare but treatable cause of atypical neurologic manifestation such as myoclonic movements. due to the possibility of an effective treatment, it is important to rule out this diagnosis, moreover in women with other associated features of aps (foetal losses, livedo reticularis, thrombosis). a.-s. korganow cnrs , strasbourg, france b lymphocytes from patients with systemic lupus erythematosus are hyperactive and produce autoantibodies. several b cell phenotypic characteristics have been reported, as the expansion of activated populations, and of a newly investigated memory compartment. a few genes have been suggested to be implicated. one of the thing that makes these results difficult to interpret is the heterogeneity of the lupic disease, and sometimes the analysis all together of quiescent, paucisymptomatic and highly symptomatic patients, treated with immunosuppressors or untreated.we made the postulat that "intrinsic" abnormalities of b cells could be a common point in very quiescent patients. we choosed patients, with minor clinical and/or biological manifestations of their disease, for at least monthes. known of them received immunosuppressive drugs since this period. the mean sledai score was below . b cell surface markers expression was determined by flow cytometry. we analysed most of the already described and phenotypically distinctive b cell populations. we confirm the presence of activated b cells even in quiescent patients. we do not confirm the significant increase of a specific memory b cell compartment. above all, we described a decreased expression of the cd surface protein for all patients. this cd lower expression is associated with cd lower levels. it is not associated with an evident gene expression alteration and in vitro stimulation restores a control phenotype. these findings suggest some mechanisms in lupus genesis. objectives: tgf-beta is a pleiotropic cytokine with wide ranging effects in proliferation, differentiation, immune suppression and apoptosis. recent work from our group has shown that tgf-beta signalling in t-cells is protective in a mouse model of colitis associated cancer. smad ubiquitin regulating factors (smurf) are ubiquitin ligases that are involved in the regulation of tgf-beta signalling. the aim of this study was to determine the function of smurf expression in t-cells on the pathogenesis of experimental colitis associated colon cancer. methods: we could isolate a known splice variant of smurf lacking an exon in the c -domain. to analyse whether this form has a regulatory role in colon associated cancer we generated a transgenic mouse strain that overexpresses smurf in t-cells. smurf expression were analysed by qpcr. wild type (wt) and transgenic (tg) mice were treated once with the mutagenic agent azoxymethan (aom) followed by three cycles dextran sodiumsulfate (dss). after each cycle, the inflammation of the gut and the tumor growth and size of every mouse were monitored by colonoscopy. results: smurf expression was upregulated by tgf-beta stimulation in t-cells and smurf was markedly upregulated in tumor infiltrating cd + lymphocytes in aom/dss treated mice. whereas wt mice suffered from severe colitis resulting in colon tumors beginning at day , smurf transgenic mice had less colitis and were significantly protected from tumor development. interestingly, t-lymphocytes overexpressing smurf showed an upregulation of the tgfbrii and an activation of smad and as compared to wild-type t-lymphocytes, which were previously described as typical smurf targets for degradation. in addition the transfection of smurf and a caga-luc plasmid into cos-cells for smad -promotor studies yielded the same effect as shown by upregulation of the smad activity. conclusion: although, wt-smurf has been described as a negative regulator of the tgf-beta signalling pathway, our data show surprisingly that a smurf splice variant upregulates the tgf-beta receptor expression and increases tgf-beta signalling effects. due to immunosuppressive effects on t-cells smurf has beneficial effects on mucosal inflammation and tumor development. smurf thus emerges as an attractive target for modulation of chronic intestinal inflammation and colitis associated carcinogenesis. the transcription factor stat has important functions in cytokine signalling in a variety of tissues. however, the role of stat in the intestinal epithelium is not well understood. we demonstrate that development of colonic inflammation is associated with the induction of stat activity in intestinal epithelial cells (iec) both in humans and in mice. studies in genetically engineered mice showed that epithelial stat activation in dss colitis is dependent on il- rather than il- . il- was secreted by colonic cd c+ cells in response to toll-like receptor stimulation. conditional knockout mice with an iec specific deletion of stat activity were highly susceptible to experimental colitis, indicating that epithelial stat regulates gut homeostasis. stat iec-ko mice, upon induction of colitis, showed a striking defect of epithelial restitution. gene chip analysis indicated that stat regulates the cellular stress response, apoptosis and pathways associated with wound healing in iec. consistently, il- and epithelial stat was found to be important in wound-healing experiments both in vivo and in cell culture experiments in vitro. in summary, our data suggest that intestinal epithelial stat activation regulates immune homeostasis in the gut by promoting il- -dependent mucosal wound healing. stat seems dispensable for gut homeostasis under steady state conditions, but is activated upon challenge to drive tissue regeneration and protection in situations of increased demand, as during colitis and injury. map and ma infection induced an increase in both cd and tlr expression at day and day after infection. mycobacterial infection did not result in differential tlr expression as compared to uninfected cells. cd is involved in stimulating th pro-inflammatory responses, although map may interfere with cd signalling ( ) . tlr signalling elicits anti-inflammatory responses, which can contribute to bacterial replication ( ) .in conclusion, monocyte-derived macrophages from crohn's disease patients show an increase in cd and tlr receptor expression in response to both map and ma infection. as ma is a known human pathogen of immunocompromised hosts, this findings further support a role for map in the immunopathology of crohn's disease. objectives: for our understanding of the pathogenesis of human ibd, animal models of intestinal inflammation are indispensable. most of them are based on a compromised intestinal barrier, and a deregulated immune response against components of the flora is considered to be critically involved in the development of ibd. the occurrence of extraintestinal manifestations suggests that cross-reactions against hitherto undefined auto-antigens could be responsible for the activation of the adaptive immune system. to further dissect the pathophysiological mechanisms responsible for initiation and progression of ibd and associated extraintestinal manifestations, we established a new antigen-specific model, in which the local activation of cd t cells by exogenous antigen leads to colitis. methods: eight million naïve cd + ot-i cells, transgenic for a t-cell receptor specific for an ova-derived peptide (siinfekl) in the context of h -kb, were transferred i. v. into b mice. at day and , mice were treated intra-rectally (i. r.) with % ethanol. thirty minutes later, ovalbumin (ova) or bovine serum albumine (bsa) were applicated i. r. proliferation of cfse-labelled cells was measured at day after the injection of ot-i cells. the phenotype of effector cells was evaluated at day by measuring ifng production and by in vivo cytotoxicity assay. based on histology and immunhistochemistry for cd , the severity of colitis was scored. results: local application of the exogenous antigen ova but not of bsa led to antigen-specific activation and proliferation of adoptively transferred naïve ot-i cd + t cells. these cells differentiated into fully activated effector t cells with the capacity to secrete ifng upon re-stimulation ex vivo and possessed in vivo cytotoxicity to siinfekl-loaded target spleen cells. furthermore ova treated mice displayed an inflammatory infiltrate in the colonic lamina propria with strongly elevated numbers of cd + t cells. our study demonstrates that the local activation of antigen-specific cd t cells by exogenous antigen in the colon leads to fully activated effector t cells with the capability to promote local intestinal inflammation in non-immune-compromised b mice. aims: to determine the immune system response of the greek population against helicobacter pylori (hp), given the fact that hp infection is a frequent causal factor of gastroduodenal ulcer and gastritis, and to study the distribution by age and sex, as well as the possible correlation with anemia markers (hematocrit, hemoglobin, iron, ferritin etc). the results of express qualitative detection method for igg and iga antibodies were studied of patients, ( male and female), with age average , years of age. patients who received antibiotics and excretory medicine in the last year were excluded. anemia laboratory tests were performed (hematocrit, hemoglobin, iron, ferritin), which were followed by statistical processing, using spss, x and t-test programmes. results: in patients ( , %,with age average , years of age) no antibodies were detected. on the contrary, in the remaining , male and female, ( , %, with age average , years of age), antibodies were detected. out of them, in cases the results were strong positive ( male and female) and weak positive in cases ( male and female). the statistical analysis that followed, showed no statistically important correlation with any of the anemia markers who were determined (hematocrit, hemoglobin, iron, ferritin, mcv and rdw). conclusions: it is proven, therefore, that: ) helicobacter pylori infection is relatively common in the general population ( , %). ) there is a statistically important correlation, as far as age (increased in elderly patients) and gender is concerned (clearly greater in women). ) there seems to be no correlation with anemia. it is evident, that the method is very useful, especially in elderly patients with dyspeptic complaints, (who frequently cannot undergo invasive procedures), and should not be neglected, given the fact that there is a great risk of helicobacter pylori infection in our country. abstract withdrawn by author m. durilova , t. ulmannova , k. stechova , k. tesarova-flajsmanova , v. stavikova , j. nevoral charles university, pediatrics, prague, czech republicobjective: was to analyze composition of cytokines in breast milk of mothers whose infants were diagnosed with allergic colitis and compare it to cytokine composition in breast milk of healthy controls. methods: breast milk of mothers whose infants were diagnosed with allergic colitis and mothers of healthy infants and no history of allergic disease was analyzed for presence of cytokines. breast milk samples were collected at the time of diagnosis of allergic colitis ( - weeks, average . weeks of infant's age) or at the age of weeks in control group. concentrations of the following cytokines were analyzed using elisa method: il- , il- , il- , il- , il- , ifn-gamma, tgf-beta , egf and eotaxin. man-whitney u test was used for statistical analysis, p x . was considered statistically significant.results: il- as the only cytokine was not detected in any of the tested samples in both groups. significant difference was seen in concentration of ifn-gamma, which was higher (p x . ) in breast milk of mothers whose infants were suffering from allergic colitis (range - . pg/ml, mean . pg/ml) than in control group (range - . pg/ml, mean . pg/ml). higher concentrations of il- and lower concentration of tgf-beta were observed in breast milk received by infants with allergic colitis but the difference was not statistically significant. conclusion: immunologic factors including cytokines present in breast milk passively and actively influence the developing immune system of the newborn. although their role is not exactly known, they are important in regulation of immunologic reactions and might be responsible for protective effects of breast milk from many diseases. inter-individual differences in cytokine composition of breast milk were previously found in many studies and their presence is influenced by various factors. the results of our study indicate that there might be a risk cytokine pattern in breast milk of mothers whose infants are suffering from allergic colitis. supported by national project no. - . background: ulcerative colitis is associated with excessive neutrophil infiltration into the lamina propria and intestinal crypts leading to the formation of crypt abscesses. the chemokine il- (murine homologs kc and mip- ) and its receptor cxcr are involved in neutrophil recruitment, thus blocking this engagement offers a new therapeutic strategy for inflammatory bowel disease. this study aimed to develop and characterize a pre-clinical in vivo model to test potential therapeutics targeting neutrophil migration. methods: peritoneal exudate neutrophils from transgenic b-actin-luciferase mice were isolated h post intraperitoneal injection of thioglycollate and phenotypically (facs analysis) and functionally characterized in an in vitro chemotaxis assay. four million exudate cells were injected intravenously into recipients with dextran sulphate sodium (dss) colitis followed by bioluminescence imaging of whole body and ex vivo organs at , , and h post-transfer. anti-kc antibody or its isotype control was administered at mg/mouse one hour before transfer followed by whole body and organ imaging hours post-transfer. results: facs analysis revealed % neutrophil purity, % of which were cxcr + . in vitro, the cells migrated towards kc and this was inhibited by anti-kc. in the bioluminescent imaging model, trafficked neutrophils were evident in whole body and ex vivo organ images of dss recipients at all time points. neutrophil recruitment to the colon was detected only in dss recipients and was inhibited by anti-kc, h post cell transfer. this study describes a novel in vivo model of neutrophil trafficking that can be used for pre-clinical studies to evaluate potential inhibitors of neutrophil recruitment. the human gut contains more than bacteria (known as the commensal microbiota) that are essential for normal function of our digestive and intestinal immunologic systems. the barrier function of the mucosal epithelium is reinforced by innate defense mechanisms and by immune exclusion mediated by secretory (s)iga and sigm. sigs are generated via epithelial polymeric ig receptor (pigr)-mediated transfer of iga and igm from the lamina propria to the intestinal lumen. to assess the role of sigs in colitis development, we constructed pigr knockout (ko) mice and tested them in the dextran sodium sulfate (dss) colitis model ( . % dss in drinking water for week, followed by pure drinking water for week). pigr ko mice suffered increased morbidity and mortality compared with wild type mice, but colitis was cured by depletion of intestinal commensals suggesting that one role of sigs is to prevent pathology induced by commensal microbiota. in contrast, % dss was lethal to all commensal-depleted mice, but these mice became anemic rather than suffering from bloody diarrhea. as previously documented by medzhitov and co-workers (rakoff-nahoum et al, cell ), treatment of commensal-depleted mice with the tlr ligand lps in drinking water protected against the lethality of % dss. thus, the commensal microbiota serve two distinct roles in the dss colitis model. at dss concentration of . % they may become pathogenic and drive an intestinal inflammation. at % dss commensals protect against the toxic effect of the chemical via their tlr ligands. in mice lacking sigs, due to deleted pigr, the severity of colitis induced by . % dss was greatly enhanced suggesting that one role of sigs is to prevent commensal microbiota from becoming pathogenic. ulcerative colitis (uc) is a human inflammatory bowel disease associated with chronic inflammation of the gastrointestinal tract. although uc is associated with a type immune response, current treatment strategies use broad anti-inflammatory drugs which are aspecific for the disease. in a mouse model resembling uc, oxazolone induces il- production which is an important pathological factor. neutralizing il- or il- prevents or ameliorates disease significantly. as many aspects of the mechanisms involving these th cytokines in colitis remain undefined, we used mice deficient in il- /il- or the key receptor through which they signal, il- ra, to further dissect their role in oxazolone-induced colitis. disease was exacerbated in il- ra -/mice with increased weight loss, mortality, inflammation and immunopathological symptoms. this was in contrast to il- /il- double deficient mice which were protected from colitis. removing il- production from il- ra -/mice, by using il- ra/il- double deficient mice, reversed the susceptible phenotype to protection. together these data strongly suggest that il- mediates susceptibility in an il- ra independent manor. recent evidence pc / introduction: the activation of cd + t-cells in the lamina propria play an major role in the pathogenesis of inflammatory bowel disease (ibd). whereas cd is associated with increased production of th -like cytokines, the cytokines profile in chronic uc is characterized by the increased production of several th cytokines, such as il- ,- and il- . however, the functional role of t cell transcription factors such as nuclear factor of activated t cells (nfat) in ibd is poorly understood. the aim of this study was to further analyze the role of this signal transduction pathway and its pathogenic significance in uc. cryosesctions of uc and cd patients were analysed by immunohistochemically methods. a significantly higher expression of nfatc was found in uc and cd colonic tissue compared to control specimen. transmitted to the th -mediated oxazolone-induced colitis model, nfatc -production is significantly increased in both diseases, too. nfatc deficient mice were analyzed in colitis model and are significantly protected against the development of intestinal inflammation compared to control mice, documented by loss of weight, histological score and miniendoscopy. interestingly, cyrosections of inflamed colonic tissue displayed a higher apoptotic rate in nfatc deficient mice compared to control mice, which can be observed by tunel assays, caspase and annexin v staining, as well as in lamina propria t cells. contrary, anti-apoptotic proteins, like bcl- and bcl-xl were downregulated for induction of apoptosis. this observation was associated with a reduced production of il- , ifn-gamma, il- and il- by mucosal t lymphocytes, tested by elisa assays. further studies with the oxazoloneinduced colitis model showed that nfatc regulates il- /il- in an indirect way. last, administration of il- blocked the protective effects of the nfatc deficiency in experimental colitis, suggesting that nfatc through il- signal transduction plays a direct pathogenic role in vivo. conclusion: our data define a unique regulatory role of nfatc in colitis by controlling mucosal t cell activation in an il- dependent manner. the examination of this signal transduction pathway emerges as a potentially new therapeutic target for inflammatory bowel diseases. the pivotal role of micrornas in the regulation of gene expression, in particular genes involved in the immune response, indicates that they may play an important role in the pathogenesis of inflammatory bowel disease (ibd) as well. the study of the expression of micrornas in ibd will unravel their role in this disease. in addition, micrornas by their mechanism of action, are promising new therapeutic agents or targets. a possible therapeutic application of micrornas is the introduction of novel, artificial micrornas or microrna mimics to regulate specific genes. because ibd is a heterogeneous disease in human we decided to define microrna expression in a well defined model of experimental colitis. as a result of this study we found a number of micrornas involved in different phases of experimental colitis. to study the role of mirnas in experimental colitis in mice we have used a well defined colitis model that resembles human ibd. this colitis is mediated by cd cd rb high t cells that are injected i. p. in scid mice. in control mice in addition to the cd cd rb high t cells also regulatory cd cd rb low t cells are transferred and no colitis develops. to study mirna expression we collected colonic tissue from the mice at different time points during colitis progression. after weeks a chronic progressive colitis developed characterized by a progressing wasting disease that was terminated at weeks. microrna was isolated from colons of mice in different stages of colitis progression ( , and weeks) and control mice that do not induce colitis (n= for each timepoint). from all mice we also processed a part of the colon for immunohistochemistry to determine disease progression at the various time point after induction of colitis.the rna isolation as well as the microarray analysis has been outsourced to miltenyi biotec gmbh, bergisch galdbach, germany. we used the mirxplore tm microarrays for microrna expression profiling. from micrornas that demonstrated an induction during the development of disease we selected micrornas for in situ hybridization and for a proof of principle of the efficacy in the cd cd rb high transfer model. objective: the purpose of this clinical trial (id: nct of www.clinicaltrials.gov) is to investigate whether the expansion of the thymus in adults can restore specific immune responses by administration of growth hormone (gh). methods: successfully highly active antiretroviral therapy (haart) treated hiv infected patients that failed to elicit a humoral response to tetanus toxoid (tt), or to hepatitis a (hva) or to hepatitis b (hvb) virus have been selected for the trial. growth hormone was given for months with the hope that they will reactivate thymic input and restore their specific responses to these vaccine antigens. patients have been randomized in groups: group a (n= ) receiving haart+ gh (for months) + tt+hva/b vaccines (at month post gh adminsitration); group b (n= ) receiving haart+gh but not vaccines; and group c (hiv control group, n= ) with haart+vaccines (at month ) but without gh. all patients are followed up months further. results: preliminary results show that an increase in thymic size was observed in gh recipients and not in controls. furthernore after weeks of administaring hormone the absolute numbers of cd incresase from ± to ± cells per mm (mean and sem; p x . ). in contrast, pacients who have not received the hormone but have been vaccinated showed a significant decay of the cd absolute numbers from ± to ± cells per mm (p x . ). viral load remained undetectable in all patients. despite the increase in cd counts the percentage of recent thymic emigrants (as assessed by the expression of cd ) as well as the proportion of naï ve and memory cells remained constant throught the trial in all patients. finally, specific responses to hepatitis a virus seem to be restored in a major proportion of patients treated with gh (group a) than in the other groups. conclusions: although the clinical trial is ongoing, the preliminary results seem to indicate an increase in the thymic size and some immmune restoration in patients treated with growth hormone before vaccination. a major problem of current vaccines is the requirement for cold chains to maintain vaccine potency. in the course of the eradication of small-pox, freeze-dried vaccinia virus which proved to be extremely stable was used to overcome this limitation (dryvax ® ). before usage, dryvax has to be reconstituted before vaccination using a bifurcated needle reflecting another drawback of classical vaccination -transmission of blood-borne pathogens. an alarming report by the who has estimated that as many as one-third of immunization injections are unsafe in four of its six geographical regions. each year, an overwhelming number of infections with hiv ( , - , ), hepatitis c virus (hcv; . - . million) and hepatitis b virus (hbv; - million) are thought to originate from the reuse of needles and syringes by health-care providers. in this report, we took advantage of the stability of freeze-dried vaccinia virus mva and directly applied it into the nostrils of mice without prior reconstitution. this direct mucosal application induced systemic antibody and t cell responses comparable to those achieved by intramuscular administration. importantly, mucosal application of lyophilized mva conferred protective immunity against a lethal vaccinia virus challenge. additionally, recombinant mva expressing the model antigen ovalbumin induced long-term and protective immunity against listeria monocytogenes-ova challenge. the data clearly demonstrate the potency of a simple needle-free vaccination, combining the advantages of mucosal application with the stability and efficiency of lyophilized mva. methods and results: seventeen haart-treated asymptomatic hiv- infected patients with g cd + t-cell counts and plasma hiv-rna levels of x . log copies/ml were treated with a dc-based vaccine. the vaccine consisted of autologous mature dc electroporated seperately with either sig-tat-dc-lamp, sig-rev-dc-lamp or sig-nef-dc-lamp mrna and were each administered at a distinct anatomical site. after four monthly vaccinations haart treatment was interrupted. pbmc from timepoints, before during and after vaccination, were analysed for ifn-g production (elispot), proliferation (cfse assay) and lytic capacity (fatt-ctl) in response to the antigens used in the vaccine. pbmc were screened upon ex vivo stimulation with pools of overlapping tat, rev, nef and gag peptides and ifn-g secretion was analysed using elispot ('peptide elispot'). elispot was also performed on re-stimulated t-cells with electroporated dc ('dc elispot') in vitro. responses were considered positive when the number of spot forming units per million t-cells was g and when the responses were g times the standard deviation above the mean of replicate negative controls (mock electroporated dc). / patients were screened with both peptide and dc elispot. an increase of responses to the vaccine-antigens after vaccination was found in both assays. based on the dc elispot data, we observed immune reactivity against tat in / patients before and / patients after vaccination. for rev, / patients showed a pre-existing rev specific response and / patients responded to rev after vaccination. nef was the most immunogenic antigen used in this vaccine with already / patients responding before and / patients responding after vaccination. for our control antigen gag, we observed an anti-gag response in out of patients before vaccination and / patients after vaccination. the results of the other assays correspond to the dc elispot results be it less pronounced. conclusion: therapeutic vaccination of hiv-infected patients with dc electroporated with tat, rev and nef induces and/or enhances antigen-specific t-cell responses, especially when monitored with the dc elispot. background: enterovirus (ev ) is an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes significant mortality among young children. no effective vaccine for ev is available yet. polysaccharide purified from ganoderma lucidum (ps-g) has been known to be a strong immunopotentiator, therefore, we studied the immune enhancing effect of ps-g as the possible adjuvant with ev inactivated virus. methods: mice were immunized intraperitoneally with mg inactivated virus /mouse with one of the following samples: pbs, cfa/ifa, and ps-g. each mouse received the same dose of boosters after , , and weeks. blood samples were collected at , , , and days. the total serum anti-ev igg level was determine by elisa, and the neutralization assay was also done. to evaluate the cellular immune responses, spleens were harvested from all mice for splenocyte proliferation assay. cytokines assay regarding ifn-g and il- from splenocytes was also measured. results: immunization with ev /ps-g showed that the anti-ev igg levels were significantly increased compared with ev alone or ev /cfa/ifa in balb/c mice. neutralization assays demonstrated an effective protection of ev /ps-g group compared to ev alone. the splenocyte proliferation test showed that production of ifn-g significantly increased in ev /ps-g-immunized mice compared to those of ev or ev /cfa/ifa-immunized mice, indicating a th cells response elicited by heat-inactivated ev vaccine with ps-g adjuvant. conclusions: vaccine design is important for the development of immune response for pathogen, and adjuvants play the very important role to enhance the effect of vaccine. the results here suggested that ps-g can be used as a novel, safe and natural vaccine adjuvant. objectives: the search for a vaccine against hepatitis c virus is hampered due to the lack of an animal model to study vaccine-efficacy other than chimpanzees. here we describe the differential modulation of cd + t-cell responses induced by a dna prime mva boost vaccine regimen in four individual chimpanzees and their association with viral clearance. methods: an ex vivo expansion protocol was used to map peptide specific cd + t-cell responses against hcv-ns , studying induction of il- , ifng and tnfa cytokine production as well as killing capacity. to assess the killing capacity and mhc restriction of the peptides, a non-radioactive killing assay was designed. peptides that induced both il- and ifng production by cd + t-cells were tested for their competence to induce killing of transfected target cells that expressed chimpanzee mhc class i molecules. introduction: high levels of hiv- -recognizing cd + cytotoxic t lymphocytes (ctl) with a widespread specificity, especially against conserved epitopes, are considered to play an important role in the control of hiv- replication, and for the prolonged survival of infected individuals. a potential immunotherapeutic strategy would be the adoptive transfer of t cells, which are reprogrammed by introduction of an hiv-specific t cell receptor (tcr). up to now, such ctl were generated by retroviral transfer of tcr-encoding genes, which harbors several challenges (i. e., activation/inactivation of genes, life-long autoimmunity). methods: therefore, we investigated the transfer of tcr-rna into cd + t cells by electroporation, and chose tcrs which were able to recognize the hla-a restricted hivpol-peptide iv , or the hivgag-peptide sl . results: t cells, reprogrammed with these receptors, released the pro-inflammatory cytokines il- , tnf, and ifng simultaneously, and showed up-regulation of the activation marker cd , after stimulation with peptide-loaded target cells or target cells (i. e. ebv-transformed b cell and cd + t cells) presenting the naturally processed epitope. furthermore, these cells maintained their ability to proliferate after stimulation. more importantly, killing assays demonstrated that the tcrreprogrammed cd + t cells were capable to specifically lyse target cells (for at least three days) loaded with the cognate peptide, or presenting the naturally processed epitope. a comparison of our reprogrammed t cells with the parental ctl showed that the transfected t cells were only one order of magnitude lower in avidity than the parental ctl. also, the parental clone's recognition pattern of mutant peptides was preserved in tcr-rna-transfected t cells. the transfection of tcr-encoding rna into cd + t cells, may represent a simple, secure, and more flexible alternative to retroviral transduction, but has the benefit that a better evaluation of the transferred tcrs is possible. background: dendritic cells (dcs) are able to capture, internalize, and process antigens leading to potent activation of antigen-specific cellular immunity. the aim of this study was to investigate the capacity of splenic dcs that ingest antigen coated magnetic beads to induce hcv cellular immune responses. methods: splenocytes of flt l pretreated balb/c mice were incubated for hrs with hcv ns -coated magnetic beads. the cells were harvested and cells that contained beads were purified by passage over a magnetic column. the isolated population contained g % dcs and was used for immunization. dc expression of the maturation markers cd , cd and cd was determined before and after ingestion of ns -coated beads, showing a significant increase of all maturation markers induced by phagocytosis. cellular immunity was assessed using a conventional ctl assay, a cfse-t-cell proliferation assay, intracellular cytokine staining and tumor challenge (with stably ns expressing sp / cells). results: in immunized animals, the ctl activity was increased -fold compared to mock immunized mice. accordingly, tumor challenge with ns expressing tumor cells showed a significant reduction in tumor growth. the number of cd + ifn-g + cells was increased g -fold and the number of cd + il- + increased g fold in the dc-ns -bead immunization group. these results paralleled the proliferative response of splenocytes to ns protein obtained from immunized animals with the most significant response in the cd + population of dc-ns -bead immunized animals. the use of ns coated beads combines three important aspects of dendritic cell based immunization in a single step: targeting of the antigen, enrichment and maturation of dendritic cells. the induction of a strong th biased t cell response makes this approach a promising new tool in therapeutic immunization in chronically hcv infected patients. the success of anti-dec- antibody as a stimulator of strong inflammatory immune responses depends on the coadministration of non-specific dendritic cell maturation factors. in their absence, anti-dec- induces antigen-specific tolerance rather than immunity. we hypothesize that regulatory t-cell epitopes contained in anti-dec- promote a tolerogenic reaction that is only overcome through the co-administration of non-specific immuno-stimulators. this hypothesis is based on our recent discovery of a set of natural regulatory t-cell epitopes derived from human immunoglobulins that induce tolerance by stimulating regulatory t cells. we have verified experimentally that these epitopes generate an expansion of regulatory t cells and suppress inflammatory immune responses. here, we embarked on a proof-of-principle demonstration that a pro-inflammatory and non-tolerogenic anti-dec- antibody can be developed. we screened the anti-dec- sequence computationally for putative hla dr -restricted, regulatory t-cell epitopes as targets for mutations that will reduce epitope binding affinity for hla. amino acid substitutions predicted to interfere with hla binding were identified and are being incorporated into an array of anti-dec- antibody variants recombinantly fused to a test antigen, hiv gag. variant antibodies that do not interfere with dendritic-cell targeting will be evaluated for reduced tolerogenicity, as well as for enhanced gag immunogenicity, in terms of cellular and humoral responses. we predict that the modification of regulatory t-cell epitopes will significantly diminish tolerogenicity, enabling the use of modified anti-dec- as a hiv antigen-delivery system that obviates the dangers associated with non-specific activation of the immune system. supported by nih r ai a live oral vaccine based on human adenovirus (ad) has proved safe and effective in us military recruits for nearly years. in these experiments, we have investigated whether replication-deficient ad can be an efficient potential vaccine carrier for oral vaccination. ad vectors were used throughout to provide a benchmark for efficacy. we generated novel ad and ad vector systems based on dna recombineering to facilitate manipulation of the vector backbone and high throughput transgene insertion (http://adz.cf.ac.uk). egfp was inserted with a hcmv ie promoter as a model transgene. preliminary in vitro studies on bloodderived human and murine cells demonstrated that primary lymphocytes are less susceptible to transduction with ad than ad . ad routinely infected and provided transgene expression in˚ % of human cd + and cd + t cells. stimulation of the hcmv ie promoter post infection increased detection of egfp to - % of cd + t cells present, showing that ad infected a surprisingly large proportion of t cells. in comparison, ad provided egfp expression in x % of either cell type, even after t cell activation. in contrast, infection rates and transgene expression in dendritic cells of both human and murine origin with ad and ad vectors were comparable. preferential infection of dcs is likely to be beneficial in the context of a vaccine. in vivo, we observed that following oral delivery both ad and ad induced restricted yet strong expression in the intestine. the vectors were rapidly taken up into the peyers patches, with optimal expression detected day after dosing, and transgene expression being reduced below detectable levels by day . interestingly, when delivered together ad and ad vectors targeted the same subset of cells. together, these data show that ad is a viable alternative to ad -based vaccines which may also avoid unwanted infection of activated t cells. aims: monoclonal antibodies (mabs) represent some of the most promising agents for anti-cancer and anti-viral immunotherapies ( recently commercialized; g in clinical trials). to date, their therapeutic antiviral efficiency has mainly been measured by their direct effects on viral spread. however, their indirect effects on long-term immune control of viral replication through their immunoregulatory properties upon interaction with other components of the immune system has hardly been assessed. as induction of long-term protective antiviral immune responses still remains a paramount challenge for treating chronic viral infections, we asked whether neutralizing mabs, in addition to blunt viral propagation, may also modulate the endogenous immune response. methods: we have developed a preclinical mouse model of fatal leukemia induced by the frcas e murine retrovirus. mice were infected with frcas e and treated, or not, with a neutralizing mab (the mab). viral propagation, survival and development of immune responses were followed up for several months. results: using this model, we have shown that -treated/infected mice develop a long-lasting protective humoral immune response as well as a strong and sustained cellular immune response with high cytolytic activity which are not observed in leukemic non-treated/infected mice. these results show that neutralizing mabs act as immunomodulatory agents capable of inducing long-term protective immunity ( g months) with both humoral and cellular contributions, despite the fact that they were administered over a short period of time (gros et al, ; gros et al, ; michaud et al. submitted) . although the initiation and maintenance of this long-term immunity is multi-factorial, we have demonstrated the crucial importance of the uptake of antibody-coated, infected cells by dendritic cells in the development of enhanced primary and memory antiviral t-cell responses. conclusions: our results show that infected-cells/antibody immune complexes play an important role in the induction and maintenance of protective antiviral immunity through enhancement of primary and memory antiviral t-cell responses. our observation might have important consequences on the design of antiviral mab-based immunotherapies. objectives: we have analyzed the potential of virus-like particles (vlps) from rabbit hemorrhagic disease virus (rhdv) as a delivery system for foreign t-cell epitopes. to accomplish this goal, we generated chimeric rhdv vlps incorporating a cd + t-cell epitope (siinfekl) derived from chicken ovalbumin (ova). the ova epitope was inserted in the capsid protein (vp ) of rhdv at two different locations: ) the n-terminus, predicted to be facing to the inner core of the vlps (rhdv-vlp- ), and ) a novel insertion site predicted to be located within an exposed loop (rhdv-vlp- ). both constructions correctly assembled into vlps and we analyzed the immunogenic potential of both chimeric rhdv vlps in vitro and in vivo. in vitro, dendritic cells (dcs) were able to process and present siinfekl peptide in the context of mhc class i from chimeric rhdv vlps for cd + specific recognition in a dose-and insert position-dependent manner. moreover, chimeric rhdv vlps activated dcs for tnf-alpha secretion.in vivo, mice immunized with the chimeric rhdv vlps without adjuvant were able to induce specific cellular responses mediated by cytotoxic (ctl) and memory t cells. although both chimeric rhdv vlps were able to induce specific ifn-g-producing cell priming, insertion of the siinfekl peptide at the amino-terminal position (rhdv-vlp- ) was more immunogenic than insertion at position for induction of ctls and anti-viral immunity.more importantly, immunization of mice with chimeric rhdv vlps at the highest dose tested was able to control an infection by a recombinant vaccinia virus expressing ova in target organs. in addition, immunization with chimeric rhdv-vlp- at the highest dose tested was able to resolve vv-ova infection. conclusion: our data demonstrated that immunization with chimeric rhdv vlps was able to protect mice from a viral challenge, suggesting the potential suitability of these constructions for new vaccine development against animal and human viral infections. objectives: a major issue pertaining to use of dna vaccines is that despite successful proof of principle results in small animal models, low efficacies have been reported in human clinical trials and large doses are required to induce protective immune responses. in this study, we describe the targeting of antigen-encoded dna directly to dendritic cells (dcs) through a pathway that results in internalisation and transfection using a cationic lipopeptide containing arginine residues and the lipid dipalmitoyl-s-glyceryl cysteine, a known tlr- ligand. methods: agarose gel electrophoresis was used to confirm the electrostatic binding of lipopeptide to dna encoding for green fluorescent protein (gfp), influenza hemagglutinin (ha) or nucleoprotein (np). transfection efficiencies of dcs using these complexes were determined by flow cytometry using specific antibodies for each encoded protein. stimulation of t cells by np-transfected dcs was also investigated by measuring their ability to induce in vitro cytokine secretion by influenza virus-specific cd + t cells. subsequently, vaccine immunogenicity was ascertained by immunisation of mice via the intra-nasal route. results: electrostatic binding of the lipopeptide to dna plasmids was confirmed by gel electrophoresis where the positively charged amino acids of the lipopeptide were able to neutralise the negative charged phosphate groups within the dna backbone and retard its ability to migrate towards the anode. high levels of gfp, ha or np were detected in murine spleen-derived cultured dcs following transfection with these complexes concomitant with the upregulation of surface mhc class ii molecules compared to when dna alone was used. the ability of transfected dcs to stimulate cd + t cells from influenza virus-infected mice was also investigated. subsequently, vaccination by lipopeptide-dna complexes resulted in the induction of higher numbers of ifn-g producing np - specific cd + t cells in the spleen and lymph nodes of mice compared to those that received dna alone. conclusion: altogether these results demonstrate that the use of a tlr- targeting lipopeptide-based system that can facilitate the delivery of dna by directly targeting and concurrently activating antigen-presenting cells, such as the dc, could prove to be advantageous in enhancing cellular responses induced by dna vaccination. the level of interferon in blood serum of non-infected mice was determined by elisa kit and by the cytopathic test in the l cell culture after aplication of ridostine and ridostine ointment. the effect of the preparation on phagocytic activity of macrophages was evaluated in the monolayer peritoneal cell culture. the statistical processing of the data was carried out by the student t-criterion. ridostine was administered to patients once or twice in the case of high temperature. the clinical signs were recorded (temperature, rhinitis, headache etc). for prophylactic and treatment purposes the ridostine ointment was intranasally applied twice per day during days. the effectiveness of the preparation was evaluated by clinical sings and the level of cd +, cd +, cd +, cd + t -lymphocytes. results: ridostine significantly decreased accumulation of the virus in lungs of infected mice at the initial stage of influenza. ridostine and ridostine ointment stimulated synthesis of interferon and phagocytic activity. ridostine in clinical practice decreased the duration of influenza, attenuated clinical signs and was more effective at an early stage of the infection. prophylactic intranasal application of ridostine ointment by healthy volunteers (nurses and doctors) resulted in a high degree of protection during the whole epidemic season and an activation of t-cell immunity. application of ointment at an early stage of disease markedly activated t-cell immunity, reduced the duration of the disease and the intoxication syndrome by , - -fold. conclusion: interferon inducer based on natural dsrna stimulates some reactions of innate immunity and resistance to influenza virus. the drugs based on dsrna show promise for treatment of influenza. objectives: the creation of gene engineering vaccines against hepatitis c virus (hcv) based on recombinant proteins is one of relevant approach. since the immunogenicity of these proteins is low as a rule, the choice of adjuvant is very important. the aim of this work was to evaluate immunogenicity of covalent conjugates between nonstructural ns and ns a hcv proteins and immunomax ® , an acid peptidoglycan of plant origin, which displays immunomodulating activity. objectives: ifn-g takes part in the development of an anti-infectious reaction of the organism, which is connected with the peculiarities of its immunomodulating action. a/h n influenza virus inhibits the ifn-g synthesis (mibayashi et al., ) and causes a decrease in cd + and cd + t-lymphocytes content in lung and lymphoid tissues associated with an impairment of this cytokine production (tumpley t. m. et al., ) . thus, ifn-g is a promising drug for prophylaxis and treatment of avian influenza under conditions of monotherapy or complex therapy. an essential defect of this cytokine is its fast degradation in blood. the goal of this work was to study immunomodulating properties of an ifn-g structural analog with increased proteolytic resistance when it was used alone or in combination with double-stranded ifn inducers. methods: a recombinant human ifn-g analog deltaferon is distinguished by a amino acid deletion at the c-end of the molecule and substitutions of amino acids in positions - (tat'kov c. i. et al., ) . deltaferon was i. p. administered to male non-inbred mice in doses of - * iu once or twice at an interval of hours, alone or in combination with double-stranded yeast rna preparation ( mg/kg). the content of ifn-a and ifn-g in mouse blood serum was determined by the immunoenzyme method, proliferative activity of splenocytes -by mtt-test (mosmann t., ) . results: when deltaferon was administered in doses of - * iu alone it did not influence the content ifn-a in blood, but caused a transient increase in the level of ifn-g. the injection of the preparation in a dose of * iu led to a an increase in both spontaneous and conconavalin a-induced proliferation of splenocytes. the two-fold administration of deltaferon in the maximal dose increased a level of ifn-g and inhibited cell proliferative activity. the combined administration of deltaferon ( * iu) and dsrna markedly increased the level of ifn-a and enhanced splenocyte proliferation. the recombinant human ifn-g analog is able to enhance ifn-g synthesis, splenocyte proliferation and to modulate the effect of ifn inducer. these data suggest that the studies of the preparation as an antiviral agent during influenza are perspective. by using a combined approach of routes of immunization and vaccine delivery systems such as poly-lactic acid (pla) biodegradable nanoparticles, we have dissected the intensity and quality of both cellular and humoral immune responses in mice. we showed that the amplitude and quality of the immune response (humoral, cellular) at systemic and mucosal sites (blood, vagina) could be largely influenced by the choice of a pertinent cutaneous route of vaccination (intradermal (id), transcutaneous (tc), subcutaneous (sc)). while id and tc route remain mostly efficient for the induction of antigen-specific cd responses (tetramer+ hiv- gag p cells), the quality of humoral responses (igg, iga) remained distinct between the two routes. in addition, sc route is less efficient than id and tc routes for the induction of cd responses after pla-p immunization. we have also shown that a lower antigenic charge of pla particles was needed when pla-p were injected using id and tc routes of immunization. currently, we are dissecting innate cellular mechanisms that gave rise to distinct quality of immune responses. this unique possibility to modulate the quality of the immune response according to the skin route of immunization paves the way for new vaccine design strategies and highlights the capacity of nanoparticles-based vaccine delivery system. b. dí az-freitas , c. prego , s. vicente , m.j. alonso , a. gonzález-fernández university of vigo. area of immunology, department of biochemistry, genetics and immunology, vigo, spain, university of santiago de compostela, nanobiofar group, department of pharmacy and pharmaceutical technology, santiago de compostela, spainobjectives: the design of effective vaccine delivery nanovehicles is opening up new possibilities for making immunization more equitable, safe and efficient. in this work, we purpose polysaccharidic-based nanocarriers as delivery structures for virus-like particle antigens, using recombinant hepatitis b surface antigen (rhbsag) as a model. our aim was to evaluate in a murine model if these nanocarriers induce an immune response after intramuscular and intranasal administration. materials and methods: loaded chitosan-based nanocarriers were prepared by cross-linking the polysaccharide chitosan, in the presence of the stabilizer poloxamer , with a counter ion, tripolyphosphate, containing the free rhbsag. the immunogenicity of these polysaccharidic-based nanocarriers with or immunizations to balb/c female mice ( weeks old) was assessed following intranasal or intramuscular immunizations. blood samples from the mouse maxillary vein were collected at different intervals (from day to post-primary immunization). specific igg antibodies levels directed against rhbsag in serum were measured by indirect elisa in miu/ml. results: chitosan-based nanoparticles with particle size in nanometric range, positive zeta potential and an important rhbsag loading were prepared. the results of the specific igg levels achieved following intramuscular administration of the antigen-loaded nanocarriers, and also of the alum-adsorbed vaccine showed the significant adjuvant effect of the nanocarriers. the response elicited was delayed respect to the alum based vaccine, and very interestingly, igg concentration was much higher using antigen-loaded nanocarriers than with the conventional vaccine. after intranasal administration, chitosan-based nanoparticles generated a lower immune response compared with the intramuscular route, but increasing over the time, showing an interesting slow release of the antigen. the igg titers elicited were enough to induce full seroprotection against the disease ( miu/ml). polysaccharidic-based nanocarriers with interesting properties for improving vaccination with complex antigens were designed and in vivo behavior of these nanocarriers suggests their potential utility as controlled release vehicles for reducing the number of intramuscular doses administered. more studies are currently underway to fully validate the potential of these nanocarrier prototypes and to optimize alternative routes of immunization such as the intranasal route. the success of immunotherapeutical approaches strongly relies on specific antigen targeting to dendritic cells (dcs) in an environment that provides optimal immunostimulatory signals. in our research group a bio-safe coronavirus-based vaccine vector platform that delivers multiple antigens to professional antigen- background: infection with human immunodeficiency virus type (hiv- ) is characterized by dysfunction of hiv- -specific t cells. to control the virus, antigenloaded dendritic cells (dcs) might be useful to boost and broaden hiv-specific t-cell responses. poly electrolyte microcapsules are potent protein delivery vehicles which can be tailored with ligands to stimulate maturation of dendritic cells. we investigated the immune stimulatory capacity of dendritic cells loaded with these microcapsules, containing both p antigen from hiv- and the tlr ligand poly i:c as a maturation factor. methods: monocyte-derived dc (mddc) from healthy subjects were cultivated with polyelectrolyte microcapsules containing, poly i:c. potential maturation of dc was evaluated by flow cytometry. mddc from hiv-infected patients under highly active anti-retroviral therapy (haart) were similarly pre-incubated with p microcapsules containing p and poly i:c. these antigen loaded mddc were used to directly stimulate autologous peripheral blood lymphocytes (pbl) in elis-pot. they were also co-cultivated for days with autologous pbl to evaluate the immunogenic capacity. potential expansion of specific t cells was measured by comparing elispot responses of pbl before and after coculture, using a pool of overlapping p peptides. intracellular staining of interferon-gamma (ifn-g), interleukin- (il- ) and cd a after p stimulation was also performed. results: mddc from hiv(-) subjects, incubated for hour microcapsules alone did not induce maturation of dc, but when poly i:c was present the dc did mature. mddc from haart treated hiv-infected individuals, cultivated with p containing microcapsules with poly i:c, were able to efficiently expand and broaden autologous effector memory t cells which contain and secrete ifn-g and il- , upon p peptide restimulation. objectives: we aimed at investigating whether and how the distance of a cytokine from the vlp surface impacts on its function and whether the relative distance towards a co-presented antigen is critical for its biological activity. methods: we inserted one, two or four ig-like domains of hcd b between our model cytokine il- and the minimal gpi-anchor acceptor sequence. subsequently, we compared particle production by western blotting for p gag, targeting of cytokines to lipid rafts and and vlp upon isopycnic membrane separation and biological activity in il- dependent proliferation assays of il- variants. results: murine il- attached to either of the four fusion partners was biologically active in vitro as shown by induction of proliferation of the il- dependent cell line ht- . we found that the membrane anchors comprising one and four ig-like domains (il- :: iggpi and il- :: iggpi) resulted in severely reduced vlp production by producer cells and despite of an increased targeting of il- :: iggpi to vlp, a reduced stimulatory capacity of producer cell crude supernatant, when compared to il- fused to the minimal gpi-anchor acceptor sequence of hcd b (il- ::gpi). il- :: iggpi, however, showed comparable particle production and biological activity in vitro when compared to il- ::gpi. furthermore, il- fused to ig::gpi showed an increased capacity to co-stimulate primary p tcr transgenic t-cells specific for lcmv-gp - in the context of h -d b . conclustions: besides the minimal gpi-anchor acceptor sequence we have identified one additional membrane anchor, which displays superior capacity to target cytokines to vlp. ig::gpi has a biological activity in vitro, which is comparable to the minimal gpi anchor. moreover, il- ::gpi displays increased co-stimulatory potential in the context of specific mhcp complexes. this work was supported by grants sfb f -b of the austrian science foundation, the austrian research promotion agency (forschungsförderungsgesellschaft) bridge grant & biomay ag, and the christian doppler laboratory for immunomodulation. a. roemhild , interdisciplinary transplant laboratory charite berlin, insitute of nephrology and medical immunology, berlin, germany immunosuppressive treatments, e. g. after transplantation are often followed by an impaired or dysfunctional immune system. missing viral immunity, particularly against ebv, is an essential key player in the development of severe infections and posttranplant lymphoproliferative disorders (ptld). ptld affects - % of solid organ transplant recipients, depending on the organ transplanted. healthy individuals control ebv infection by ebv-specific cytotoxic t lymphocytes (ctls), but some patients under immunosuppression are unable to do so. in these cases, immunotherapy is increasingly used as a new approach for re-establishing a functional immune response by retransferring in-vitro expanded autologous virusspecific t cells into the patient. currently these t cells are generated by repetitive stimulations with ebv-infected autologous lymphoblastoid cells (lcls). due to a generation time of - months, many patients suffering from missing viral immunity and subsequent severe viral disease are excluded from therapeutic benefit. therefore, shortening the generation time would be an important step to make adoptive immunotherapy available for more patients. t cell lines were generated with two different protocols. in the first protocol t cells are generated by repetitive stimulation with ebv-infected autologous lcls. the second protocol is based on stimulation with different overlapping ebv peptide-pools and immunomagnetic cell isolation. expanded t cells were analysed using multicolour flow cytometry. cells were stained for diverse surface markers and intracellular cytokine production. cytotoxic capacity and specificity was determined by a calcein release assay. our group developed a new protocol for the production of ebv specific t cells, thereby shortening the generation time from , month to days. t cell lines are composed of cd and cd cells with a mainly effector memory like phenotyp. after restimulation the cells produce more tnfa than ifng. depending on the generation protocol t cells specifically recognized and lysed autologous lcls alone or loaded with ebv-peptides. the detailed characterization of ebv-specific t cell lines should help to further improve the adoptive immunotherapy and its outcome. the novel, short time generation protocol did not affect phenotyp and cytokine production of the t cells. nevertheless their therapeutic potential in vivo has to be tested in further experiments. s. s. schmucker , m. assenmacher , a. richter miltenyi biotec gmbh, r&d cell biology, bergisch gladbach, germanyadoptive transfer of virus-specific t cells provides a promising treatment of infection in immunocompromized patients. as expansion of virus-specific t cells from antigen-experienced donors is feasible, no reliable protocols for generation of antigen-specific t cells from naive hosts exist. in this study we established a cell culture system for priming of highly rare naive cmv pp -specific cd + and cd + t cells from cmv-seronegative donors in vitro.magnetically isolated naïve (cd ro -cd -) cd + and cd + t cells from pbmc of cmv-seronegative donors were co-cultured with autologous mature monocytederived dc loaded with cmv pp peptide pool and cd -depleted autologous pbmc as feeder cells in the presence of il- , il- , and il- . already - days after primary activation pp - /a -tetramer + cd + t cells were detectable for hla-a + blood donors. to analyze cd + t cells having other specificities than for the peptide pp - as well as probably primed cd + t cells, we looked for the production of cytokines after a second stimulation. we found ifn-g secretion in up to . % of the cd + t cells and up to . % of the cd + t cells after restimulation with pp peptide pool, but not with either irrelevant ie- peptide pool or without antigen, in each of eight donors tested. for generation of t cell lines, we magnetically enriched the primed t cells according to their ifn-g secretion. subsequent cultivation for days led to a - fold expansion of pp -specific t cells, defined by their sustained capability to produce ifn-g. evaluation of the antigen-specificity of the expanded t cells also showed upregulation of the activation markers cd and cd only if restimulated with the pp peptide pool. further cytokine analysis of the cells revealed co-production of ifn-g, tnf-a, and il- , indicating the functionality of the in vitro primed and expanded t cells.in conclusion, we established a cell culture system, which enables the in vitro priming and expansion of cmv-specific cd + and cd + t cells derived from the naive compartment. this should extend the application of adoptive t cell therapy to patients for whom immune donors are not available. a. i. wolf , k. mozdzanowska , l. otvos , j. erikson the wistar institute, philadelphia, united states, temple university, philadelphia, united statesthe influenza virus a matrix protein ectodomain (m e) sequence has remained highly conserved among various human influenza a strains and is therefore a promising target for a protective vaccine. based on previous work using a synthetic m e-based multi-antigenic peptide vaccine (mozdzanowska at al., vaccine ; virology journal ), we generated a novel peptide and investigated its efficacy in inducing an anti-m e antibody (ab) response and its ability to confer protection against viral challenge.objectives: cytomegalovirus (cmv) causes significant morbidity and mortality in patients after haematopoietic stem cell transplantation (hsct). due to limitations of current antiviral therapies, alternative approaches, involving transfer of donor-derived cmv specific cd + t cells, have been considered. clinical data confirm a crucial role for antiviral cd + t cells inversely correlating with the incidence of cmv reactivation and disease. cmv specific cells have to reach protective levels in order to be effective. levels of such cells correlating with protection against cmv infection and disease have only been reported in patients expressing hla-a* and hla-b* previously. considering other frequent hla alleles cmv specific cd + t cells were monitored longitudinally in hsct patients in this study to establish the cell number thresholds at which patients are protected from cmv reactivation. methods: we have correlated the pattern of different ex vivo cmv peptide specific cd + t cell responses (frequency analysis using tetramer staining and interferon gamma elispot analysis) with the cmv viral load (dnaemia) and clinical status in patients. different response groups were compared using the mann-whitney-u test.results: our results demonstrate that the presence of different cmv specific cd + t cells inversely correlates with the ability to detect of cmv reactivation in patients at different cell number thresholds. we show that the cell number thresholds for hla-a* /pp ( - ) ( . x cells/l) and hla-b* /pp ( - ) ( . x cells/l) specific cd + t cells are significantly lower than those for hla-a* /pp ( - ) ( . x cells/l) and hla-a* /pp ( - ) ( . x cells/l) specific cd + t cells in hsct recipients post transplant. this difference is also evident in healthy cmv seropositive volunteers. conclusion: these findings suggest a differing efficiency of the responses restricted by the two sets of alleles. the data merit further studies using larger patient cohorts and are important for considerations regarding the epitope restriction and quantities of ag specific t cells to be monitored after therapeutic strategies for cmv in hsct patients. ( , - mcg) . no adverse effects were indicated during trials (up to month of observation).hiv-specific antibodies were induced by dose-dependent manner, the most prominent response was detected after th immunization with mcg of vichrepol.no differences were detected in cd + and cd + t cell counts and cd +/cd + ratio, so there was additional safety issue concerned to the possible sensitivity of vaccinees to hiv infection. the results of phase i clinical trials of vichrepol vaccine were approved by who authorized russian national control institution and transition to phase ii immunogenicity trials was recommended. objectives: to improve the vaccination efficiency of adenoviral vectors for anti-retroviral vaccination, we constructed adenoviral nanoparticles by fusion of the vaccine antigen to the adenovirus capsid protein pix. the adenoviral nanoparticle vaccine was evaluated in the friend virus (fv) mouse model and compared to conventional adenoviral vectors. methods: adenoviral nanoparticle vectors were constructed by deletion of pix from the adenoviral genome and insertion of the fusion protein encoding sequence as transgene. for vaccination against fv, that is a retrovirus complex of friend murine leukemia virus (f-mulv) and spleen focus forming virus, we constructed fusion proteins of pix and the f-mulv surface env protein gp or gag. to elucidate underlying mechanisms we produced displaying-only nanoparticles and plasmid dna encoding either pixgp or gp alone. conventional adenoviral vectors were used that express full-length f-mulv env and gag. the vaccines were tested in cb f hybrid mice that are highly susceptible to fv infection and develop viremia and splenomegaly after fv infection. results: vaccination of cb f mice with adenoviral nanoparticles expressing fusion proteins containing gp resulted in protection from viremia and splenic enlargement after fv challenge that was superior to vaccination with conventional vectors. immunological analyses showed that the adenoviral nanoparticle vaccine induced a significantly higher number of f-mulv env-specific cd + t cells and higher antibody titers than a conventional adenoviral vaccine expressing the vaccine antigen. we could show that for the beneficial effect it is necessary that the fusion protein is incorporated into the adenoviral particle and it also has to be expressed from the adenoviral vector in vivo. conclusion: adenoviral nanoparticles are a useful tool for the induction of antibody and cd + t cell responses that are superior to conventional adenoviral vectors. this new type of adenovirus-based vaccination vector combines genetic and protein vaccination and should make adenoviral vectors even more interesting for vaccination purposes. . antibody levels were monitored by elisa and hemagglutination inhibition assay, viral excretion in nasal washes was assessed by quantitative rt-pcr, and cellular production of ifn-gamma was measured via flow cytometry. results: we found that animals vaccinated with caf exhibited higher levels of serum igg and mucosal iga than the ones which received the vaccine alone, and that they excreted - % less virus. animals that received only vaxigrip were producing ifn-gamma after challenge, a sign of infection by low virulence influenza strains, whereas the animals that received also caf did not show any increase in their levels of ifn-gamma. conclusion: caf enhances the protection conferred by the commercial inactivated vaccine against strains matched by the vaccine. evaluation of the t-cell specific immune response is very important for global eradication of measles and rubella. peripheral blood lymphocytes (pbl) from children aged - years old ( boys and girls) -group , and children ( boys and girls) - years old -group were isolated on a gradient of density before vaccination ( or revaccination) with priorix, week, and months after and incubated with cfse. then million/ ml pbl were incubated in rpmi- supplemented with % fcs (the negative control), at presence of mcg/ml pha (the positive control) or at presence of the measles or rubella viruses antigens in a humidified atmosphere containing % cÎ at °c within day. intensity of a fluorescence estimated on fl by flow cytometr facscalibur (bd biosciences, usa). cytokines production was measured in the same cultures by bioplex technology (biorad, usa). in the negative control % pbl in both groups did not enter mitosis. in the positive control % of cells have passed one and more mitoses. in group measles or rubella antigens did not induced lymphocytes to enter mitosis, like in negative control, before the vaccination and in a week, however in months - % of lymphocytes demonstrated antigen-specific proliferation. in group , on the contrary, before the vaccination the most part of cells ( - %) has not entered division, but - % of cells have passed and more mitoses. in a week specific lymphocyte proliferation decreased and in months it was increased up to - %. production of the interleukin (il) , ifn-g, tnf-a, il- , il- was more informative than il- , il- , il- , il- . measles and rubella antigens induced cytokines production in pbl of immune children and did not influence on pbl of intact children. thus, it was shown, that both methods can be applied to revealing the specific cellular immune response to measles and rubella antigens. objectives: broadly neutralizing human monoclonal antibodies (mab) and patients' sera recognizing functionally conserved epitopes on hiv envelope (env), such as the gp cd -binding site (cd bs), appear to be uncommon. therefore, new approaches are needed to elicit the humoral response on these conserved epitopes. here we describe the generation of two anti-idiotype (ai) murine antibodies recognizing human anti-hiv- neutralizing polyclonal iggs directed against the cd bs. the mabs were shown to react with an anti-cd bs human neutralizing mab (b ), to elicit antibodies that recognize the gp molecule and an anti-hiv- neutralizing response in rabbits, confirming them as cd bs mimotopes. these mabs were also used as probe to detect the expression of clonally distinct anti-gp antibodies in sera of hiv-infected individuals. methods: broadly neutralizing sera were collected from long-term non-progressor patients. anti-cd bs iggs were purified and used to immunize mice for hybridoma generation. mabs reacting in elisa with the anti-cd bs igg fraction were used to immunize rabbits. rabbit sera were then tested for anti-gp titer and hiv neutralizing activity by pseudovirus-based neutralization assay. sera from hiv-infected individuals at various clinical stage of infection were studied to validate an immunoenzymatic assay able to detect the reactivity to the ais. serial dilution of b in sera from healthy hiv-negative donors were used to determine elisa sensitivity. results: two clones (p and p ) reacted in elisa only with the cd bs-directed igg fraction. the clones were also recognized in elisa by b . p and p -immunized rabbit sera showed a strong anti-gp titer. in the pseudovirus assay the ais-immunized rabbits showed a neutralization activity against virions bearing hxb strain glycoproteins. in particular, / rabbits in the p group and / in the p group showed an % hiv neutralization at dilutions ranging from : to : . the immunoenzymatic assay used, allowed to detect a p and p reactivity in hiv-positive sera and was able to detect a b concentration equal to ng/ml. conclusions: these data demonstrate that immunogens designed on the idiotype of broadly neutralizing abs are feasible and could help in the design of effective anti-hiv vaccines or diagnostic assays. yellow fever vaccines ( d and dd) are well tolerated, with a very low rate of severe adverse events (yf-sae), such as serious allergic reactions, neurotropic (yf-and) and viscerotropic (yf-avd) diseases. viral and host factors have been postulated to explain the basis of yf-sae, especially those able to modify the host immune response to the yf vaccine. however, the mechanisms underlying the occurrence of yf-sae still remain unknown. in the present investigation, we present a detailed immunological analysis of a -year-old us citizen female patient, who developed yf-and characterized by encephalitis associated with pancreatitis and myositis following d- vaccination. our findings highlighted that yf-and exhibited decreased expression of fc-g-r in monocytes (cd , cd and cd ) along with increased levels of nkt-cells (cd + cd +/-cd +/-/cd + ratio) and activated t-cells (cd + and cd + ) and b-lymphocytes. enhanced levels of plasmatic cytokines (il- , il- , il- , il- and il- ) besides exacerbated ex vivo intracytoplasmic cytokine pattern, mainly observed within nk-cells (inf-g + , tnf-a + and il- + ), cd + t-cells (il- + and il- + ) and b-lymphocytes (tnf-a + , il- + and il- + ). the analysis of cd + t-cells revealed a complex profile with increased frequency of il- + and ifn-g + and decreased percentage of tnf-a + , il- + and il- + cells. depressed cytokine synthesis was observed in monocytes (tnf-a + ) following in vitro antigenic stimuli. these results support the hypothesis that a robust magnitude of the adaptive response and abnormalities in the innate immune system may be involved in the establishment of yf-sae. this is the first case report of yf-sae investigated by members of the international laboratory network for yellow fever vaccine associated adverse events. g. mester , h.-g. rammensee , s. stevanović eberhard-karls-universität tübingen, department of immunology, tübingen, germany adenovirus (adv) is a widespread pathogen in humans and can persist in its hosts after infection. persistent virus is an important cause for severe disease in immunocompromised individuals, e. g. bmt recipients, with high rates of mortality. however, the cellular immune response against adv is poorly characterised, and very few t cell epitopes have been published up to now. thus, our aim was to detect dominantly immunogenic adenoviral cd t cell epitopes by analysing the responses of healthy blood donors who have overcome infection. we have predicted possible cd t cell epitopes for the frequent mhc class i alleles a* , a* , and a* from the proteins pii (hexon), pviii, and e a of adv strains ad and ad by using the syfpeithi software developed by our group (www.syfpeithi.de). subsequently we performed a -day recall stimulation of pbmcs from at least healthy donors with synthetic peptide followed by ifn-g elispot screenings to identify naturally occurring t cell responses and assess their frequency in the population. tetramer and intracellular cytokine stainings were also carried out to confirm the presence of specific cd t cells. we could identify new peptides eliciting ifn-g responses, several of which were confirmed as novel cd t cell epitopes. amongst others we found at least one immunodominant epitope recognised by more than half of the healthy donors for each examined hla restriction as well as, to our knowledge, the first adenoviral epitope derived from a protein other than hexon. these findings will be helpful to identify frequently immunogenic and thus promising candidate peptides for in vitro t cell priming or expansion preceding adoptive transfer, which has been proven to be a valuable therapeutic approach in the treatment of persistent viruses in immunocompromised patients. methods: sle ( ara criteria) was diagnosed in a -year-old african female patient with hiv- (clade c) infection. good initial response occurred on hydroxychloroquine and steroids followed by disease flare and drop of cd t-cell count x cells/mm . initiation of mg mmf bid was associated with biological and clinical remission of sle and cd t-cell increase. no opportunistic infections or cancers were noted during a -year follow-up and the patient remained always naive to art. hiv- -specific cd and cd t-cell responses were analyzed after months of mmf by ifn-g elispot assay and polychromatic flow cytometry assessing ifn-g, tnf-a and il- production following stimulation with a panel of hiv- -derived optimal epitopes ( / -mers) covering various hiv regions and a pool of hiv- -derived peptides ( -mers overlapping by aminoacids) encompassing the entire gag protein. all peptides are derived from hiv- consensus strain iiib. results: highly polyfunctional hiv- specific cd and cd t-cell responses against gag were detected. epitope-specific cd t-cell responses were identified: except for one response restricted by hla a* and another one by hla cw* , all the others were restricted by hla-b alleles and mostly by b* (n = ). seven out of responses were strong enough to be further analysed with regard to their functional profile and shown to be highly polyfunctional (i. e. ifn-g+, tnf-a+ and il- +) regardless of the viral region and hla restriction. conclusion: strong, broad and polyfunctional hiv- specific cd and cd t-cell responses known to be associated with nonprogressive infection were detected during mmf treatment.we therefore suggest that mmf use in the context of sle-hiv is not detrimental to the establishment or preservation of protective hiv- t-cell immunity. the rabies virus was propagated in the vero cell line. virus was titrated by focus fluorescent units. virus preparations having a titer of dl /ml were inactivated with b-propiolactone. aluminium hydroxide gel or squalene, at different concentrations were adsorbed to the inactivated rabies virus. male mice of the strain cf- of - g and no less than four weeks age, were distributed in six groups for intraperitoneal immunization, group a was immunized with virussqualene, group b with virus-aluminium hydroxide, group c with the antigen alone, group d with saline buffer-squalene, group e with saline buffer-aluminium hydroxide and group e was inoculated with mock-infected cell culture supernatant. mice were boosted at the th day. all mice were properly bled to prepare preimmune sera and hyper-immune sera. at the end of the immunization protocol the igg raised against the rabies virus was tested by an indirect elisa. results: the highest titers of neutralizing antibodies were obtained with similar concentrations of either squalene-or aluminium hydroxide-based vaccine formultaions. there was a significant difference in the neutralizing antibody titers produced by mice immunized with the antigen (inactivated rabies virus) adsorbed to the adjuvant, as compared to those obtained from mice immunized with the antigen alone, as expected, no neutralizing antibodies were detected on mice inoculated with saline buffer or mock-infected vero cell supernatant. conclusions: the use of either squalene or aluminium hydroxide as adjuvant in the canine antirabic vaccine formulation increases immunogenicity, almost to the same extent. aluminum hydroxide adsorbed to the antigen seems to be a better option, since squalene is more expensive than aluminium hydroxide. supported by: concyt- , cofaa and cgpi- . . state of vaccine-induced measles immunity was determined by means of elisa in - , - and - years since revaccination with live measles vaccine (lmv) before and after tuberculosis chemoprophylaxis. statistic data were processed with t-, w-and u-criteria. results: during the first three years since lmv revaccination igg level was middle (children with negative and long-term positive mt) and high (children with conversion and hyperergic mt). in - years since lmv revaccination uninfected and long-term infected children showed a significantly decreased (p p . ) measles immunity and antibody level much lower (p p . ) than among children with mt conversion. in - years the comparison group kept decreased (p p . ) measles immunity, the majority ( ± . %) of persons had minimal protected igg level, but the observation groups were characterized by average immunity level, which was higher (p p . ) than in the comparison group. comparing measles immunity level before and after tuberculosis chemoprophylaxis demonstrated the following: measles igg level among long-time infected children on completion of chemoprophylaxis decreased (p p . ), the majority ( . ± . %) of persons lost protected antibody level; among children with mt conversion in - years since lmv revaccination immunity state didn't change, but in further periods antibody level decreased (p p . ) to low values; among children with hyperergic mt igg level decreased (p p . ) and reached low (in - years), minimal protected (in - years) and lower than protected (in - years) values. -at the early stage of tubercular infection process measles immunity was higher compared to uninfected with mycobacterium tuberculosis persons, which fact is connected with immunomodulatory action of low-molecular peptide of bacterial cell wall -muromildipeptide.-in remote periods since lmv revaccination and on completing preventive tuberculosis treatment decreased measles immunity was observed.-in countries with high tuberculosis morbidity chemoprophylaxis level among children with latent infection is high, which can indirectly influence population measles immunity. objectives: to evaluate the balance of ifn-gamma and il- producing cells in lungs during the immunotherapy of tuberculosis with the dna vaccine encoding the heat-shock protein (dnahsp ). methods: balb/c female mice were infected by intra-tracheal route with h rv mycobacterium tuberculosis. immunotherapy with endotoxin free dnahsp genetic vaccine was done at days , , and post-infection. each dose consisted of micrograms of dna vaccine in the quadriceps. intracellular cytokine staining of cd +, cd + and gamma-delta t cells from lungs were determined and days after the end of the therapy. bacilli loads, histopathological and morphometric analysis of lungs were also evaluated. differences of p x . were considered significant (t test). results: at day after the end of the immunotherapy, dnahsp treated mice exhibit increased numbers of absolute cd + and gamma-delta t cells when compared to non-treated animals. the percentage of ifn-gamma and il- producing gamma-delta t cells were the same between treated and non-treated animals. in contrast, dnahsp treated mice showed more ifn-gamma producing cells in both cd + and cd + cell populations. at day after the end of the therapy, the main observation in mice which received dnahsp treatment was the augment of all three populations producing ifn-gamma. although non-treated animals also increased the frequency of cd + and gamma-delta t cells positive for ifn-gamma, they did not increase the numbers of ifn-gamma cd + cells, together with a more frequency of gamma-delta t cells producing il- . finally, the immunotherapeutic effects of dnahsp vaccination also included the diminution of bacilli loads in lungs, spleen and liver and the reduction of inflammation in lungs as determined by the histopathological and morphometric analysis. the results presented here indicates that cd + cells producing ifn-gamma and the reduction of the frequency of gamma-delta t cells secreting il- , are the main effects of dnahsp immunotherapy of murine tuberculosis. furthermore, these results have important implications since they indicate the importance of an appropriate balance of il- and ifn-gamma levels for the combat of the bacilli and the reduction of the immunopathologic damage in lungs. the detection of quantitative changes in mrna expression levels are currently being performed using either genome-wide (microarray) or single gene (real-time pcr) screening methods. because these techniques are technically challenging and too costly to be applied on a routine basis in resource poor settings, we have developed a reverse-transcriptase multiplex ligation-dependent probe amplification (rt-mlpa) method. rt-mlpa is a reliable, robust, low cost and user friendly technique permitting rapid mrna expression profiling of as many as loci in a single reaction. genes of interest can be selected on a tailor-made basis. the assay is highly reproducible, has an extensive dynamic range of - log depending on the genes of interest, and a pcr amplification step within the rt-mlpa ensures assay sensitivity, which is an essential prerequisite for the relative quantification of scarcely expressed genes. since this assay is relatively high throughput ( -well format), requires only ng rna per sample, and allows mrna profiling in direct ex vivo whole blood samples (from e. g. pax-gene tubes), it is an exceptionally suitable technique for performing semi-large scale gene expression analyses in human cohort studies. to illustrate this, we have been able to successfully implement this assay in different laboratories in sub-saharan africa. thus far we have applied rt-mlpa to characterize the human immune response to mycobacterium tuberculosis, with particular emphasis on the expression of genes associated with protective host cellular immunity and human disease susceptibility. a particularly useful application of the rt-mlpa is the identification and monitoring of host-biomarker profiles that predict (protection from) tuberculosis (tb) disease in latently infected household contacts or (in)adequate responsiveness to therapy in active tb patients. initial data sets already probe differences in immune reactivity in populations, yielding new candidate biomarkers associated with tb disease. these biomarkers may provide new and relevant information that can be applied in future tb studies for rapid, easy, semi-quantitative and reliable detection of host immune biomarker profiles. preclinical m. leprae infection is a major source for leprosy transmission. therefore, early detection of individuals infected with m. leprae is crucial. however, to date there are no diagnostic tests available that can identify preclinical leprosy. such tests will contribute to the prevention of leprosy disability and its further transmission by otherwise undiagnosed and untreated index cases.newly developed hla based bio-informatic tools combined with comparative genomics have created novel opportunities to help design improved tests for early detection of m. leprae infection.using this post genomic approach, we were able to identify candidate proteins and peptides unique to m. leprae containing predicted t cell epitopes restricted via several major hla-class i and ii alleles. since the selected genes were of unknown function, their expression in m. leprae bacilli was assessed.evaluation of the immunogenicity of these m. leprae proteins in pbmc from a brazilian population showed that candidate antigens induced significant ifn-g levels in m. leprae infected individuals but not in healthy controls from an endemic area.importantly, among exposed healthy controls % had no detectable igm antibodies to the m. leprae specific pgl-i, but instead responded to one or more m. leprae antigen(s). to further improve the diagnostic potential of these m. leprae sequences, synthetic peptides spanning all m. leprae proteins were analyzed similarly. determination of cumulative t cell responses towards of these peptides that activated pbmc of leprosy patients increased the sensitivity compared to single peptides to % in pb, % in rx and % in hhc, without compromising specificity.since diagnostic tools should be applicable in several populations regardless of the genetic background, these m. leprae antigens are also tested in populations on the african (ethiopia) and asian (nepal) continent.in addition, we have applied these antigens in a new user-friendly ucp-lf assay to detect different cytokines. this assay proved to be more sensitive than elisa for detection of ifn-g and can be easily applied in field sites. tuberculosis, an infectious disease caused by mycobacterium tuberculosis (mtb), affects millions of people. m. bovis bcg is the vaccine against tuberculosis but its efficiency is variable for the pulmonary form of the disease. paratuberculosis, an enzootic bacterial disease in ruminants, due to mycobacterium avium subsp. paratuberculosis (map), has a significant economic impact on livestock production, and moreover, map infection may be one of the microbial triggers of crohn's disease in humans. map vaccines can delay apparition of clinical symptoms, but they do not prevent infection and they have a confounding effect in the skin-test based bovine tuberculosis control programs. cd l, a co-stimulatory molecule preferentially expressed on activated cd + t cells, is the ligand of cd . cd -cd l interaction induces the production of il- and the initiation of a th -type immune response. several studies show that cd l is required for the activation of macrophages and the maturation of dcs. moreover, cd l enhances the capacity of cd + t cells to produce ifn-g and to lyse mtb-infected monocytes. in this study we attempt to improve existing tb and map vaccines with a recombinant bcg expressing cd l. we have constructed the recombinant bcg strain expressing cd l (rbcg ) by electroporation of bcg with pgfm /signalsequenceag b-cd lec and an another recombinant strain with empty vector pgfm (rbcg ) as a control. the expression of cd l has been evaluated by western blotting. balb/c mice were vaccinated with the recombinant bcg vaccines. bcg growth kinetics were compared by counting viable bacteria (cfu) in spleen and lungs. the immune response was evaluated by measurement of th type cytokine secretion of splenocytes after in vitro restimulation with immunodominant antigens and selected peptides. two months post vaccination, mice were challenged with mtb and map and protection was evaluated. preliminary results show normal persistence of the two recombinant bcgs. analysis of the immune response shows an effect of cd l weeks after vaccination but not at and weeks. rbcg seems to be more protective against paratuberculosis than rbcg , but not against tuberculosis. another vaccination experiment is required to confirm these results. the effects of bcg-cd l on cultured dcs in vitro will further be explored. objectives: tuberculosis is a major health problem globally and it is of critical importance to develop an effective vaccine to prevent further spread of the disease. iron is a key nutrient for both mycobacterial infection and for a successful protective immune response by the host. the regulation of iron availability within the host involves the intracellular iron-binding protein ferritin and it is proposed here that the regulation of ferritin is tightly controlled in the host immune response to tuberculosis. methods: using the guinea pig model of mycobacterium bovis bacillus calmette-guérin (bcg) vaccination, populations of immune cells were isolated and restimulated ex vivo over a time-course study using purified protein derivative (ppd) of mycobacterium tuberculosis or infected with bcg or m. tuberculosis. the expression of ferritin in co-ordination with key immuno-regulatory proteins, tnfa, ifng and il- a, was examined using real-time pcr. to determine whether immuno-regulatory proteins are involved in the regulation of ferritin, cytokine cascades were inhibited in the ppd re-stimulation studies by the addition of guinea pig specific tnfa and ifng antibodies. results: a typical pro-inflammatory immune response was observed with significant up-regulation (p x . ) of tnfa, ifng and il- a after re-stimulation with ppd and mycobacteria. of interest was a trend in ferritin down-regulation after re-stimulation with ppd and bcg and this was significant (p x . ) after restimulation with m. tuberculosis. the down-regulation of ferritin was also affected by the addition of tnfa antibody in the ppd re-stimulation study. conclusions: ferritin is important in the storage and management of intracellular iron and its regulation must be tightly controlled to restrict iron availability from invading mycobacteria from sequestering free iron. the data indicate that the regulation of ferritin is very subtle and is affected by cytokine cascades that involve tnfa. these results contribute to our understanding of the role of iron and intracellular ferritin in developing a protective immune response to mycobacteria in the guinea pig model of tuberculosis. this work is funded by health protection agency phd studentship award. methods: anti-cd mab and ag a were chemically treated with sata and sulfo-smcc respectively, in order to produce a stable crosslinker between both proteins. crosslinking was confirmed by western blotting and cd binding on cd transfected l fibroblasts. the conjugates were tested in vivo in wild type and cd + cell-depleted mice for the induction of specific anti-ag a serum antibodies. splenocytes were challenged ex vivo with ag a and were examined for their ability to produce th -related cytokines. elispot assays were performed to determine ifng production and flow cytometry was used to analyse intracellular cytokine staining for tnfa, ifng and il- . we developed a method to successfully crosslink anti-cd mab to ag a. serum antibodies against ag a were detected after immunisation with this conjugate vaccine in both wild type and cd + cell-depleted mice. t cells derived from mice immunised with conjugate vaccine, and stimulated ex vivo, showed an increase in ifng production (elispot), when compared to mice vaccinated with ag a alone. production of two other th -related cytokines, tnfa and il , was also increased in these t cells as shown by intracellular cytokine staining. conclusion: our results suggest that anti-cd conjugate vaccines could provide a new way to increase vaccine efficacy. this new conjugate vaccine may be able to by-pass the need for cd + t cell help in the production of specific antibodies, which would be a major benefit of any therapeutic vaccine to be used in immunocompromised patients. h. schäfer , r. burger robert koch-institute, cellular immunology, berlin, germany, robert koch-institute, infectious diseases, berlin, germanyobjective: immunity against mycobacterial infections is mediated by both cd -positive and cd -positive t-lymphocytes. cd -positve cells respond to peptides derived from cytosolic proteins and presented on mhc class i molecules of antigen presenting cells (apc) via the endogenous pathway. some apc however, are able to take up extracellular antigens and present peptides thereof on mhc class i molecules. this process has been termed cross-presentation and has been shown to be of importance in the immune response against intracellular bacteria. to define the contribution of cross-presentation to activation of cd + t cells in the response against mycobacterial antigens, we analyzed the secondary immune response in the guinea pig. methods: purified t lymphocytes from guinea pigs immunized with bcg or complete feund's adjuvant were labeled with the intracellular fluorescent dye cfse and incubated with ppd and/or apc for days. surface phenotype and proliferation of t-lymphocytes were analyzed by flow cytometry.results: up to % of lymph node t lymphocytes form immunized guinea pigs proliferated after in vitro restimulation with ppd-pulsed macrophages. no difference was observed between bcg-(living mycobacteria) and freund's adjuvant (heat killed mycobacteria)-immunized animals. the responses of both t cell subsets were equally strong, although the killed immunogen should primarily target the exogenous pathway of antigen presentation and therefore preferentially prime cd + t-lymphocytes in vivo. similarly, the cd -positive subpopulation should primarily respond to soluble antigens presented on mhc class ii molecules. proliferation of both the cd + and the cd + subpopulation depended on the presence of apc. stimulation oft cd + cells as a consequence of direct loading of peptides onto mhc class i molecules was ruled out by using mhc-class i-positive fibroblast cells instead of professional apc, which did not lead to proliferation of primed t-lymphocytes. conclusion: cross-presentation of soluble antigens to cd -positive t cells is a highly effective means to stimulate the response of cytotoxic t cells against mycobacterial antigens even without direct contact to infected cells. therefore cross-priming might represent an important mechanism for the induction of the cellular immune response against intracellular pathogens and should be useful for the rational design of vaccines against mycobacterial diseases. objectives: due to broad antigenic cross reactivity of purified protein derivatives (ppd) with bcg vaccine strains and environmental mycobacteria, results of currently used tuberculin skin test (tst) is not reliable to evaluate the specific anti-tuberculosis immune response. therefore, new tools are required to improve mycobacterim tuberculosis (tb) diagnosis and treatment, including enhanced ability to compare new treatment strategies. among different antigens early secretory antigenic target (esat- ) protein is highly specific for tb complex and elicit strong t-cell response in human. in order to monitor the immune response against the pathogen, iranian and afghan adults ( patients with sputum smear and culture positive tuberculosis, recovered patients during months after full course of chemical treatment and healthy individuals) were recruited to quantify the frequency of esat- and ppd specific t-cells in their peripheral blood by home made elispot ifn-gamma assay. results: considering cut off of spot forming unit ( g spots per million), we found detectable response to esat- in almost % of patients with active disease. this frequency among treated patients after disease recovery was not significantly different and % of these individuals had detectable esat- specific response even after six months completing treatment. neither of healthy individuals showed such response. t cell response against ppd was identified in %, % and % of healthy participants, active patients and healing individuals, respectively. conclusion: elispot ifn-gamma assay showed a sharp induction of th immune response, against esat- , in tb patients which persists after successful treatment and full recovery. these results may show potential application of tuberculosis-specific elispot testing as a proxy measure of tb diagnosis and treatment. bcg is the only available vaccine today to fight tuberculosis, but it has been reported to be variably efficacious in the field. both environmental and genetic host factors as well bcg strain variability and virulence of the intruding m. tuberculosis strain have been suggested to affect the efficacy of bcg vaccination. in mouse and bovine models it has been shown that pre-exposure to mycobacterium spp. negatively affected protective efficacy of bcg. we use non-human primates (nhp) for the evaluation of new tb vaccine candidates and possible identification of immune mechanisms of protection. however, in naive rhesus macaques a variable efficacy of bcg reminiscent of the clinical situation was revealed. by meta-analysis we compared immune response parameters measured after vaccination using bcg strain danish in the context of protection measured by gross pathology evaluation after experimental infection with a constant m. tuberculsosis strain erdman. although numbers of animals used are relatively low, data suggest that both breeding origin as well as the immune status of monkeys impact on the efficacy of bcg. most remarkably, while bcg induced levels of ifng secretion did not correlate with protection, kinetics of secretion monitored after in vitro stimulation of peripheral blood lymphocytes did correlate. our findings would suggest that, in accordance with mouse and bovine experimental data and epidemiologic observations, possible pre-exposure to mycobacterial antigens beyond the current sensitivity of tb diagnostics for nhp, negatively affects the protective efficacy of bcg. together, these results are relevant for evaluation and interpretation of tb vaccine tests in nhp and support further research into the identification of (mechanisms of) protective immunity in the primate host. p. s. nagpal , p.k. upadhyay national institute of immunology, pdc- , delhi, indiaobjective: study was aimed for the preparation of dry powder formulation containing live mycobacterium indicus pranii for pulmonary immunization against tuberculosis. pulmonary delivery evokes both systemic as well as mucosal immune response against the antigen. secondly, pulmonary delivery is a needle-free delivery system, long desired for vaccine delivery. dry powder aerosol one such method, in which vaccine can be directly delivered to lung without any kind of invasion and it has an edge over liquid formulation being feasibility of storage at room temperature, long term shelf stability and higher drug content per unit mass as compared to liquid one. method: sodium alginate solution with suspended mycobacterium indicus pranii was aerosolized using laboratory modified nebulizer assembly. aerosols so generated were entrapped in cacl solution with poly-vinyl alcohol (pva) as surfactant. particle so formed collected by centrifugation and lyophilized for dry powder formulation. pva and alginate concentration varies the size, surface and shape of the particles. formulation so prepared was delivered to c bl/ mice directly into lung by endotracheal intubations of mice. proliferation index (pi) of spleenocytes of immunized mice was measured after in-vitro stimulation with mycobacterium tuberculosis antigen. result: . % alginate, . % pva concentration gives particles with size of - micrometer as confirmed by particle size analyzer and scanning electron microscopy. viability of the mycobacterium indicus pranii was best achieved with % trehelose and . % pvp (poly vinyl pyrollidone). there was fold increase in proliferation index of spleenocytes and releases pico-gram of interferon gamma after week of immunization. formulation also induces the activation of dendritic cells after their in-vitro incubation as shown by % increase in cd and . % in ccr expression as compared to blank alginate formulation. bacterial exopolysaccharides (epss) are heterogeneous polymers containing a wide array of homo-or hetero-carbohydrates as well as organic and inorganic substituents. epss are produced by many bacteria and play a critical role in helping these microorganisms to cope with adverse environmental conditions. some epss contain sulphate groups as inorganic substituents. the presence of these groups contributes to the biological activity of epss, which have been shown to have anticoagulant, insulinotropic, antiviral, antitumoral and immunomodulatory properties, among others. b is a constitutively sulphated eps produced by halomonas maura, a recently discovered halophilic bacterium. in preliminary experiments we found that modification of its eps by adding sulphate groups to the native polymer (thus obtaining b s) resulted in vigorous antiproliferative activity in several haematopoietic tumour cell lines. at the same time we found that other epss produced by closely related strains had only a very limited antiproliferative effect on these same tumour cells. it was therefore of interest to determine whether the antiproliferative activity of b s was mediated by the induction of apoptosis, and if so, to dissect the pathway triggered by b s. by cell cycle analysis we determined that b s is able to induce apoptosis in up to % of jurkat and molt- t cell leukemias. the examination of a large panel of haematopoietic and nonhaematopoietic cell lines revealed that apoptosis induced by b s is restricted to cells of the haematopoietic lineage and that leukemic t cells are particularly sensitive to death induced by b s, but that untransformed cells are not. a time-course of caspases activation indicated that caspase is the first to be cleaved, followed by caspases and , thus suggesting that b s triggers apoptosis through the mitochondrial pathway. it is noteworthy that b s also induces vigorous apoptosis in primary leukemic t cells obtained from the peripheral blood of patients. therefore, b s may well provide a satisfactory therapeutic alternative to patients with acute t cell leukemias, since current antitumoral drugs are very inefficient in the treatment of these types of cancer. particulate antigen delivery tools have been shown to enhance the induction of immune responses by targeting dcs. polyelectrolyte microcapsules form a new class of microcapsules generated by the sequential adsorption of oppositely charged polyelectrolytes onto a sacrificial spherical template which is consequently dissolved, yielding a hollow microcapsule surrounded by a thin shell. this layer-by-layer approach allows an efficient incorporation of macromolecules under nondenaturing conditions. by using the biopolyelectrolytes dextran-sulphate and poly-l-arginine, biodegradable microcapsules can be obtained. in this study, we have chosen the lungs as a non-invasive route for vaccine delivery. as demonstrated by flow cytometry and confocal imaging, dextran-sulphate/poly-l-arginine microcapsules were readily taken up by local pulmonary apcs and transported to the mediastinal lymph nodes, making them excellent tools for antigen targeting towards apcs. microcapsule instillation also affected the pulmonary apc activation status, indicated by the emergence of an apc population expressing increased levels of mhcii, the co-stimulatory ligands cd , cd and cd , and of the inflammatory cytokines il- , il- and mcp- . using ovalbumin (ova) as a model antigen, we have analysed the adjuvant properties of these polyelectrolyte microcapsules. analysis of the alveolar infiltrate, cd t cell and antibody profiles revealed that polyelectrolyte microcapsules display different adjuvant properties than the standard th and th /th skewing adjuvants alum and complete freund's adjuvant (cfa). in response to ova aerosol exposure, microcapsule based vaccination resulted in an alveolar infiltrate dominated by monocytes, while alum and cfa respectively induced typical eosinophilic and neutrophilic inflammations. striking differences were also observed on the level of cd t cell responses. microcapsule based vaccination resulted in a marked induction of il- secreting th cells, without inducing strong th (cfa) or th (alum) responses. these differences were also reflected on the level of the humoral immune response, with microcapsules being the sole adjuvant producing antibodies of all isotypes tested (igg , igg c and ige).in conclusion, polyelectrolyte microcapsules allow an efficient targeting of antigens to lung apcs, and possess immune stimulating activities distinct from alum and cfa. due to their capacity to generate th responses, polyelectrolyte microcapsules may become interesting tools to combat fungal and bacterial infections. pneumolysin is an important virulence factor produced by virtually all clinical isolates of streptococcus pneumoniae. the protein binds to cholesterol in cell membranes and creates transmembrane pores, leading to cell lysis. published findings have proposed that, at sublytic concentrations, the toxin causes a range of effects including activation of host complement, activation and chemotaxis of cd + t cells and increased production of pro-inflammatory cytokines in immune cells. in this study we investigated the interaction of pneumolysin with murine dendritic cells (dc). we found that pneumolysin induced the activation of dc, reflected in the enhanced expression of the costimulatory molecules cd , cd and cd and mhc class ii molecules. the toxin alone was found to be a poor inducer of cytokine production by dc but it did enhance the secretion of tlr agonist-induced cytokines such as il- and tnf-a. previous published findings have shown that pneumolysin activates peritoneal macrophages in a tlr -dependent manner. however, we found that pneumolysin was capable of activating dc from both wildtype and tlr -defective c h/hej mice, by inducing cell maturation and synergising with tlr agonists to enhance cytokine secretion. importantly, we also found that pneumolysin is a strong inducer of il- b secretion by dc, through its effects on caspase- processing, which is also tlr -independent. the results suggest that pneumolysin is a potent stimulus for dendritic cell activation and that this does not require tlr signalling. objectives: transmission of immune competence from mothers to newborns during pregnancy and lactation is crucial for education of neonate immune system in order to develop optimal protection against early life infections. the objective of the present study was to assess whether maternal supplementation with probiotics may enhance neonatal responses to measles immunization. methods: pregnant balb/c mice were supplemented with placebo (maltodextrin) or probiotics (lactobacillus paracasei ncc (st ) or lactobacillus rhamnosus ncc (lpr), each at x cfu/day), suspended in the drinking water, throughout the gestation period and up to the weaning of pups. at weaning, pups were immunized with live attenuated measles vaccine (mv-s, aventis-pateur). weight evolution of pups was followed from week to week of life. fresh feces were collected at , and weeks of life for determination of iga levels (assessed by elisa). pups were bled and weeks after immunization for determination of measles-specific igg and igg a antibodies. analysis of microbiota composition (plating on semi-selective agar media) was performed on fresh feces collected one week after weaning. results: all newborns grew normally and no significant differences in the weight were observed between the groups all along the trial. fecal iga production increased progressively in all pups from weaning, reflecting a normal development status. nonetheless, feeding mothers during pregnancy and lactation did not significantly affect post-weaning s-iga production in pups. lpr supplementation of the mothers significantly potentiated post-weaning measles-specific antibody responses in pups in comparison to control group. interestingly, no significant effect was observed in the st -fed group. finally, a modification of the microbiota composition was observed in pups of supplemented mothers. particularly, there was a significant increase in lactobacilli in pups from the lpr group as compared to controls. conclusion: this study supports the benefit of perinatal intervention with probiotics during pregnancy and lactation on immune maturation in the offsprings. moreover, these first results seem indicate that the effects are strain specific. chitosan, ( - )- -amino- -deoxy-beta-d-glucan, is a deacetylated form of chitin, an abundant biodegradable, positively charged natural polysaccharide. chitosan (chi) is used for antigen delivery through mucosal barrier due to its ability to disrupt tight junctions. here we studied the ability of chitosan nanoparticles to form complexes with proteins of different size and charge. nanoparticles (chi-np) were prepared from kda chitosan by ionotropic gel formation. bovine serum albumin (bsa) and myoglobin, human immunoglobulin g and superoxidedismutase (sod), and chicken lysozyme were fitc labeled. chi-np were preincubated with proteins at : ration and washed times. after washing chi-np containing bound proteins were run by denaturating gel electrophoresis. all proteins were able to form complexes. most effective binding was shown for bsa, sod, and lysozyme. the stability of chi-np complexes with proteins was studied in vitro on macrophage cell line raw . by confocal microscopy. for this chi was labeled with rhodamine and nanoparticles were coincubated with fitc labeled proteins before addition to the cells. we showed co-localization of chi and fitc for all proteins studied. these results demonstrate that chi-np form stable complexes which are internalized by macrophages. the family euphorbiaceae consists of a large group of plants whose compounds have been documented to possess anti-inflammatory activities, however, their effects as modulators of innate or acquired immunity has not been described yet. in the present study, different aspects of the immunomodulatory activity of extracts from euphorbiaceaes on peripheral blood mononuclear cells (pbmc) from healthy individuals were evaluated. the pbmc were exposed to the extracts w/o phytohaemagglutinin a (pha), cycloheximide (chx) or lipopolysaccharide (lps) as agents that induce proliferation, apoptosis and cytokine production in pbmc. the lymphoproliferative activity of pbmc was evaluated by thymidine incorporation and cfse dilution assay using flow cytometry. the mitochondrial membrane depolarization (as an early apoptosis indicator) was measured using dioc /propidium iodide staining by flow cytometry and tnf-a secretion in the culture supernatans by elisa. we found that up to euphorbiaceae's extracts had the ability to modulate one or more of the immune parameters evaluated in this study. however, only the bark extract of croton spp. insoluble in hexane:diclorometane:methanol (hdm) and the latex extracts of euphorbia cotinifolia and euphorbia tirucalli induced strong proliferation, apoptosis and also tnf-a production in pbmc. these extracts were subfractioned by sephadex column chromatography obtaining three subfractions with enhanced activity in comparison to the crude extracts. additionally, we started with the characterization of the specific immune effects of these subfractions on pbmc. all three subfractions induced proliferation predominantly on cd + cells. these effect was also observed in isolated t cells indicating that accessory cells are not necessary for the subfractions'activity. the lymphoproliferative activity of these subfractions was also not inhibited by the carbohydrates d-(+) galactose or a-methyl-mannopyranoside. these results demonstrate the presence of immunomodulatory compounds in plants from the euphorbiaceae family and suggest an antigen-presenting cell-and carbohydrate-independent mechanism of the subfractions to exert their effects. we found significant increase on lymphocytes and eosinophils populations obtained from lps + p. acnes-treated group in relation to control group.on th day, we detected a significant negative correlation between eosinophils absolute number and fec. both il- and ige serum levels were increased on animals from group i when compared to control.the enhancement on th immune response pattern induced by lps and p. acnes treatment diminished drastically parasitic load. conclusion: our findings support the idea of the use of immunostimulant as a helminthiasis control strategy in sheep, which stimulate non-specific mechanisms of resistance and therefore can act against nematodes infections. vaccines based on partially purify populations from the organism or recombinant subunits proteins have been recently developed and are often not sufficiently immunogenic by themselves due to the lack of innate immune stimuli. indeed, current influenza a vaccines do not generate significant immunity against serological influenza a virus subtypes and would thus be ineffective in the face of a pandemic novel variant. hence adjuvant usually needs to be added to those types of vaccines. here we show that wittycell compounds significantly augment cellular and humoral immune responses to commercial seasonal influenza vaccines. experiments performed in mice showed induction of specific cd + ctl cells against conserved proteins that were accompanied by the induction of ifn-g producing cd +t cells, following single immunisation. in addition increased hi titres and higher levels of specific igg a and igg b antibodies were found even long periods after single vaccination with reduced doses of vaccines. consequently, protection from lethality was observed following challenge with homologous or heterogonous influenza viruses in vaccinated animals. this promising finding on the improvement of seasonal influenza vaccines by wittycell compounds in these preclinical studies strongly provides support for the careful evaluation in phase i clinical trials in humans. s. lindgren , , n. almqvist , a. lönnqvist , s. Östman , c. rask , e. telemo , a.e. wold university of göteborg, department of clinical bacteriology, göteborg, sweden, university of göteborg, department of rheumatology and inflammation research, göteborg, swedenobjective: dietary antigens normally evoke immunological tolerance. a prerequisite is their processing by intestinal epithelial cells, which leads to the appearance of a tolerogenic form in the serum of fed animals that confers antigen-specific tolerance when transferred to naï ve recipients. the gut microbiota may influence the handling of dietary antigens as atopic diseases have increased in western societies in parallel with reduced complexity of the infantile commensal microflora. we have observed that children neonatally colonized with s. aureus in the gut seem protected against development of food allergy. here we examine whether a s. aureus toxin affects tolerogenic processing by the intestinal epithelium. methods: mice were given s. aureus enterotoxin a (sea; . mg/ml) in the drinking water for days and, days later, mg ovalbumin per os. one hour postfeeding, serum was transferred to naï ve recipients, whose tolerance to ovalbumin was tested in a model of allergic airway inflammation (sensitization followed by intranasal challenge with ovalbumin). results: recipients of serum from sea pretreated ovalbumin-fed donors exhibited increased tolerance compared to recipients of serum from ovalbumin-fed donors not pretreated with sea. this was demonstrated as reduced ovalbumin-induced airway inflammation with diminished influx of eosinophils into the lungs and reduced antigen-induced production of interleukin- and interleukin- . examination of gut sections from sea treated donor mice revealed increased density of cd a + intraepithelial lymphocytes. our results show that sea promotes oral tolerance induction, possibly by facilitating tolerogenic processing of soluble antigens by the absorptive intestinal epithelium via activation of intraepithelial lymphocytes. abstract withdrawn by author to develop an efficient vaccine against cp pneumoniae we cloned chlamydial genes encoding proteins of the outer membrane like ompa, omcb, and pmp , proteins of the inclusion membrane like incc, secreted proteins like cpaf, and the heat shock protein groel. cpg-dna , a highly stimulatory oligonucleotide for apcs, was used as adjuvans. subcutaneous co-injection of ompa, omcb, pmp , or groel together with cpg-dna reduced the chlamdial burden in nasally infected mice. however, symptoms like substantial loss of body weight were not influenced. in contrast, a low dose infection with cp. pneumoniae almost completely prevented the loss of body weight upon challenge. to improve the efficacy of the vaccine we used poly-dl-lactide-co-glycolide microspheres loaded with the protein ompa or pmp together with cpg-dna . the microsphere based vaccine offers the advantage that antigen and adjuvans are delivered to the same apc. intranasal but not subcutaneous vaccination of mice with ompa or pmp microspheres efficiently lowered chlamydial burden upon challenge and prevented loss of body weight. pmp microspheres induced protective ifng-secreting cd + t-cells and raised pulmonary pmp -specific iga levels in vivo. also, pmp microspheres caused lower il- serum levels upon administration than the injection of pmp together with cpg-dna , indicating fewer side effects. objectives: staphylococcus (s.) aureus superantigens are highly potent t cell mitogens and the causative agents of toxic shock syndrome (tss) and food poisoning. most s. aureus have superantigens and patterns are highly variable. to date, the role of superantigens in bacteraemia is not well defined.to analyse whether superantigens play a role in bacteraemia, we investigated s. aureus strains and anti-superantigen antibody responses in cases of s. aureus bacteraemia in iv drug users and cases in nonaddicts. a rise in neutralising antibody titers indicates that superantigens are produced during infection. the study comprised iv drug users with positive s. aureus blood culture and an equal number of age-and sex-matched nonaddicts from the original fintrova and finlevo trials (ruotsalainen ).all s. aureus isolates were analysed by sequence-based genotyping (spa-typing), and multiplex-pcr was applied to determine the superantigen gene pattern. sera from patients were obtained at diagnosis (day ) and four weeks thereafter (day ). neutralising capacity of the sera was tested against the superantigen cocktail produced by the respective infecting strain as well as a panel of representative recombinant superantigens.results: genetic analysis confirmed our previous observation that most strains harboured superantigen genes, which were linked to staphylococcal lineages (holtfreter ) . there were no major differences in superantigen gene patterns in isolates from iv drug users and nonaddicts. interestingly, the staphylococcal lineage st (spa-type t , agr , and sea, seb, sek and seq) was much more prevalent among bacteraemia strains from iv drug users than from nonaddicts (p= . ).most iv drug users had neutralising antibodies against enterotoxins already at onset of bacteraemia, likely due to previous encounters with the infecting strain. we frequently observed a rise in antibody titers during infection. surprisingly patients with st strains did not show any elevations in neutralising antibody levels. conclusion: s. aureus bacteraemia induces an antibody formation against staphylococcal superantigens. this indicates that superantigens are produced during infection. however, the action of superantigens is frequently modulated by specific neutralising antibodies. this and the special behaviour of s. aureus st strains need further investigation. objectives: down syndrome (ds) is associated with recurrent infections, hematological malignancies and auto-immune diseases, suggesting immunological changes. to test for more severely disturbed specific antibody response we investigated the antibody response to the highly immunogenic protein antigen tetanus toxoid (tt), which is part of the dutch immunization program. methods: after booster vaccination at and years of age, quantitative (titer) and qualitative (avidity) tt responses were investigated in and ds children, respectively. samples were taken before and - weeks after vaccination. tt-specific igg and igg-subclass antibodies were measured in serum by quantitative enzyme linked immunosorbent assay (elisa), avidity of igg -anti-tt by an avidity elisa. the results were compared with reference values from the laboratory. results: at years, post-vaccination anti-tt-titers were decreased (geometric mean total igg, igg , igg and igg ). at years, ds children had lower postvaccination geometric mean igg anti-tt-titers only. post-vaccination igg -anti-tt avidity levels were decreased in / and / ds children at four years and nine years of age, respectively. the quantitative and qualitative anti-tt-responses in both ds groups are shifted downwards compared to the reference values. although the anti-ttresponse increases towards normal titers with increasing age, the avidity (qualitative response) is still abnormal at that age, showing that ds children have profound and lasting difficulties with specific anti-tt antibody formation. kda; , kda; , kda; , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] kda and , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] kda were expressed by a majority of examined strains independently of the associated diseases. we assume that these omps could be conservative proteins of h. pylori. conclusion: considering omps as potential targets in the search for disease-related biomarkers and potential vaccine antigens, the identification of h. pylori omps as well as the elucidation of their role in modifying the host immune responses seems to be very important research subjects. the increasing cases of severe diarrhoea and invasive lethal infections in children caused by salmonella typhimurium are a major public health problem in mexico. the rapid dissemination of multidrug-resistant s. typhimurium, and the lack of a licensed vaccine against non-typhoidal infections reduce the possibilities of an effective treatment. the objective of this study was to evaluate if the high incidence of non-typhoidal multidrug-resistant salmonella infections was associated with a reduced in anti-salmonella immunity. a cohort of families, from a mexican agricultural community with a high incidence of endemic salmonella infections, was followed prospectively for an -month period. sera were obtained from healthy subjects from the same community ( months to years of age). the highest incidence of salmonella-associated diarrhea, / , occurred in children under years of age. the lowest incidence, / , was observed in the population aged to . whereas serum from individuals ranging - years of age showed maximum igg , igg and iga anti-s. typhimurium titres, children less than years-old did not show detectable igg and igg titres and had weak igg , iga and igm antibody levels; only their igg levels were comparable to those detected in adults. moreover, the levels of igg and igg antibodies were lower in adults with a diarrheal-associated episode. interestingly, s. typhimurium yuhs - , a commonly isolated human strain from this endemic area, resisted the complement-fixing activity of antibodies although it was sensitive to opsonisation and to fc-mediated phagocytosis by human monocytes. these data contributes to define the protective immune response involved in anti-s. typhimurium immunity. diseases caused by the yeast of candida genus are a serious clinical and social problem. despite this fact, there is no effective prevention against these opportunistic pathogens yet. although c. albicans is the major cause of the mycoses ( %), the number of the multiresistant non-albicans isolates increases. c. dubliniensis, which was described only recently as serious human pathogen, belongs to the group of these resistant isolates. the surface mannan of candida cells is component of the cell wall mannoprotein complex and participates in an initial contact with its host and subsequently with the host defense mechanisms. because of complexicity of this homopolymer it is necessary to identify a subunit of the mannan that is most effectively recognized by the immune system and thus influences the specificity of the induced antibody response (immunodominant epitope). in our study we prepared oligosaccharides from acid-stable part of mannan c. dubliniensis by conventional acetolysis. this procedure specifically cleaves the a- , linked mannopyranose units of mannan backbone and releases the side oligomannosyl branches. obtained oligosaccharides were used in inhibition elisa and spr (surface plasmon resonance) measurements. the reason of these measurements was to quantify interactions of these oligosaccharides with anti-mannan antibodies present in rabbit serum after -fold immunization with mannan-hsa conjugate injections in week intervals as well with inactivated c. dubliniensis whole z. neščáková , s. bystrický institute of chemistry, slovak academy of sciences, bratislava, slovakiaour newest approach to sub-cellular vaccine against gram-negative bacterial pathogens exploits detoxified lipopolysacharide (detoxified lps) as the target antigen. this is achieved by conjugation of carbohydrate to a protein carrier which secures the t cell dependent immune response. the goal of the immunization with this conjugate is to generate the effective production of memory b cells. here we prepared subcellular conjugate with the detoxified lps from vibrio cholerae strain o using polymer carrier and a protein. the cell immunity induced by the vaccination with the conjugate was evaluated in mice, namely their peripheral blood and the spleen. activation and differentiation of b-cell populations in the time-dependent manner was determined by flow cytometry analysis of these samples. a single-platform approach based on flow cytometry and defined number of fluorospheres was used to count b cells. however in our hands this method, previously used in humans, had to be adapted for mouse blood samples first. the protocol allows quantifying cells simultaneously with cytometric immunophenotyping without cell loss or other cell preparation steps. like pan-b cell marker cd , expressed almost on all blood and tissue b cells, was used. here we investigate the characteristics and development of antibody (iso)types after secondary immunization with mencc or plain polysaccharide and the possible role of certain antibody responses in maintaining immunity after vaccination. methods: volunteers, age - years, were immunized with mencc or received a secondary immunization with mencc or plain menc ps. blood samples were obtained before and seven time-points after immunization. igg, iga, igm, igg , igg and avidity were assessed by a multiplex immunoassay. functional antibodies were determined by a serum bactericidal assay. results: high levels of antibodies were still present years after primary mencc immunization. secondary immunization resulted in increased igg and sba titers after to days. in primed individuals, igm was still present, and this only increased following a secondary immunization with plain ps. in addition, immunization with ps induced a higher igg response compared to mencc immunization. discussion: secondary immune responses are quiet slow. the composition of the ig (iso)type distribution is different between mencc and plain ps and might be of influence on functional titers. although this study indicates that immunological memory was previously induced by a single mencc vaccination, it highlights the importance to sustain protective antibody levels against a rapid invasive organism such as n. meningitidis. the immunological effectiveness of these two semi-synthetic immunogenic conjugates was established according to antigen-specific titers of igg, iga and igm isotypes and by phagocytic and respiratory metabolic activities of granulocytes. results: prime-boost immunization strategy resulted in enhanced production especially dlps-specific igg and igm isotype antibodies in both experimental groups (peak titers : ). igm-igg isotype switch was more pronounced with o-sp. peak values of dlps specific iga isotype were signicantly lower than igg and igm ones ( : vs. : ). flowcytometric simultaneous determination of phagocytosis and stimulated oxidative burst of granulocytes revealed conjugate induced enhancement, more evident with o-specific polysaccharide and ip final boost (stimulation index was . fold of normal control). subcutaneous immunization gave a weaker stimulation: . fold of normal control. the second de-oac conjugate exerted different pattern of stimulation, sc intervention was more effective. our results are indicative for immunological effectiveness of novel dlps derived glycoconjugates; thus promising further application in cholera subcellular vaccine. this work was supported by apvv - and vega / / grants of the slovak grant agencies. background: the yeast candida albicans is an opportunistic pathogen that causes infections in immunocompromised individuals with a high morbidity and mortality levels. a long-acting, effective and safe vaccine that protects against medically important candida species should significantly reduce the incidence of various forms of candidasis by these etiologic agents. mannan, polysaccharide component exposed at the most external layer of the fungal cell wall, contains a backbone consisting of a- , -linked d-mannopyranose units and many branches composed of a- , a- , and/or b- , -linked mannopyranose units that are connected to the backbone. investigation of oligosaccharides immunomodulatory functions could be considered as an important part of their protective immunity against fungal diseases. objective: in this study, for mice immunization, synthetically prepared oligosaccharide (heptamannoside) conjugated to protein carrier (bovine serum albumin, bsa) was used. methods: in order to study the immunogenicity of heptamannoside -bsa conjugate as inducer of hummoral and cell-mediated responses, balb/c mice were subcutaneously immunized without adjuvant ( mg oligosaccharide per one conjugate dose) two times in days intervals and then intraperitoneally or subcutaneously boosted. cell-mediated and humoral responses were analyzed on day after injections by flow cytometric immunophenotyping of peripheral blood leukocytes and by measuring the levels of mannan specific antibodies presented in serum using elisa. results: prepared conjugate was immunogenic and re-injection elicited increase of mannan specific serum antibodies levels. intraperitoneal boost elicited significantly higher igg and igm levels than subcutaneous boost. immunization also induced changes in proportions of major lymphocyte subpopulations in peripheral blood. introduction: bulgarian immunomodulator respivax enhances the natural resistance of organisms and specific immunity towards the most frequent respiratory pathogens. it is composed of killed bacterial bodies and lysates of six microbial species (streptococcus pneumoniae, branhamella catarrhalis, streptococcus pyogenes, haemophilus influenzae, staphylococcus aureus, klebsiella pneumoniae). the immune response in respiratory tract includes not only systemic immunity in lungs, but also balt as a part of common mucosal immune system. most animals develop balt after antigen stimulation and this tissue plays a central role in antigen uptake and local immune response regulation. therefore, immunostimulation of balt may contribute to more efficient mucosal immunity in respiratory tract. aim: to study balt development in different terms after oral application of respivax in guinea pigs. methods: male guinea pigs ( g- g) were treated orally with mg respivax five consecutive days. after the last application, on days , , , , and six animals on each term were sacrificed and lungs were removed. morphological changes were evaluated on mm thick serial sections, stained with hemalauneosin. the populations of cd , cd and b cells were identified on cryosections by using indirect peroxidase immunostaining. zio technique was used to detect intraepithelial dendritic cells (dc). results: balt was not identified in control animals. in the treated group on day subepithelial lymphocyte infiltrates and diffuse lymphocytes in lamina propria were found. the following two terms were presented by hyperplasia of lymph epithelium with massive complexes of intraepithelial lymphocytes. they were composed mainly of cd positive cells, which number reached maximum at the end of the second week. on day b cell lymphoid follicles with different size were found. lamina propria was presented by abundant lymphocyte infiltrates, composed of cd and cd positive cells. on days and the morphological reaction in the airways was reduced, characterized with small size lymphocyte accumulates. numerous intraepithelial dc's were detected in treated animals, comparing to controls in which only a few were identified. conclusion: oral administration of respivax in guinea pigs resulted in significant immunomorphological reaction in the airway mucosa presented by increased number of dc and balt development. g. gupta , s. majumdar bose institute, molecular medicine, kolkata, indiavisceral leishmaniasis (vl) caused by the protozoan parasite, leishmania donovani, is characterized by the loss of ability of the host to generate an effective immune response in the form of free radicals and proinflammatory cytokines. chemokines, particularly cc chemokines, have been shown to render protection against leishmania infection. there is no clear understanding about the immunoprotective role of cxc-chemokines in vl.in the present study, the comparative potential of cxc chemokines, interferon gamma inducible protein- (ip- ) and interleukin- (il- ) in restricting leishmania donovani infection via the release of nitric oxide (no) and proinflammatory cytokines was studied in an in vitro model. no, a crucial mediator for ip- mediated leishmanicidal activity, was found to be dependent on inducible nitric oxide synthase (inos ) expression and was linked to the mapk signaling pathway via antagonistic regulation of p mapk and erk / . further, ip- was also able to abrogate the survival of leishmania in an in vivo model of vl by restoration of th cytokines and no. thus this study strongly demonstrates that ip- , like cc chemokines, is involved in rendering a protective response in vl via upregulation of proinflammatory mediators. african trypanosomiaisis (at), known as sleeping sickness, is an orphan and extremely debilitating disease in human, cattle and domestic animals. at is caused by the protozoan trypanosoma brucei and at the present, there's no safe or efficient pharmacology intervention. the dna vaccines could be the answer for this disease by being able to induce production of igg antibodies and induce of th /th cytokines mediated by cd + t cells and activating cd + t helper cells. in this study, we shows that balb-c mice immunized intramuscularly with a single dose of plasmids encoding three antigenic candidate genes from trypanosoma brucei, named invariant surface glycoprotein (isg), trans-sialidase (tsa), and fosfolipase c (plc) are able to produce igg antibodies anti-trypanosoma. this immunization process was able to control the mortality level when mice were submitted to challenger assay with trypanosoma brucei brucei parasites. in mice co-infected with s. ratti and l. major (nl) neither clearance of l. major nor strongyloides infection was changed. mice co-infected with s. ratti and p. yoelii (nl) showed the same course of parasitaemia as single infected mice. these results suggest a strictly compartmentalized and successful immune response in both murine co-infection models, s. ratti and l. major or p. yoelii. if this compartmentalization is also observed in the antigen specific cytokine response of ex vivo prepared lymphocytes will be the topic of further investigations. in the present study ,we evaluated tsa -encoded dna vaccine against l.major in balb/c mice. igg and ifn-g values were markedly increased in the immunized group ,which were significantly higher than in the control groups (p x . ) following immunization and after challenge with leishmania major. il- values were increased in all groups, but there was no statistical difference between the groups(p g . ) following immunization and after challenge with leishmania major. the immunized mice with the dna vaccine presented a considerable reduction in diameter of lesion comparing to the control mice and indicated a significant difference was observed between the immunized and the control groups (p x . ) in this regard . the survival time of the immunized mice with the vaccine was significantly higher than the control groups (p x . ) after the challenge with leishmania major. the immunized mice had significantly lower parasite load comparing to the control mice(p x . ). the findings of this study indicated that the tsa -encoded dna vaccine increased the cellular response and induced protection against infection with leishmania in the mice. the tsa -encoded dna vaccine may be an excellent candidate for future vaccine developments against leishmania. there is a lot of evidence showing that bcg vaccination at mucosal site via intranasal, intragastric and intrarectal routes are effective in conferring protection against virulent mycobacterium and several non mycobacterial infectious diseases. in this study the protective effect of autoclaved leishmania major (alm) vaccine in combination of either rectal or subcutaneous bcg on susceptible balb/c mice was evaluated.one month after bcg vaccination, balb/c mice were immunized subcutaneously twice with alm+alum at week intervals. three weeks after booster injection, × stationary phase l. major promastigotes were inoculated subcutaneously in one footpad. immunological evaluation at before and post infectious challenge, showed strong proliferative responses in the spleen cells of the rectal immunized group after stimulating with parasite lysate. high level of interferon gamma was induced in the spleen and significant increase in the serum ratio of igg a/igg was observed only in rectal immunized group. rectal immunized mice showed comparable nitric oxide production and inos induction in peritoneal macrophages .the obtained results in rectal bcg vaccinated group showed no mortality but low parasite burden in the liver and spleen and suggested protective efficacy of intrarectal bcg immunization against leishmaniasis might be due to the long-lasting induction of type immunity. methods: two groups of balb/c mice were infected by l. tropica. one group was infected subcutaneously into the left footpad and the other group intradermally into the left ear dermis. mice were challenged by l. major in the right footpad after establishment of l. tropica infection. the immune response was evaluated at two intervals: one week and one month after challenge. single cell suspensions were prepared from draining lymph nodes of mice. cells were stimulated by phorbol myristate acetate (pma). cell surface markers and cytokine production were determined by intracellular cytokine assay using flow cytometry. the following parameters were assayed in the two experimental groups: lesion development, delayed type hypersensitivity (dth) to l. major challenge, production of gamma interferon (ifn-g) and interleukin (il- ), and cellular expression of cd and cd . results: infection through subcutaneous route in comparison to the intradermal route induces significantly higher levels of dth and ifn-g, lower levels of cd + lymphocytes, and higher protection against l. major challenges. conclusion: intradermal infection of l. tropica, in comparison to subcutaneous infection, induces significantly more protective immunity in balb/c mice. therefore, we propose the route of infection as an important variable in this experimental model. this factor should be considered for development of an appropriate experimental model for human l. tropica infections. objectives: many mammals exhibit a periparturient relaxation of immunity (ppri) to gastrointestinal nematode parasites culminating in increased worm burdens. it has been suggested that the extent of ppri may have a nutritional basis as this effect on host resistance is considerably augmented when protein supply is scarce. subsequent studies have shown that increased dietary protein intake can ameliorate this phenomenon. however, this effect is often confounded with increased food intake and thus increased energy levels. here, we aim to dissect the effects of protein and energy nutrition on the immune status and resistance to gastrointestinal nematodes in the periparturient host. the nippostrongylus brasiliensis lactating re-infected rat model was utilised as a well established model for mammalian ppri. lactating rats, re-infected with , infective n. brasiliensis larvae on day post parturition, were offered one of three levels of crude protein at one of two levels of metabolisable energy (me). parasite burdens were assessed by counting worms in the small intestine at day post secondary infection. histological counting of intestinal inflammatory cells, assessment of antibody levels and measurement of cytokine mrna levels in the mesenteric lymph nodes were carried out to assess the host immune status. results: increasing cp supply, but not increased me supply, reduced worm burdens. whilst feeding treatment did not affect eosinophil and goblet cell numbers, increased cp supply increased mucosal mast cell numbers and levels of n. brasiliensis specific antibody (total igg, ige, igg and igg a). this was independent of level of me supply. feeding regime did not affect levels of the type- cytokines il- and il- . conclusion: this study effectively demonstrates that increasing protein supply per se can decrease periparturient parasite burdens. this anti-parasitic effect correlates strongly with an upregulation of immune effector mechanisms, namely accumulation of mast cells and production of antibody. this data emphasises the role of immunonutrition in combating infectious disease. protein supplementation of periparturient mammals has considerable potential as a non-chemotherapeutic method of controlling gastrointestinal nematode parasites. background: gp is the major surface glycoprotein of leishmania that exhibits protease activity and has an important role in the biology of the parasite. the aim of this study was cloning and expression of gp of l.major strain mrho/ir/ /er. methods: l.major promastigotes were grown in rpmi supplemented with % fcs. l.major rna extraction and cdna synthesis were carried out. gp gene segment was amplified by specific primers and cloned into ptz r to construct ptz r/gp . the presence of gp into ptz r was confirmed by pcr. then, ptz r/gp was sent to determine the sequence of its nucleotides. after that the gp gene segment was sub-cloned into pet a (+) expression vector and transformed into e.coli bl (de ) plyss and gp protein was expressed in presence of mm iptg. objectives: the development of a vaccine against malaria caused by plasmodium falciparum is an urgent public health priority. influenza virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. at appropriate antigen doses, seroconversion rates of % were achieved against two synthetic malaria peptide-mimetics in malaria naï ve volunteers (genton et al., plos one, ) . the aim of this clinical trial is to proof that virosomes are a suitable delivery system for malaria peptide-mimetics in malaria semi-immune subjects. objectives include demonstration of safety and tolerability of virosome formulated malaria peptide-mimetics and determination of the humoral and cellular immune responses against these malaria peptide-mimetics. particularly, boosting of pre-existing naturally acquired anti-malaria immunity will be investigated. the study design was a single centre, randomized, controlled, double-blind, age deescalating trial including volunteers. male volunteers ( and years) for the adult group, and children of both sexes ( - years) were enrolled. subjects received virosomal formulations containing mg of ama -c (pev t), an apical membrane antigen- derived synthetic phospatidylethanolamine (pe)-peptide conjugate and ug of uk (pev t), a circumsporozoite protein derived synthetic pe-peptide conjugate. comparator groups received the influenza vaccine inflexal v. volunteers received two injections at study days , and . results: safety and tolerability defined as occurrence of local and systemic adverse events and incidence of clinically significant hematological and biochemical abnormalities are assessed. this vaccine showed a very good safety and tolerability profile in all study participants. curcumin dissolved in dmso when administered orally to p.berghei infected mice has been shown to have antimalarial activity, enabling % of the treated mice to survive till days after infection by which time all of the untreated mice had died. under such condition we found that bioavailability of curcumin was only . % of the amount fed and it remained in circulation in the blood only for minutes post feeding. we therefore prepared curcumin bound to chitosan nano particle to improve it's delivery and found that oral feeding of such particles not only increased its bioavailability to . % ( of the amount fed but it's circulation was sustained till hrs post feeding. under such conditions when mgm of curcumin bound to mgm of chitosan nano particles were fed one time daily for days post infection to plasmodium yoelii infected mice % of mice were cured and survived atleast for days without any infection and were resistant to reinfection with the same parasite. curcumin under such condition accumulated preferentially in infected erythrocytes, the quantity increasing with increase in parasitemia and fluorescence microscopy revealed that it was bound to the parasite. like chloroquine, curcumin inhibited hemozoin formation in vivo and heme polymerization in vitro in a dose dependent manner. we believe that it is one of the ways by which curcumin may be killing the parasite. among immune cells, nk and gamma-delta t cells are suspected to play a critical role in the early control of plasmodium falciparum parasitaemia and to influence malaria adaptive immunity. gamma-delta vgamma vdelta t cells, a non-conventional t cell subset specific of primates, are activated and expanded during primary p.falciparum infections in response to malaria non-peptidic phosphoantigens, and they are an important source of ifn gamma. furthermore these cells inhibit in vitro growth of p.falciparum blood stages by a granule exocytosis-dependent cytotoxic pathway and granulysin -an nk and t cell specific cytotoxic molecule has been incriminated. so far, the precise mechanism of the parasite inhibitory capacity of those cells, as well as the parasite blood stages involved remains unclear. to further investigate the anti-parasitic activity of gamma-delta t cells an rnai strategy based on a lentiviral vector approach was undertaken. we demonstrate that granulysin, but not perforin is essential for the anti-parasitic activity of gamma-delta t cells. concerning parasite blood stages, we show that both mature infected red blood cells and the free invasive form (merozoite) trigger gamma-delta degranulation and granulysin release, but noteworthy merozoites were the only stage affected by gamma-delta t cells. in addition, we also provide evidence that such a mechanism may occur in infected patients. altogether these data highlight a new mechanism by which gamma-delta t cells might directly contribute to malaria immunity opening new perspectives based on gamma-delta t cells to prevent or cure malaria. the immune system has a number of mechanisms to prevent self-destructive responses. amongst these, regulatory t cells (treg) have the ability to actively suppress effector responses. many questions surround the issue of antigen specificity of treg, since selective inhibition of only the pathogenic response, leaving the rest of the immune system intact, is the ideal therapeutic goal. the purpose of the project is to develop a model of robust, highly specific regulation operating in vivo that can be studied to understand the underlying mechanisms. such a model is provided by murine autoimmune hemolytic anemia (aiha) induced by immunisation with cross-reactive rat red blood cells (rbc). mice recover from disease due to the development of regulation with exquisite specificity, which suppresses only responses to self-epitopes whilst selectively allowing those to rat-specific determinants to be boosted. the re-establishment of tolerance is associated with the loss of t-cell proliferative responses, and emergence of il- responses, to epitopes on the dominant rbc autoantigen, anion exchanger- (ae- , or band ) protein, and protection can be transferred by injecting splenocytes from recovered mice into naï ve recipients. here we show that transfer of tolerance to naï ve recipients is dependent on ido mediated immunosuppression as mice receiving previously tolerised splenocytes under the cover of methyl tryptophan, an inhibitor of ido, were refractory to tolerance and developed hemolytic disease. induction of ido is therefore an important process in antigen-specific tolerance, and initiators of ido activity, including ctla- + regulatory t cells or soluble forms of ctla- , may also be crucial components of this regulatory pathway. consequently, this finding has important implications for our understanding of tolerance processes in autoimmune disease. objectives: it was shown alpha-fetoprotein (afp) induced immunosuppression of cell-mediated immunity in vivo. our previous work discovered afp-activated mice bone marrow hematopoietic stem cells (hscs) suppressed effector reactions of cell-mediated immunity in vitro. we investigated relationship existed between afp-induced hscs suppressor activity and immunosuppression of cell-mediated immunity during afp-produced teratocarcinoma development. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, methods of molecular biology and rna interference (rnai) were used in this work. results: as a result, there was a negative correlation (r medium =- . ) between dynamics of hscs suppressor activity elevation in spleen and inhibition of nk cells, nkt cells and cd + t cells cytotoxic activities ex vivo during tumor growth. besides, the inhibition of spontaneous and induced cytokines productions such as ifng, tnf-a and tnf-b from these types of immunocompetent cells negatively correlated with increasing of suppressor factors expression such as tgf-b (r medium =- . ) and il- (r medium =- . ) in isolated splenic hscs ex vivo. analysis of effector cd + t cells in spleen showed decrease of t h cells quantity and simultaneous t h cells number increase during teratocarcinoma development. moreover, it were found elevated numbers of cd + cd + ctla- + -and cd + cd -il- + il- regulatory t cells in spleen as well as increasing suppressor activity of isolated regulatory t cells ex vivo. number boost kinetics of t h , t h and regulatory t cells were correlated (r th =- . , r th = . and r th = . and r tr = . ) with kinetics of hscs suppressor activity level. in addition, dynamics of regulatory t cells activity were linear (r th = . and r tr = . ) to hscs suppressor activity level in spleen during tumor growth. quantities of tgf-b -and il- -produced hscs in spleen were correlated (in some cases negatively but in other positively) with cell-mediated immunity effector reactions alteration during teratocarcinoma development also. however, inhibition of afp expression by rnai caused to inhibition as immunosuppression activity of hscs and their appearance in spleen as well as normalization of cell-mediated immunity effector reactions. conclusion: thus, hscs suppression activity is correlated with changes in cell-mediated immunity during endogenous afp productions by teratocarcinoma cells and may play a role in afp-mediated dysfunction of normal immunoregulation during afp-produced tumor development. syphacia obvelata, a murine pinworm gastrointestinal nematode, is common even in well-managed animal colonies. although often considered as irrelevant, pinworm infections were shown to alter hosts' immune responses and to interfere with the experimental settings. our studies showed that naturally aquired s.obvelata infection also influences the hosts' hematopoietic responses, inducing the increased production and release of the cells of granulocyte-macrophage, as well as of erythroid lineage from the bone marrow of the infected cba mice. while the enhanced myelopoiesis compensates the increased peripheral demand for a larger supply of tissue neutrophils and macrophages, the cause of stimulated erythropoiesis is less obvious, but as infection consequence clearly underscores the disturbed and altered hematopoiesis. beside cellular changes, we also evaluated the impact of the s.obvelata on mitogen-activated protein kinases (mapk) signaling in bone marrow cells and found that infection upregulated all three mapk families, p , jnk and erk. additionally, s.obvelata enhanced the expression of mrna for the inducible nitric oxide synthase (inos). to evaluate how this pinworm infection modifies hematopoietic cells' reactivity, we also examined the influence of interleukin- , t cell-derived cytokine implicated in the regulation of hematopoiesis and inflammation, on the bone marrow cells. bone marrow myeloid and erythroid progenitors from s.obvelata-infected mice displayed altered sensitivity to il- , as compared to non-infected controls. the infection also altered the effect of il- on mapk activation by preventing its stimulating effect on p mapk. moreover, in s.obvelata-infected animals il- markedly down-regulated the expression of both inos and constitutive, endothelial (e)nos, not affecting the low basal nitrite production, which was opposite to the effect previously observed in noninfected mice, i. e. il- induced no production through the activation of both inos and enos. besides highlighting the importance of working under pinwormfree conditions when using experimental murine models for immunohematopoietic investigations, the data obtained pointed to the multiple layers of modulatory ability of this pinworm parasite and confirmed that the overall orchestration of the host response to the parasites is a complex process still being unraveled at both the cellular and molecular level. .-validation of the method: in order to determine linearity, analytical range, and reproducibility, three different sera with previously identified mc were serially diluted from ⁄ to / with a normal serum pool. .-implementation as a standard method for analysis of the mc in patients with paraproteinemia. the method showed good linearity: r g . . the analytical range was from g/l to g/l. the coefficient of variation (cv) was x % for [mc] n g/dl, and x % for [mc] x g/dl. this procedure was successfully implemented to quantify the mc in serum samples between march and february, . among these samples, we have quantified light chains, heavy chains, igd and biclonal paraproteinemias. conclusions: . we have developed a simple, reproducible and low-cost method to quantify the mc using standard analyses of serum protein electrophoresis (spe), serum albumin, and densitometric quantification of mc and albumin regions. . the procedure allows monitorization of the mc in patients at diagnosis, after therapy, and evaluation of complete remission. objectives: direct influence of alpha-fetoprotein (afp) on immunosuppressor factors synthesis as well as immunosuppressor activity of bone marrow hematopoietic stem cells (hscs) was detected in our previous work in vitro. we investigated possible role of endogenous-produced afp in induction hscs immunosuppressor activity at tumor-bearing mice. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, inhibition assay, colorimetric elisa, methods of molecular biology and rna interference (rnai) were used in this work. results: our results demonstrated afp endogenous synthesis adduced to elevation of immunosuppression activity of hscs in bone marrow. this afp effect becomes developed from day and reached plateau level after day of teratocarcinoma insertion. moreover, cd + cd cells showed in spleen and main lymph nodes from day and achieved plateau level after day of teratocarcinoma growth. however, immunosuppression activities of purified hscs from spleen and lymph nodes discovered at day and had a maximum pick at day of teratocarcinoma inoculation. besides, immunosuppression activity of hscs from spleen and lymph nodes was more than , times lower than in bone marrow in the same period of tumor development. isolated hscs from bone marrow, spleen or lymph nodes produced similar spectrum of suppressor factors such as tgf-b , il- , pge and no. inhibition of such suppressor factors lead to levelling of hscs immunosuppression activity ex vivo. kinetic of hscs quantity and activity had significant correlation (r cell number = . and r activity = . ) with afp level dynamics in blood serum. in addition, inhibition of afp expression by rnai caused to diminishing of hscs immunosuppression activity as well as hscs appearance in spleen and lymph nodes. conclusion: therefore, afp plays role not only specific inducer of hscs immunosuppression activity but also as a factor of activated hscs penetration into spleen and lymph nodes during afp-produced tumor development. cd -ligand molecules -that are powerful immunomodulators -are strongly expressed by activated platelets; membrane-associated cd l is cleaved to soluble (s)cd l. we sought to examine the levels of scd l in platelet concentrates (pcs) having led to an acute transfusion reactions (atr), and to test for its biological effect on b-lymphocytes. we recorded atr episodes that could lead to investigation of residual platelets in container. two fractions of aliquots from each pc (and controls) were prepared, one for assay of individual supernatant fractions and one of corresponding lysates of platelets; scd l -along with other products -were assayed by quantitative elisa. levels of il , cd p and pdgf-ab in pc supernatants and lysates from pcs associated with atr were similar to that in controls. supernatants of pcs associated with an atr contained higher scd l levels compared to controls, and -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd l . to examine if scd l was biologically active, we stimulated purified b cells recovered from healthy blood donors and exposed those normal b cells to supernatants and cell lysates of pcs implicated in atr, or control material, and we measured il- secretion. the il- concentration was consistently below - pg/ml in pc supernatants and lysates, and unstimulated b cells did not secrete detectable levels of il- . the addition of supernatant from atr-associated pc samples to purified b cells consistently resulted in sustained il- production over control (p x . ) at d after the onset of the culture, while -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd l (p x . ). pre-incubation of b cells with cd -blocking antibodies substantially abrogated il- secretion, unlike isotype-matched control. the partial blocking of cd binding on cd + b cells strongly suggests a potentially synergistic role in b cells for cytokines other than scd l (under investigation) and indicates a sustained role for pc-derived scd l. these data prompt us to investigate a larger series of events and controls to delineate on the one hand if certain factors can be responsible for an enhanced production of scd l by collected/stored cpa. objectives: there is accumulating evidence for a role of natural killer (nk) cells in the antitumor response against hematological malignances. nk cells exert their action by means of a large panel of structurally distinct activating receptors that recognize their ligands on target cells. analysis of activating receptor pathways in nk cells has revealed a dominant role for natural cytotoxic receptors (ncrs) and dnax-accessory molecule- (dnam- ) in the lysis of acute myeloid leukemia (aml) blasts. here, we investigate the expression of these activating receptors on nk cells from aml patients stratified by age. methods: we analyses by flow cytometry peripheral blood mononuclear cells (pbmcs) from aml patients before specific anti-leukemia therapy and healthy donors. all results were analyzed statistically using spss version . results: aml patients under years showed a significant reduction in the expression of dnam- , nkp and nkp compared with age-matched controls. both healthy individuals and aml patients older than years showed a reduction of these receptors compared with young donors. in contrast, we have found that nkp expression was increased in some patients of aml. on the other hand, the analysis of ligands for these activating receptors on leukemic cells showed a high variability that was not correlated with age or fab subtype. in addition, an inverse correlation in the expression of dnam- on nk cells and its ligand cd on aml blasts has been found in aml patients under years. to analyze if leukemia cell were involved in the modulation of these receptors we have performed in vitro cocultures of leukemic blast and healthy nk cells. the initial recognition of aml cells by nk cells may represent a crucial process to prevent tumor development. here we described for the first time, a decrease of dnam- expression on aml patients and confirm previous reports showing a significant decrease of nkp and nkp on aml-nk cells. altogether, these alterations of the major receptors involved in nk cell-mediated cytotoxicity of leukemic cells represent an important mechanism of immunoescape that may correlate with disease progression and patient survival. a. stelmaszczyk-emmel , e. gorska , u. demkow , m. wasik medical university of warsaw, department of laboratory diagnostics and clinical immunology of developmental age, warsaw, polandglucocorticosteroids are often used in leukemia treatment. their therapeutic use is limited due to several side effects. one of them is multidrug resistance phenomenon, which causes lack of patients response for treatment. dexamethasone is used in schedule of children's all-b treatment and the response on glucocorticosteroids therapy is very important. the aim of this study was to examine whether dexamethasone changes multidrug resistance of lymphoblasts in all-b and their tendency to begin apoptosis. the study involved children with all-b. bone marrow cells isolated by centrifugation on histopaque at the day of diagnosis were cultured for or hours with or without dexamethasone in concentration - m. analysis of: p-gp surface expression, p-gp function (rhodamine test), phi (carboxy-snarf) and apoptosis test (annexin-v and pi test) were performed with the use of flow cytometer coulter epics xl. for statistical analysis nonparametric wilcoxon test was used.the results showed that p-gp expression on lymphoblasts was , %± , . after hours of lymphoblasts incubation with or without dexamethasone any statistically significant changes were observed. average percentage of lymphoblasts with rhodamine efflux, which characterized p-gp activity, was , %± , . after hours of cells incubation with dexamethasone there was seen significantly higher percentage of cells able to eliminate rhodamine ( , %± , , p= , ). average phi in lymphoblasts was , ± , . acidification of cells incubated hours with dexamethasone was seen in - % percentage of cells ) . rest of lymphoblasts showed alkalization (phi - , ).the percentage of lymphoblasts in early stage of apoptosis after hours incubation with dexamethasone (annexin-v test) was higher than in control cells ( , % vs , %; p= , ). we concluded that dexamethasone does not influence surface p-gp expression on lymphoblasts of patients with all b but significantly increases activation of this protein. functional test should be performed to evaluate multidrug resistantace of leukemic cells, because surface expression of p-gp is not identical with its activity. moreover, dexamethasone alkalizes cytoplasm of lymphoblasts and induces early stage of their apoptosis. those effects may contribute to the treatment outcome. . however, small numbers of clonal pc can also be detected in the peripheral blood (pb) of the majority of patients and, in a minority of cases, mm is transformed into pc leukaemia (pcl). here, we describe that tumoral pc can express cd d/cd integrin with high or, sometimes, low affinity states, which is associated with their retention in or release from the bm, respectively. objectives: to evaluate the activation state of cd on malignant pc from bm and pb, as well as its regulation. methods: pc and active integrin expression on these cells were detected with anti-cd and anti-cd (clon huts ) moab, respectively, by flow cytometry. to study the integrin activation with divalent cations and pc index proliferation (brdu+ cells), we used short-term pc cultures ( hours). results: cd active form was expressed in the majority of normal and tumoral bm pc from healthy subjects ( . ± . , n= ) and mm patients in the early stages of the disease ( . ± . , n= ). in these cells, huts epitope was clearly upregulated by mn + . in contrast, circulating pc were almost all huts negative, and levels did not significantly augment when these cells were exposed to mn + . moreover, not only pb but also bm malignant cells from pcl patients were also huts negative and divalent cation refractory. it was also observed that pc from pcl patients showed an increased proliferative index ( . ± . brdu+ cells, n= ) in comparison to pc from mm patients in the first stages of disease ( . ± . brdu+ cells, n= ). these results suggest that the active form of cd must be expressed on pc to retain these cells within the bm environment. moreover, its downregulation is associated with increased numbers of circulating pc and disease progression. multipotent mesenchymal stem cells derived from (hucb) represent promising candidates for the development of future cellular therapy strategies. they are able to self-renew and they terminally differentiate into multiple lineages, including bone, cartilage, muscle, bone marrow, fat and other diverse connective tissues. in the first part of this study, we compared different protocols for the expansion of human mesenchymal stromal cells (hmsc) starting from diagnostic samples of bone marrow aspirates and the cord blood (cb). the protocols differed in the presence of either % fetal bovine serum (fbs) with and without fgf ,or % human platelet lysate (hpl), %hpl, ( % fbs + % hpl), ( % fbs + % hpl). we obtained a significantly better expansion with hpl, compared to cells with a selected batch of fbs and in fewer days.in the second part of this study, we focused on proteins that were differentially expressed during osteogenic, adipogenic and vascular muscular differentiation by western blotting. we compared the quality and the quantity of protein expression before and after differentiation (day ). two bm and two cb differentially expressed spots were observed between the two groups (before and after differentiation).we noted the low pourcentage of hmsc in cb samples: in ten samples, only two made msc colonies as in bm samples. we were also interested to the different coloration: osteogenic diffentiation was determined by alizarin red s staining, for adipogenic differentiation, the cells were stained with oil red o to visualize lipids droplets. background: inherited bone marrow failure syndromes (ibmfs) comprise a group of genetic disorders characterized by single or multiple cytopenias, as well as distinctive clinical features and varied molecular pathways. activation of p tumor suppressor pathway leads to cell cycle arrest and initiates apoptosis. we studied the presence of p dna (as a marker of cell cycle dysregulation); in bone marrow of children with fanconi anemia (fa) and those with acquired aplastic anemia (aaa). subjects and methods: this is a cross sectional study that involved: ) ten cases with fa diagnosed on the basis of dna breakage analysis, ) ten cases with aaa, and ) ten normal control cases. the presence of p dna was measured in both bone marrow and peripheral blood samples using a real-time quantitative pcr by taqman assay. results: p dna was demonstrated in bone marrow of % of children with fa, compared to % in children with aaa (p x . ), while, no p dna was seen in normal control. a positive correlation between dna breakage and presence of p dna was seen in bone marrow from fa (p x . , r . ). the presence of p tumor suppressor gene by real time pcr in bone marrow of fa may represent an early indicator of significant dna genetic alteration in those patients. key: cord- - dannjjm authors: nan title: research abstract program of the acvim forum denver, colorado, june – , date: - - journal: j vet intern med doi: . /j. - . . .x sha: doc_id: cord_uid: dannjjm nan clinics Ãà also see infectious disease abstracts id- -id- (thursday, june , : pm - : pm) Ãà also see pharmacology abstracts p- -p- (thursday, june , : pm - : pm) Ãà also see gasteroenterology abstracts gi- - june , hypertrophic cardiomyopathy (hcm) is the most commonly observed myocardial disease in cats. beta-blockers and calcium channel inhibitors are frequently administered drugs to cats with preclinical hcm despite the fact that neither drug category has been proven to slow disease progression or improve survival. ivabradine (procorolan s , servier, france) is a novel negative chronotropic agent used in the treatment of ischemic heart disease in people. little is known about its efficacy and safety in cats. the purpose of this study was to determine the short-term effects of ivabradine on heart rate (hr), blood pressure, left ventricular (lv) systolic and diastolic function, left atrial (la) performance, and clinical tolerance in healthy cats after repeated oral doses. ten healthy laboratory cats were involved in the present study. physical examination, systolic blood pressure measurement, and transthoracic echocardiography were performed in all cats at baseline and after oral administration ( weeks each) of ivabradine ( . mg/kg, q h) and atenolol ( . mg/cat, q h; . - . mg/kg) in a prospective, double-blind, randomized, active-control, fully crossed study. a-priori non-inferiority margins for the effects of ivabradine compared to atenolol were set at % (f . ) based on predicted clinical relevance, observer measurement variability, and in agreement with fda guidelines. variables were compared by use of -way repeated measures anova. ivabradine was clinically well tolerated with no adverse events observed. hr (ivabradine, po . ; atenolol, po . ; ivabradine vs. atenolol, p . ) and rate-pressure product (ivabradine, p o . ; atenolol, p . ; ivabradine vs. atenolol, p . ) were not different between treatments. at the dosages used, ivabradine demonstrated more favorable effects than atenolol on echocardiographic indices of left ventricular (lv) systolic and diastolic function and left atrial performance. ivabradine is non-inferior to atenolol with regard to effects on hr, rate-pressure product, lv function, la performance, and clinical tolerance. clinical studies in cats with hcm are needed to validate these findings and further assess safety. the aim of this study was to compare outcome from cpa in dogs following initial administration of either epinephrine or vasopressin during cardiopulmonary resuscitation (cpr). dogs having cpa in the er or icu of a university hospital were randomized to receive either iv epinephrine ( . - . mg/kg) or vasopressin ( . - . u/kg) in a blinded fashion immediately following establishment of iv access and again three minutes later. a standardized cpr protocol was followed. other vasopressors were not permitted during the six minute study period, at the end of the study period additional cpr interventions were at the discretion of the managing clinician. the primary end point was return of spontaneous circulation (rosc) within the study period; secondary end points included rosc at any point, survival to minutes, and survival to one hour. sixty dogs completed the study, received epinephrine and received vasopressin. rosc within six minutes was % ( vasopressin, epinephrine p . ), rosc at any time was % ( vasopressin, epinephrine p . ). survival to minutes was % ( vasopressin, epinephrine p . ), survival to one hour was % ( vasopressin, epinephrine p . ). five dogs survived to hours, one survived to hospital discharge. of animals dying after rosc, / were euthanized and / rearrested. no advantage of routine substitution of vasopressin for epinephrine was seen for rosc, a small survival advantage at one hour was seen in the group receiving epinephrine. the study also demon-strated that prospective clinical cpr research in animals is both possible and practical. three dogs were evaluated in phases. phase- : single-dose diltxr at approximately mg/kg po. phase- : same dose q h for . days. phase- : after a day wash-out the single-dose protocol was repeated using cut tablets to assess affect on extended release properties. blood pressure (bp), -lead ecg, echocardiogram, and h-ambulatory ecg were performed at baseline, and conclusion of phase- . blood samples and bp was obtained , , , , , , and h after final dosing. peak median plasma diltiazem concentrations (mcg/ml) measured by hplc for each phase were . , . , and . respectively. diltiazem concentrations were below the limit of detection in the majority of samples in phase- . median diltiazem concentration reached purported therapeutic concentrations ( . - . mcg/ml) by h post-pill in phase- and h post-pill in phase- . therapeutic concentrations were maintained for h in phase- , but only h in phase- . median bp (mmhg) was . at baseline and . at peak concentration in phase- . median heart rate (bpm) was . at baseline. h-ambulatory ecg analysis revealed the median hourly heart rate was . at baseline and during phase- . median heart rate at peak concentration in phase- was . . lack of detectable plasma diltiazem during phase- may be due to up-regulation of drug metabolism via p-glycoprotein (abcb - ) mutations. ongoing data collection and analysis will include mutation testing. adiponectin (adpn) is a cytokine produced by fat cells which has been shown to be correlated with adverse cardiac conditions in humans. in the heart, adiponectin activates several pro-survival reactions, including the ampk pathway and cox receptors, which protect the heart following ischemic injury. recent studies have shown that higher levels of adpn influence cardiac remodeling signaling, inhibiting protein synthesis and suppressing pathological cardiac growth. in humans, adpn plasma levels rise with decreased activity of the sympathetic nervous system and b-adrenergic agonists inhibit adpn at the level of gene expression. in contrast c-reactive protein (crp), a marker of systemic inflammation is elevated in humans with congestive heart failure (chf) and correlates to the severity of disease. first, we hypothesized that dogs with chf would have reduced adpn and elevated crp compared to normal dogs and that cytokine concentrations would predict severity of chf. second, we hypothesized that adpn receptor- (r ) and adpn protein would be elevated in the myocardium of chf dogs reflecting a compensatory process. we collected serum from dogs ( healthy and chf). circulating adiponectin and crp levels were quantified using a mouse/rat adiponectin elisa and a canine crp elisa. we found lower mean crp concentrations in normal dogs ( . ae . mg/ ml) than dogs with chf ( . ae . mg/ml), however, the results were not statistically significant due to the large variability seen among the chf dogs (p . ). we found greater mean adpn concentrations in normal dogs ( . ae . mg/ml) than chf dogs ( . ae . mg/ml) (p . ). in general, the greater the severity of the heart failure, the lower the level of serum adpn. when the tests the purpose of this study was to determine if there are any clinically important differences between the approaches (including devices) used in non invasive transvascular (interventional) closure of patent ductus arteriosus (pda) in dogs in our institution. initial and follow up records from all dogs (n ) that underwent attempted transvascular pda occlusion from january -december were examined. dogs were placed into groups depending on the device and route of vascular access (transvenous or transarterial). group : amplatz s canine ductal occluder (acdo) (transarterial) - dogs; group : gianturco s or mreye flipper s detachable embolization (flipper) coil (transarterial) - dogs; group : amplatzer s vascular plug (avp) (transarterial) - dogs; group : flipper coil (transvenous) - dogs. statistical comparisons were made using the kruskal-wallis test with mann-whitney tests to compare pairs of groups when significance was detected. p o . was considered significant. there was no significant difference in ages between the groups. there was a significant difference in body weight between groups with dogs receiving a coil either transarterially or transvenously (groups and ) being significantly smaller than dogs receiving an acdo or avp. this was by design since the acdo and avp cannot be used in small dogs. overall, the success rate of the total procedure (including vascular access and satisfactory pda occlusion) was high ( %) with success rates being comparable between groups ( - %). there was a significant difference in complication rate between groups (p o . ) with the acdo group having a markedly lower complication rate than the remaining groups ( % for acdo versus - % for the other groups). total fluoroscopy time ranged from - minutes (median minutes). fluoroscopy time for the transvenous method was significantly longer (median minutes; range - minutes) than in the remaining groups (median minutes; range - minutes) (p o . ). number of dogs with residual flow immediately following the procedure and hrs later was significantly less in the acdo group than in the remaining groups ( dogs from group and from group had moderate persistent flow while dog from group and from group had severe persistent flow hours after the procedure). the acdo appears superior in ease of use, complication rate, completeness of occlusion and fluoroscopy time than other devices. the remaining limiting factor with this device is patient size. until a smaller acdo device is marketed, coils remain the only choice for interventional closure in very small dogs ( o . kg). previously presented at the university of california davis, house officers seminar day. subvalvular aortic stenosis (sas) is one of the most commonly reported canine congenital heart defects and is inherited in newfoundland dogs and human beings. the golden retriever and rottweiler are breeds over-represented in dogs with subvalvular aortic stenosis; however, a genetic cause of this disease in these breeds has not been described. we performed genome wide association analysis in both normal and sas affected rottweilers and golden retrievers to identify chromosomal regions of interest that could implicate a causative mutation by high density single nucleotide polymorphism (snp) array. ( unaffected/ affected) adult golden retrievers and ( unaffected/ affected) adult rottweilers were included in this study. criteria for affected included a subcostal continuous-wave doppler aortic velocity ! . m/s and presence of a left basilar systolic ejection murmur; criteria for unaffected included a doppler aortic velocity . m/s. dna samples were obtained from anticoagulated blood. genotypes were obtained using high density ( , ) snp arrays, and genome wide association with sas was evaluated for each breed. significance cut-off was set at p  À , and all snps meeting this criterion were plotted within each breed and compared across breeds using plink. affected golden retriever data implicate the most significant region of genetic variation on chromosome at location (p .  À ; odds ratio . ) with other significant surrounding snps . affected rottweiler data also implicate the most significant region of genetic variation on chromosome at location (p .  À ; odds ratio . ) with other significant surrounding snps . other regions of statistical significance were on chromosomes and in the golden retriever and and in the rottweiler. genome wide association with subvalvular aortic stenosis in the golden retriever and rottweiler implicate overlapping chromosomal regions of interest for causative mutations on chromosome . the different secondary chromosomal regions of interest (chr , in golden retrievers and , in rottweilers) supports the known familial nature of this disease within different breeds and may suggest the presence of multiple mutations or breed specific disease modifiers. these data highlight the need for candidate gene evaluation on chromosome in golden retrievers and rottweilers with sas. heart valves share developmental signaling pathways with bone and cartilage. degenerative aortic valve disease in humans is characterized by valve stenosis and calcification. recent evidence suggests that degenerative aortic valves are undergoing pathologic processes that mimic osteogenesis. degenerative mitral valves in dogs and humans are characterized by valve regurgitation, and rarely undergo calcification. we tested the hypothesis that canine and human degenerative mitral valves might be undergoing pathologic processes that mimic chondrogenesis. to test this hypothesis, expression of bone morphogenic protein (bmp ), a chondrogenic growth factor; sox , a chondrogenic transcription factor; aggrecan, a proteoglycan abundant in cartilage; and type ii collagen were evaluated utilizing immunohistochemistry. normal canine mitral valves, different stages of canine degenerative mitral valves (early, intermediate, and late), and late-stage human degenerative mitral valves were studied. canine and human degenerative mitral valves showed focal areas that co-expressed all four markers of chondrogenic signaling and phenotype. valve interstitial cells and surrounding extracellular matrix in these focal areas adopted a morphologic appearance reminiscent of cartilage. focal chondrogenesis was present in all stages of canine degenerative mitral valves, but not normal canine mitral valves. focal areas of chondrogenesis did not coincide with nodular areas of glycosaminoglycan accumulation on the leaflet edge, but rather seemed to occur at points of chordae attachment to leaflets. in conclusion, canine and human degenerative mitral valves undergo pathologic processes that mimic chondrogenesis. this finding suggests that mitral valve degeneration may be recapitulating developmental signaling pathways shared by heart valves and cartilage. the triggering events for chondrogenesis in mitral valves remain unknown; as does the reason why aortic and mitral valves appear to be undergoing different pathologic processes. the fact that humans exhibit degeneration of both the aortic and mitral valve, and that dogs commonly exhibit only the latter could eventually provide insight into both processes. arrhythmogenic right ventricular cardiomyopathy (arvc) is a familial cardiomyopathy characterized by right ventricular fibrofatty infiltration and ventricular ectopy of left bundle branch block morphology (vpc) . a deletion in the striatin gene has been associated with arvc in at least some boxer families. syncope and sudden death (sd) occur in some affected dogs, although many affected dogs survive for years. the objective of this study was to define clinical characteristics of arvc in boxers that experienced sd, and compare them to those of a contemporaneous group of arvc boxers that had not died suddenly (nsd). data for both groups were collected from adult boxers enrolled in a long term prospective study of arvc in which echocardiograms and hour ambulatory ecg (aecg) are evaluated annually. aecg are quantitated for vpc numbers and arrhythmia grade ( - ). arvc diagnosis requires at least vpcs/ hours in the absence of other disease. forty three adult boxers that entered the study had died suddenly at the time of analysis (sd defined as the absence of observed clinical signs within hours prior to an unexpected and unexplained death). striatin genotype was available for of the sd dogs ( heterozygotes, homozygotes); were female ( intact) and were male ( intact). sd occurred at a mean age of years (range, - ); sd dogs ( %) had no prior history of syncope. twelve sd dogs ( %) were on antiarrhythmics at the time of death (metop-p oooooooooooprolol ( ), sotalol ( ), amiodarone ( ), procainamide ( ), mexiletine & atenolol ( ), atenolol ( )). eleven sd dogs ( %) had decreased myocardial systolic function defined by a shortening fraction (%fs) o % (range - , mean ) on the most recent echocardiogram prior to sd. median vpcs/ hours on annual aecg was , (range - , ) with a median arrhythmia grade of (range - ). twenty one contemporaneously entered arvc boxers that had survived to at least the median age of the sd group with nsd were available for comparison; / were genotyped ( heterozygous, homozygous, negative), were female ( intact) and male ( intact). twelve nsd dogs ( %) had no prior history of syncope. median nsd group age was years (range, - ); / ( %) were on an antiarrhythmics (sotalol ( ), mexiletine & sotalol ( ), mexiletine & atenolol ( )). one nsd dog had decreased %fs (nsd group %fs range - , mean ). the nsd median number of vpcs was , (range - , ), median arrhythmia grade was (range - ). striatin genotype was significantly associated with sd. no significant differences were found between groups with respect to vpc numbers or arrhythmia grade. shortening fraction was significantly lower in the sd group (p o . ). sd in arvc appears to be associated with the presence of the striatin mutation and reduced % fs, it does not appear to be associated with number of vpcs or arrhythmia grade. coughing in the small breed dog may be related to cardiac causes associated with myxomatous mitral valve degeneration (mmvd) including pulmonary edema and compression of the mainstem bronchus by a severely enlarged left atrium, or due to respiratory causes such as tracheal and/or bronchial collapse or chronic bronchitis. the purpose of this study was to evaluate the association between left atrial enlargement and large airway collapse in dogs with mmvd and chronic cough. we hypothesized that airway collapse was independent of degree of left atrial enlargement. twelve dogs with mmvd and a chronic cough in the absence of congestive heart failure were prospectively evaluated with thoracic and cervical radiography, echocardiography, fluoroscopy, bronchoscopy and bronchoalveolar lavage (bal). group dogs (n ) had moderate to severe left atrial enlargement based on an echocardiographically calculated left atrial:aortic surface area [la:ao(a)] . group dogs (n ) had no to mild left atrial enlargement [la:ao(a) ]. the site and severity of airway collapse was graded on bronchoscopy and bal cytology was assessed for evidence of inflammation or infection. the occurrence of bronchoscopic abnormalities was compared between groups using fisher's exact test. p o . was considered significant. age and body weight did not differ between groups. left atrial size was interpreted radiographically as moderately to severely enlarged in of dogs in group and as moderately enlarged in of dogs in group . fluoroscopy revealed variable degrees of airway collapse during normal respiration and induced cough in both groups. radiography and fluoroscopy were not accurate in identifying site and degree of collapse in either group when compared to bronchoscopy. cervical tracheal collapse was identified during bronchoscopy in both group ( of ) and group ( of ) dogs but was subjectively less severe in group dogs. bronchial collapse % was evident at multiple sites in both groups of dogs with no difference between groups. all dogs had suppurative and/or lymphocytic inflammation on airway cytology. infection was not present in either group of dogs, although non-specific light bacterial growth was detected in of group dogs and of group dogs (p . ). preliminary results failed to identify an association between left atrial enlargement and airway collapse in dogs with mmvd but did suggest that airway inflammation is common in affected dogs. further studies are needed to identify factors contributing to airway collapse in dogs with and without mmvd. atenolol is often used empirically in cats with asymptomatic hcm, even though clinical and experimental evidence of efficacy is lacking. cardiac biomarkers play a critical role in the early detection of subclinical cardiac disease, in the prediction of long-term prognosis, and in monitoring the response to therapy in humans. we hypothesized that serum concentrations of the biomarkers, nterminal pro-brain natriuretic peptide (nt-probnp) and cardiac troponin i (ctni), would improve following the chronic administration of atenolol po to asymptomatic cats with hcm. six maine coon or maine coon cross cats with severe hcm from the research colony at ucdavis were administered atenolol ( . mg po twice a day) for days. no cat had severe left ventricular dynamic outflow tract obstruction due to systolic anterior motion of the mitral valve. the concentrations of nt-probnp and ctni were assayed prior to drug administration and on the last day of drug administration. there was no statistically significant difference identified in nt-probnp [median before: pmol/l (range: - pmol/l), median after: pmol/l (range: - pmol/l); p . ] or ctni [median before: . ng/ml (range: . - . ng/ml), median after: . ng/ml (range: . - . ng/ml); p . ] concentrations before and after drug administration using the wilcoxon matched pairs test. the ctni finding suggests that atenolol does not reduce chronic myocyte death in cats with hcm. the lack of improvement in nt-probnp suggests that atenolol does not improve myocardial wall stress in cats with hcm. a clinical trial is warranted to confirm or refute the findings from this study. therefore, leptin-gene expression was investigated in blood samples of dogs with congestive heart failure (chf; n ) in comparison to dogs presented for cardiac screening (n ) without abnormalities. additionally myocardial samples (interventricular septum, right and left atrium and ventricle) of dogs with no cardiac abnormalities (controls), seven dogs with acquired and three with congenital cardiac diseases were investigated using quantitative rt-pcr. leptin blood levels were significantly higher in dogs with chf in comparison to dogs without diseases (p . ). there was an association with gender with higher myocardial leptin levels in female dogs with cardiac diseases (p . ). differences between cardiac regions were present (p o . ) and cardiac diseases resulted in an increase in atrial leptin levels in both sexes (p . ). interestingly, a significant reduction of myocardial leptin was present in dogs with congenital cardiac diseases (p . ), whereas acquired cardiac diseases resulted in an increase in leptin (p . ) in comparison to controls. these results suggest that the heart might be a target of leptin action in the dog and myocardial leptin production might play a role in regulating cardiac function in an auto-and paracrine manner. predicting risk of chf in asymptomatic dogs with mitral valve disease (mvd) is challenging. we examined ability of nt-probnp to identify asymptomatic dogs at high risk for chf. dogs with isachc- b (la:ao . ) were prospectively recruited; dogs with current or previous chf or diuretic therapy were excluded. physical examination, radiography, echocardiography, and nt-probnp were performed at - mo intervals for dogs (median follow-up d, range, - d). thirty-one patients reached a study endpoint of radiographic pulmonary edema; remained asymptomatic. parameters from the visit immediately previous to onset of chf (future-chf) or prior to the most recent visit without chf (remain-asympt) were analyzed. median nt-probnp of future-chf ( pmol/lpmol/l, iqr - ) was significantly different from remain-asympt ( pmol/l, - ; p . ). median time to chf of future-chf was d (iqr, . groups also differed in median la:ao (future-chf . [ . - . ]; remain-asympt . [ . - . ], p . ); vhs (future- ]; remain-asympt . [ . - . ], p . ); and lvidd:ao ]; remain-asympt . [ . - . ], p . ). roc analysis to predict if chf would occur prior to the next visit yielded auc . ( %ci, . - . ). sensitivity was . % or . % and specificity . % or . % for nt-probnp pmol/l or pmol/l, respectively. mean increase in nt-probnp between penultimate visit and two visits prior to endpoint: future-chf . pmol/l vs. remain-asympt . pmol/l. within mo, . %, . %, . %, and . % of dogs with nt-probnp o , , and pmol/l developed chf. nt-probnp and heart size helped assess risk of chf in asymptomatic mvd. increasing the assay's upper limit of detection would likely improve utility of nt-probnp. piiinp is a serum biomarker of collagen biosynthesis and is described as a marker of myocardial fibrosis in human patients. we hypothesised that piiinp concentrations would vary according to the degree of remodelling demonstrable in dogs with naturallyoccurring myxomatous mitral valve disease (mmvd). serum piiinp concentrations (mg/ml) were measured in dogs with mmvd and healthy controls using a validated commerciallyavailable radioimmunoassay. results are reported as (mean ae sd). non-normally distributed variables were logarithmically transformed. comparisons of continuous variables were made between groups using t-tests and one-way anovas with tukey's post-hoc comparisons. univariable analyses were used to evaluate associations between piiinp and clinical characteristics (age, breed [cavalier king charles spaniel (ckcs) yes/ no], sex, weight, heart rate [measured from ecg], treatment with acei [yes/ no], treatment with diuretics [yes/ no] and echocardiographic measurements [la/ao, lvedd/ lvfwd, lveddn, lvesdn]). multivariable analysis was initially performed with all dogs included and then repeated excluding all dogs receiving therapy. dogs with mmvd were divided into those with no cardiomegaly (nc) (la/ao o . and lveddn o . ), those with cardiomegaly (la/ao ! . and/ or lveddn ! . ) but no clinical signs (c) and those dogs with cardiomegaly requiring treatment for congestive heart failure (chf). one hundred and fifty-four dogs with mmvd and control dogs were studied. there was no difference in age (p . ) or weight (p . ) between the mmvd and control groups. there was a significant difference in serum piiinp (p . ) between normal ( . ae . ), nc ( . ae . ), c ( . ae . ) and chf ( . ae . ) groups. post-hoc comparisons demonstrated a difference between nc and chf groups (p . ). there was no difference in serum piiinp between genders (p . ). in the univariable analysis ckcs (yes/ no) (p . ) was positively associated with serum piiinp. age (p o . ), log (la/ao) (p . ) and lveddn (p . ) were negatively associated with serum piiinp. in the multivariable model including all dogs, lveddn (p o . , b À . ( %ci À . to À . )), age (p . , b À . ( %ci À . to À . )) and ckcs (yes/ no) (p . , b . ( %ci . to . )) were independently associated with serum piiinp. in the multivariable model including only dogs not receiving therapy (n ), lveddn (p . , b À . ( %ci À . to À . )), age (p . , b À . ( %ci À . to À . )) and ckcs (yes/ no) (p . , b . ( %ci . to . )) were independently associated with serum piiinp. in conclusion, serum piiinp decreases with age and with increasing lveddn. ckcs have higher serum piiinp measurements independent of age and lveddn, which may reflect a difference in collagen turnover in this breed. left atrial (la) chamber dilation and congestive heart failure (chf) are common consequences of cardiac conditions in cats. in some cases chf is manifest as right-sided chf (r-chf) or pleural effusion, in other cases chf manifests as left-sided chf (l-chf) or pulmonary edema. it is not always readily apparent as to which cats will develop what form of chf. a general impression has been that la enlargement is associated with the average burden of elevated filling pressures, but little attention has been paid to the function of the la chamber itself. since chf is classically preceded by abnormal atrial chamber dilation and alterations in atrial chamber function, we want to understand how these changes may help us manage or predict chf in the cat. we hypothesized that cats manifesting r-chf have la failure with the la acting primarily as a conduit, resulting in greater pulmonary hypertension, whereas l-chf cats maintain some booster pump and reservoir function. we measured la maximum and minimum areas from right parasternal long axis four-chamber views on d echo, and la m-mode dimensions at maximum, minimum, and beginning of atrial contraction. la area change, fractional shortening, active emptying fraction, and expansion index were calculated from these measurements. right ventricular internal diastolic diameter was also measured on m-mode views. preliminary data revealed that maximum left atrial size is not significantly different between r-chf and l-chf cats on d or m-mode measurements due to high variability. however, total left atrial fractional shortening is significantly reduced in r-chf cats ( . % ae . ) compared to l-chf cats ( . % ae . )(p . ), and r-chf cats have reduced left atrial active emptying fraction ( . % ae . ) as compared to l-chf cats ( . % ae )(p o . ). left atrial expansion ability is poorer in r-chf cats ( . % ae . ) than in l-chf cats ( . % ae )(p . ). these findings may suggest that atrial stiffness and poorer atrial function is associated with a greater degree of pulmonary venous and thus secondary pulmonary arterial hypertension resulting in pleural effusion (r-chf). right ventricular diameter on m-mode was increased in r-chf cats ( . mm ae . ) when compared to l-chf cats ( . mm ae . )(p . ) and normal cats ( . mm ae . )(p . ), which may also be evidence for a greater degree of pulmonary arterial hypertension in these cats. episodic weakness and syncope are common in boxer dogs. reported causes include rapid ventricular tachycardia (vt) and exertion-excitement triggered neurally-mediated bradycardia (nmb) .the purpose of this retrospective study is to describe the features of presumed nmb in boxers. to be included in the study, each dog must have been overtly healthy with a history of exertion-excitement triggered syncope or presyncope; had a normal echocardiogram (ec); had absence of vt and fewer than ventricular premature complexes (vpc) on an initial and subsequent hour holter recordings; and been alive and overtly healthy for at least six months following the initial evaluation. a total of boxers were identified. sixteen were male and were female. most ( %) dogs were either less than or more than years of age. most dogs had multiple, but infrequent, episodes and heart rhythm was documented at the time of an episode in only ( %) and only once (bradycardia) on the first holter recording. owners were instructed to attempt to precipitate episodes. bradycardia related episodes were subsequently recorded in : during the nd ( ), rd ( ) or th ( ) day of hour holter recordings and during the th day of an event recording ( ). collapse and bradycardia were documented during auscultation in additional dogs. the heart rate during syncope was never documented in ( %) dogs. a presumptive diagnosis of nmb was based on the absence of initial and follow-up of ec abnormalities and the presence of no or few vpc during extended ecg monitoring. multiple holter recordings ( - hours) were performed in of ( %) dogs and event monitoring of days ( ) and days ( ) was performed in additional dogs. in conclusion, documentation of the heart rhythm during episodes of collapse was difficult, accomplished in only % and was unlikely during the first holter recording. in boxers with suspected nmb, extended ecg monitoring and implantable loop recorders may be best for hr documentation. arrhythmogenic right ventricular cardiomyopathy (arvc) is an inherited myocardial disease with high prevalence in the boxer dog population, and is associated with sustained monomorphic ventricular tachycardia, sudden cardiac death, and replacement of myocardium with fatty or fibro-fatty tissue. though several genes have been linked to the disease both in humans and in boxers, the etiology of arvc is still unclear. several mechanisms for the development of arvc have been suggested, including dysfunction of the canonical wnt pathway, which results in an arvc phenotype in the mouse. the canonical wnt pathway has been linked to many cellular functions, including growth and differentiation of adipocytes. with the recent discovery that the gene encoding striatin, a protein involved in wnt signaling, may be involved in the development of boxer arvc, we hypothesized that changes in the wnt pathway may also play a role in the etiology. here, we show changes in the localization and decreased amount of proteins affiliated with the canonical wnt pathway. afflicted boxers were identified by -hour holter monitoring and histopathological examination of the heart. samples from the right ventricle rv) of arvc afflicted boxers, and unafflicted dogs ( beagle, mongrels, and german shepherds) were collected, fixed in % formalin, processed, treated with antibodies recognizingcatenin, striatin, and calnexin, and examined using confocal microscopy. western blots were performed on unafflicted rv samples, and arvc afflicted rv samples. frozen tissue samples were homogenized in laemmli buffer, and mg of protein was loaded into each well of a - % gradient gel. -catenin, an integral modulator of the wnt pathway, and striatin were colocalized with the endoplasmic reticulum (er) marker, calnexin. in the unafflicted animals, -catenin localized at sites of cell-to-cell apposition, and striatin localized in a diffuse intracellular pattern, with no detectable localization in the er. in contrast, in the arvc boxers, bothcatenin and striatin were colocalized with calnexin in an er pattern. in the afflicted samples, -catenin and striatin were not visualized to the intercalated disc and intracellular space, respectively. western blots of striatin and -catenin revealed no changes in the amount of protein. interestingly, a western blot for the wnt protein revealed a decrease in the amount of protein in arvc samples, compared to unafflicted samples. our preliminary data suggest that disturbances of the canonical wnt pathway may play an etiological role in the development of arvc in the boxer dog. there are numerous benefits of omega- fatty acid supplementation in human heart disease, including reduction in arrhythmias, decreased incidence of sudden death, and improved survival in heart failure. antithrombotic effects of omega- fatty acids have been demonstrated in people, which may have particular benefit in cats given their risk of thromboembolic complications with cardiac disease. benefits also have been found in canine heart disease, and reduced serum concentrations of the omega- fatty acids, eicosapentaenoic acid (epa) and docosahexaenoic acid (dha) have been found in dogs with congestive heart failure (chf) secondary to dcm. to the authors' knowledge, no studies to date have investigated fatty acid concentrations in cats with cardiomyopathy. the purpose of this study was to measure serum fatty acid concentrations in normal cats and cats with cardiomyopathy. serum fatty acid concentrations were measured in normal cats and cats with cardiomyopathy using gas chromatography. cats with cardiomyopathy and at least mild left atrial (la) enlargement (la to aortic ratio . on two-dimensional echocardiography from a right parasternal short axis view) were candidates for study. normal cats had a normal history, physical examination, echocardiogram, packed cell volume, total solids and platelet count. cats with evidence of other systemic disease or those receiving anticoagulants were excluded from the study. normally distributed and skewed data were compared between the cardiomyopathy and control groups with independent t tests or mann whitney u tests, respectively. statistical significance was set at p o . . thirty cats with cardiomyopathy ( neutered males and neutered females) and healthy controls ( neutered males and neutered females) were enrolled. median age was yr (range, - yr) in the cardiomyopathy group and yr (range, - yr) in the control group (p . ). cats in the cardiomyopathy group were classified in the international small animal cardiac health council stage b (n ), (n ), a (n ) and b (n ). compared to control cats, cardiomyopathic cats had higher concentrations of palmitic acid (p . ) and dha (p o . ), and lower concentrations of linoleic acid (p . ). among cats with cardiomyopathy, there was no significant correlation between any serum fatty acid concentration and left atrial size or age. these findings warrant further investigation into the role of fatty acids in cats with cardiac disease. platelet mapping is an application of thromboelastography that relies on the generation of at least three tracings: ma thrombin (maximum platelet activity),ma fibrin (fibrin activity only), an-dma aa or adp (platelet activity not inhibited by arachidonic acid or adp receptor antagonists, respectively). using these three tracings, the % inhibition of platelets can be calculated. the purpose of this study was to evaluate the platelet mapping assay in normal cats and assess platelet inhibition in cats receiving clopidogrel. employee-owned cats with normal history, physical exam, echocardiogram, thromboelastography, packed cell volume, total solids and platelet count were eligible. clopidogrel ( . mg po q h) was administered for days with platelet mapping performed on days and . platelet mapping values were compared using a paired t test, with significance set at p o . . seven cats ( fs, cm, aged - years) were enrolled. compared to day , ma adp (p o . ) and ma fibrin (p o . ) were lower on day . the latter unexpected result prompted measurement of fibrinogen concentrations at day and in the last of these cats. fibrinogen was not different from day to in these cats. these results suggest that platelet mapping may be a simple, outpatient clinical tool to measure antiplatelet activity in cats receiving clopidogrel. this clopidogrel dose resulted in significant platelet inhibition as measured by ma adp in all cats. further studies correlating antiplatelet effects measured by platelet mapping with clinical outcomes in cats with cardiomyopathy are warranted. this study investigated the hemodynamic effects of application of an itd in a canine model of cardiopulmonary arrest. laboratory beagles which were part of a separate terminal study were anesthetized and instrumented for continuous measurement and recording of right atrial pressure, arterial pressure and carotid blood flow. following euthanasia, cpr was performed for one minute, then a pause of one minute followed by a second one minute period at a compression rate of - /minute, ventilation with % oxygen was delivered at eight breaths/min and ml/kg tidal volume. cpr was performed with the itd in place (itd-cpr) and without the itd (s-cpr) for one period each in a randomized fashion with the rescuer blinded to its application. baseline, s-cpr and itd-cpr data were assessed for normality, a kruskal-wallis one-way anova on ranks was used (baseline v cpr). when appropriate a pairwise multiple comparison procedure (dunn's method) was used. percentage of baseline s-cpr v itd-cpr was assessed using the student t-test. the right atrial diastolic pressure was significantly more negative with the itd attached than without (p . ), the coronary perfusion pressure and carotid blood flow were significantly higher during cpr with the itd than standard cpr (p . , p . ). no significant differences in diastolic, mean or systolic arterial pressure or end tidal co were seen. application of the itd resulted in significantly improved hemodynamics during cpr in dogs. clinical evaluation of the device is warranted to examine whether this translates into improved success in resuscitation and survival. left ventricular (lv) systolic dysfunction is a common problem in dogs and can be due to a variety of etiologies. one potential etiology for systolic dysfunction is persistent or paroxysmal tachyarrhythmias, leading to tachycardia-induced cardiomyopathy (ticm). in humans, ticm carries a relatively good prognosis in that remodeling may be reversible with normalization of heart rate. differentiating between primary and secondary tachyarrhythmias in dogs with systolic dysfunction is critical for prognostic purposes as primary tachyarrhythmias may be associated with a better outcome. the goal of our study was to describe a population of dogs with ticm and to determine if treatment of arrhythmias was associated with reversible cardiac remodeling as indicated by standard echocardiographic parameters. medical records of dogs referred to the cardiology service of ksu vmth from to were reviewed. ticm was defined as the presence of severe tachyarrhythmias that were reversible with treatment, systolic dysfunction or ventricular enlargement that improved with treatment of the arrhythmia, or dogs with severe tachyarrhythmias and systolic dysfunction of breeds with that are atypical for idiopathic dilated cardiomyopathy. exclusion criteria were dogs with congenital heart disease, severe mitral regurgitation, and endocarditis. transthoracic echocardiography, thoracic radiographs and electrocardiography (ecg) were performed in all dogs. ventricular enlargement and systolic dysfunction were defined according to standard echocardiographic parameters. arrhythmias were confirmed with a holter monitor in dogs. a total of dogs were included in the study. mean age was years (range - years) with males ( intact, castrated) and females ( spayed, intact). four dogs had pulmonary venous congestion or pulmonary edema and two dogs had ascites. at initial presentation, the meanaesd values were as follows: heart rate ae bpm, m-mode fractional shortening (fs) . ae . %, ejection fraction (ef) using the area-length method . ae . %, and left atrial to aortic root ratio (la/ao) . ae . . initial meanaesd m-mode derived lv internal dimensions corrected for body weight were as follows: diastolic . ae . and systolic . ae . . at least one of the following tachyarrhythmias were identified in each dog: atrial fibrillation ( ), supraventricular tachycardia ( ), junctional tachycardia ( ), and ventricular arrhythmias ( ). ten dogs were available for follow up. seven dogs improved in at least one of the following parameters: resolution of tachyarrhythmia ( ), improved systolic function ( ) antidiuretic hormone (adh) has been shown to be elevated in humans with congestive heart failure (chf). recently, antidiuretic hormone antagonists were successful during investigational treatment of refractory congestive heart failure in humans. adh levels have been only modestly investigated in dogs with cardiac disease, primarily due to the technical difficulty in measuring adh levels via radioimmunoassay. based on the homologous structure of canine and human adh, we aimed first to determine the feasibility of measuring adh in dog plasma using a human elisa kit, and secondly to investigate the level of adh in dogs with congestive heart failure due to acquired cardiac disease. elisa assay kit validation was performed using six healthy dogs with normal clinical and echocardiographic examinations. pooled canine plasma was spiked with synthetic adh and intra-assay precision, dilutional parallelism, and linearity were assessed. to address the second aim of the study, samples were collected from normal dogs and dogs with heart failure due to one of two types of acquired cardiac disease: chronic degenerative valve disease (cdvd) or dilated cardiomyopathy (dcm). patients underwent clinical, radiographic, and echocardiographic examination to confirm diagnosis, assess severity of disease, and determine presence of pulmonary edema. whole blood was collected into edta tubes containing protease inhibitors, cold centrifuged, and plasma was stored at À until analysis. following ether extraction, plasma adh in each sample was measured in duplicate using a human elisa kit. statistical analysis included a d-agostino and pearson test for normality; group results were compared using a nonparametric mann-whitney test. adh was measured in canine plasma using the human elisa kit with acceptable intra-assay precision, linearity, and dilutional parallelism. intra-assay coefficient of variation was %. twenty-four dogs were recruited for the second phase of the study. six normal dogs and twelve dogs with radiographic evidence of pulmonary edema due to either cdvd or dcm were selected for participation. the remaining six dogs were excluded due to lack of definitive radiographic evidence of congestive heart failure. median adh values were . ae . pg/ml for the normal group (n ) and . ae . pg/ml for the heart failure group (n ). median adh values for the two groups were statistically different (p . ). our preliminary results indicate that measuring canine adh using a human elisa kit is feasible and provides results with an acceptable coefficient of variation. we also showed that dogs with congestive heart failure due to cdvd and dcm have elevated adh levels in comparison to normal dogs. our findings motivate further investigation to assess the degree of plasma adh level elevation and the possible use of adh antagonists as an adjunct treatment for refractory congestive heart failure in dogs. aortic thromboembolism (ate) occurs in both cats and dogs. whereas ate in cats is strongly associated with structural heart disease and typically has an acute catastrophic presentation; the pathogenesis and presentation of ate in dogs is less well known or understood. further, an effective antithrombotic strategy for ate in dogs has not been reported. medical records of dogs diagnosed with ate between and were examined retrospectively. diagnosis of ate was based on ultrasonography, doppler flow studies, and diminished or absent femoral pulses. dogs were treated with various acute and chronic antithrombotic therapies. the severity of ambulatory dysfunction was graded as none, mild, moderate, severe, or non-ambulatory at presentation and after therapy. a cohort of dogs in this study received a standardized protocol of chronic warfarin therapy with or without antiplatelet drugs. target international normalized ratio for warfarin therapy was to . twenty-six dogs were diagnosed with ate. all had an apparent mural aortic thrombus caudal to the renal arteries with most having evidence of embolization to the iliac and femoral arteries. none had structural heart disease at diagnosis. twenty dogs ( %) were still ambulatory at diagnosis. the median duration of ambulatory dysfunction prior to presentation was . weeks (range day - weeks). a majority of dogs ( %) had no concurrent conditions at diagnosis. nine dogs ( %) had protein-losing nephropathy. four dogs ( %) were hypothyroid. fourteen dogs were treated with a standard warfarin protocol for a median period of . months (range . - months). eight dogs were treated concurrently with aspirin, dogs were treated concurrently with clopidogrel, and dogs were treated with warfarin only. ambulatory function improved between and grades in dogs treated with chronic warfarin. the median period until clinical improvement was . days (range - days). two dogs treated with chronic warfarin therapy had documented resolution of ate, and dogs had complete resolution of ambulatory dysfunction. none of the dogs treated with chronic warfarin became nonambulatory, died, or underwent euthanasia because of ate. the median period of freedom from an adverse event was . months. no serious hemorrhagic events were reported. four dogs were treated with tpa. three of these had an unfavorable outcome. two dogs were ambulatory before tpa and become non-ambulatory after treatment. two dogs underwent surgical thrombectomy. one had a favorable outcome until ate recurred months after surgery. in conclusion, the pathogenesis of ate in dogs is not associated with structural heart disease or left atrial thrombus formation. the presentation tends to be for chronic ambulatory dysfunction. most dogs are still ambulatory at presentation. warfarin, with or without concurrent antiplatelet therapy, is an effective antithrombotic treatment strategy for dogs with ate. information is known. through previous work, investigators have encountered norfolk terriers (nt) with echocardiographically apparent dmvd in the absence of a heart murmur. in order to more fully understand dmvd in this breed of dog, we sought to characterize findings from the physical and echocardiographic exam, biochemical, biomarker, and nutritional profile, and select environmental variables from a cohort of apparently healthy nt. overtly healthy nt ! yrs old were recruited by different veterinary hospitals and underwent historical, physical, ecg, and d/color-flow doppler echocardiographic exam. anterior mitral valve length, maximal thickness, area, and prolapse were measured from d images. presence of dmvd was defined as thickened, prolapsing, or flail mitral valve leaflets in the presence of color flow doppler evidence of mitral regurgitation. blood samples were obtained for serum biochemistry and serotonin, plasma nt-probnp, amino acid profile, c-reactive protein, and cardiac troponin-i. forty-eight dogs were entered into the study (median age, yrs iqr [ - ]; gender, f, m; median bcs, ). of the dogs, ( %) had murmurs, ( %) had mid-systolic clicks, ( %) had ecg p-pulmonale, and ( %) were deemed to have echocardiographic evidence of dmvd, including nt without a murmur. seven ( %), ( %), and ( %) dogs were classified as isachc , a, and b, respectively. mean indexed echocardiographic mitral leaflet length (p o . ), thickness (p . ), prolapse (p . ), and la:aod (p . ) were significantly different between isachc a/b vs . between isachc a/ b and , there were no differences in serum amino acids, c-reactive protein, troponin, diet, or environmental factors; however different amino acids (ala, gly, phe, pro, trp, tyr) were significantly higher in isachc b vs. a. median serum serotonin was increased in dogs with a/b vs. (p . ). dogs whose diets contained some canned food (p . ) and dogs residing in suburban environments (p . ) had higher serotonin concentrations. nt-probnp tended (p . ) to be higher in isachc a/ b vs. . dmvd appears to be relatively common in nt and echocardiographic changes consistent with mild dmvd can be seen in dogs without a heart murmur. the results of this study establish a foundation of useful information upon which additional prospective studies can be developed. left ventricle (lv) evaluation is one of the most important contributions of echocardiography in the assessment of cardiac function. however, lv analysis can be made from images obtained by different modes and views of the heart. the aim of this study was to compare lv measurements, shortening fraction (sf) and ejection fraction (ef) obtained from four methods: m mode in short-axis and in long-axis, bidimensional mode in short-axis and in long-axis views of the heart. forty normal adult german shepherds were selected. echocardiographic study of lv of each animal was performed by the four methods described above. ancova test was used to examine the effects of axis, mode, weight and gender over lv measurements. isolated effect of the axis was observed for lv end-diastolic diameter (lvedd), with greater values obtained from short-axis views. there was isolated effect of mode over ef and sf, with greater measurements derived from bidimensional mode methods. weight correlated with all linear lv measures at least in one method, but not with ef and sf. weight had positive effect over lv endsystolic diameter and lv end-diastole posterior wall thickness in all methods, except from m mode in short axis in the last one. gender had isolated effect over lvedd, males showing greater values than females in bidimensional mode in short and long axis. the combined effect of axis, gender and weight was identified in interventricular septal end-diastolic thickness. we concluded that normal reference values obtained by different echocardiographic modes and planes should not be used interchangeably. abstract c- assessment of left ventricular diastolic func-tion by color tissue doppler imaging echo-cardiography in maine coon cats tested for mypbc-ap mutation hypertrophic cardiomyopathy (hcm), characterized by increased cardiac mass and diastolic dysfunction, is the most common feline heart disease. myocardial analysis by color tissue doppler imaging (tdi) is more sensitive than conventional echocardiography. this study evaluated diastolic dysfunction in various stages of feline hcm. maine coon cats (n ) were screened for the mybpc-a p mutation and examined with both echocardiography and tdi. then, were phenotypically classified in: normal (n ), suspects for hcm (n ) and hcm group (n ); and genotypically classified in: negative (n ), heterozygous (n ) and homozygous group (n ). myocardial velocities, measured in the basal and mild ventricular segment of the interventricular septal wall (ivs), left ventricular free wall (lvw) and in radial segment of left ventricular wall, was compared among different groups. a significant decreased (p , ) longitudinal em/am at the basal segment of ivs was observed in hcm cats compared with suspects and normal cats. a significant increased (p , ) longitudinal e/em at the basal segment of ivs was observed in hcm cats compared with suspects and normal cats. and a significant decreased (p , ) longitudinal sm at the basal segment of the lvw was observed in heterozygous cats compared with negative cats, both without hypertrophy. there was a significant positive correlation between summated early and late diastolic velocities (emam) and heart rate (p o , ); and a positive correlation between sm and em velocities and heart rate (p o , ). the mybpc-a p mutation is not consistently associated with ventricular hypertrophy and negatives cats can also develop hcm. the tdi alone is not able to identify cats with mutation before myocardial hypertrophy. diastolic dysfunction occurs in many cats with hypertrophic cardiomyopathy (hcm) but less is known about systolic function in various stages of hcm. myocardial strain analysis by tissue doppler imaging is a noninvasive echocardiographic method to assess systolic function. this study evaluated systolic function in various stages of feline hcm. maine coon cats (n ) were screened for the mybpc-a p mutation an examined with both echocardiography and strain. then, were phenotypically classified in: normal (n ), suspects for hcm (n ) and hcm group (n ); and genotypically classified in: negative (n ), heterozygous (n ) and homozygous group (n ). peak myocardial strain, measured in the basal and mildventricular segment of the interventricular septal wall (ivs), left ventricular free wall (lvw), left ventricular anterior wall (lvaw), left ventricular posterior wall (lvpw) and radial segment of left ventricular wall, was compared among different groups. whereas conventional echocardiography demonstrated an apparently normal contractile state based on fractional shortening, myocardial strain (at mildventricular segment of ivs) in hcm cats was significantly decreased compared with normal group (p , ). myocardial strain (at basal segment of lvaw) also was significantly decreased in heterozygous cats compared with negative group (p , ); and was significantly decreased in heterozygous cats compared with negative group, both without ventricular hypertrophy (p , ). there was a significant negative correlation between strain values and wall thickness (p o , ). this method allows detection of abnormal systolic deformation in maine coons cats with hcm mutation despite apparently normal systolic function. the abnormal systolic deformation already can be present in heterozygous cats without hypertrophy and increased with progressive ventricular hypertrophy. recently, multiple advanced resting electrocardiographic (ecg) techniques have been applied in humans for detection of cardiac autonomic and repolarisation function. this has improved the diagnostic and/or prognostic value of short-time ecg in detection of common human cardiac diseases even before onset of symptoms or changes in the standard ecg. therefore, this study investigates, if advanced ecg can predict the severity of mitral regurgitation (mr) in dogs with myxomatous mitral valve disease (mmvd) and thereby improve the diagnostic value of ecg. the study included privately owned cavalier king charles spaniels (ckcss) (age . ae . years; males and females). all dogs were examined by echocardiography and a short-time ( - min) high-fidelity -lead ecg, with the dog in a resting position and in sinus rhythm. dogs were divided into groups according to the degree of mr estimated as the percentage of the left atrium area using color doppler mapping ( %; % o jet %; % o jet %; jet %; jet % and with clinical signs of congestive heart failure). ecg recordings were evaluated via custom software programs to calculate different parameters, including heart rate variability (hrv), qt variability (qtv), t-wave complexity, wave morphology and -d ecg. one-way anova determined ecg parameters, which were significantly different (p o . ) between the dog groups. principal component factor analysis identified a factor model with . % explained variability. qrs dipolar voltage and two repolarization indices of qtv increased significantly with mr severity, whereas total power of the frequency spectrum of rr interval and the standard deviation of qtv decreased significantly with mr severity. for the selected parameters the prediction of mr jet value was tested by multiple linear regression. a correlation coefficient (r) of . indicated that the prediction value was significant (p o . ). if age was included in the multiple linear regression the prediction value was further increased (r . ). our results indicate that for a cut-off criteria of mr ! % jet the five selected ecg parameters could predict the severity of mr caused by mmvd in ckcss with sinus rhythm with sensitivity % ( % with age inclusion) and specificity % ( % with age inclusion) (p o . ). nt-probnp concentration is increased in canine patients with heart disease. relatively little is known about the stimuli for release of nt-probnp in dogs. physical activity independent of cardiac disease and the stress of being in the hospital could influence nt-probnp release and affect diagnosis and management of patients. we hypothesized that nt-probnp concentration in healthy dogs would not exceed the normal reference value ( pmol/l) following a period of exercise. the goal of this study was to examine whether physical activity could elevate plasma nt-probnp and cause false positive results in healthy dogs. the study population included healthy dogs yr of age without heart murmur or known systemic disease, and normal d/color flow echocardiographic exam. plasma samples for nt-probnp were obtained before, immediately after, and hour after a standardized -minute submaximal exercise regimen. the study included dogs with a median age of . yrs and included females and males. there was no statistical difference in median plasma nt-probnp concentration across the three time points (baseline median, [iqr, immediately post, ; p . ). the average coefficient of variation of nt-probnp concentration across the exercise regimen was . ae . %. in of dogs ( . %), nt-probnp increased from to pmol/l immediately after exercise. the results of this study demonstrate that submaximal exercise does not significantly change median nt-probnp concentration and the incidence of false positive results is low. further studies should investigate effects of exercise on nt-probnp concentrations in dogs with heart disease. obesity is an increasing problem in veterinary medicine. obese human patients are shown to present lower levels of natriuretic peptides, regardless of an increased volume and pressure load, what raises the possibility that the natriuretic response is impaired in these individuals. considering the controversial findings in obese humans, and the lack of studies reported in dogs, this study proposed the evaluation of nt-proanp concentration in obese dogs. nt-proanp concentration was determined prospectively in obese dogs ( females; males; - months) and in non-obese dogs (controls; females; males; - months) from a veterinary hospital population. obesity was determined by body condition score [ ( / ); ( / ); ( / ); ( / ); ( / ); ( / )]. dogs were excluded if they had any primary cardiac disease, renal insufficiency, endocrine disease, or if they were receiving diuretics, vasodilators, antiepileptic drugs or corticosteroids. commercial kits were used (vetsign s canine cardio screen nt-pro-anp vc -guildhay/biomedica). mann whitney test was used for group comparison. results are presented as median; interval; p and p ). nt-proanp was significantly lower in obese dogs [ . fmol/ml ( . - . ); p . ; p . ] than in controls [ . fmol/ml ( . - . ); p . ; p . ]; (p . ). results were similar to what has been found in obese humans. lower levels of natriuretic peptides are also seen in obese heart failure patients. this study provides important information regarding nt-proanp concentration in obese dogs, which should be better explored characterizing the behavior of natriuretic peptides after weight loss, and also in obese dogs with primary heart disease. left-to-right shunting patent ductus arteriosus (pda) is one of the most common canine congenital cardiovascular defects. human studies have shown that bnp and nt-probnp concentrations are elevated in patients with pda, and can be used to detect hemodynamically significant pda. the purpose of this study was to measure nt-probnp concentrations in dogs with pda, and to assess whether additional indicators of hemodynamics correlate with ntprobnp. we hypothesized that nt-probnp will serve as a simple non-invasive marker of hemodynamic significance in dogs with pda prior to and following transcatheter ductal occlusion. nt-probnp was measured in client-owned dogs with echocardiographically normal hearts. ten dogs with pda were initially evaluated with thoracic radiographs, transthoracic and transesophageal echocardiography, pulmonary capillary wedge pressure (pcwp) and nt-probnp. nt-probnp and echocardiography were repeated at day and months following ductal occlusion. pcwp was repeated at months. baseline nt-probnp was significantly higher in pda dogs compared to control ( ae pmol/l (mean ae sd), ae ; p o . ). at day and months following ductal occlusion, nt-probnp was ae and ae , respectively. the following decreased significantly from baseline: pcwp ( . ae . to . ae . mmhg; p . ), and indexed left ventricular internal dimensions in diastole ( . ae . to . ae . ; p . ) but not significantly in systole ( . ae . to . ae . ; p . ). nt-probnp is elevated in dogs with pda and transductal closure is associated with a reduction in nt-probnp, pcwp and left ventricular size. cardiac biomarkers, particularly nt-probnp, are becoming more commonly used in dogs and cats as part of a diagnostic work up. multiple studies already have documented the correlation of this peptide with cardiac disease status and potential clinical implications. in a portion of these reports the manner in which samples were handled was placement of whole blood into an edta tube, followed by centrifugation and decanting of the supernatant that was ultimately stored at À c or À c prior to shipment, either with or without protease inhibition. our objective was to compare the nt-probnp concentrations in feline plasma collected using the previously reported methods to the california animal hospital (cah) collection method using tubes containing a protease inhibitor. this study compared nt-probnp concentrations using the protease inhibitor tubes vs. edta tubes from privately owned feline patients, with confirmed cardiac disease, and control feline patients. for all study participants, we performed a full history and physical examination, a hematology and chemistry panel, thoracic radiographs, ecg, and echocardiogram. in each study participant, at least ml's of whole blood was drawn from a peripheral vein, and transferred to a plastic edta tube. the sample was centrifuged within hour after collection. ml of plasma was then transferred to a tube containing a protease inhibitor, which was stored at c until being shipped within hours of collection. the remaining plasma was placed into separate microtubes, which did not contain a protease inhibitor. one microtube was then stored and shipped as previous studies have reported (À c, styrofoam container, shipment within hours), and the second microtube was frozen at À c. all samples were shipped, received and analyzed within hours of collection. results of this study showed that no difference was found between the frozen sample methods ( pmol/l and pmol/l p . ). it was determined that both frozen methods had lower nt-probnp levels ( and pmol/l) when compared to plasma samples shipped in protease inhibitor tubes ( and pmol/l). the findings of this trial demonstrate that the nt-probnp levels are significantly different between samples placed in edta tubes vs. contain protease inhibitor (p . and p . ). utilizing protease inhibitor tubes allows more accurate measurement of plasma nt-probnp. as for its relevance for future research and publications, authors should take care to investigate the manner in which blood samples were handled and the conclusion/results of these studies should be taken in light of the methodologies used in collecting, storing, shipping and analyzing the samples. degenerative mitral valve disease (dmvd) is one of the most common heart disease and is present approximately % of the canine heart disease. although the high prevalence exists in small dogs, the underlying molecular mechanism of its pathophysiology is rarely known. dmvd is functionally and pathologically similar in humans and dogs, thus, there will be a common pathogenesis in human and dogs with naturally occurring dmvd. serotonin and serotonin-related mechanisms have been implicated as a cause of valvular disease in human and animals, including spontaneous dmvd in dogs. increased circulating ht concentration as a potential source of heightened ht signaling is demonstrated in small dogs with dmvd. the aim of this study was to investigate whether serum ht concentrations were associated with severity of naturally occurring dmvd in small dogs and to investigate potential associations of dog characteristics on serum ht concentrations in our study population. forty-eight dogs were included in this study and were classified into control and dmvd groups according to the results of physical and echocardiographic examinations. based on the la:ao ratio, dogs with dmvd were classified as follow: control (la:ao ratio . and no mr), mild (la:ao ratio . and mr), moderate ( . o la:ao ratio . and mr), severe (la:ao ratio . and mr). serum serotonin concentrations were measured by elisa. an overall significant difference (p o . ) was found among groups and ht concentrations (control, . ng/ml [ . - . dmvd, ). significantly higher ht concentrations were observed in dogs with moderate (p o . ) and severe (p o . ) dmvd, compared with concentration in control group. additionally, ht concentration in dogs with moderate dmvd were significantly higher (p o . ) than concentration in dogs with mild dmvd. also, dogs with severe dmvd had significantly higher ht concentration than dogs with mild (p o . ) and moderate (p o . ) dmvd. there was no significant association of age, platelet, and lvidd, on serum ht concentration, however, weak correlation between serum ht increased significantly and la:ao ratio (r . , p o . ) was observed. the results of this study indicate that serum ht concentrations were higher with increasing severity of spontaneous dmvd, which may be the potential cause to advance the progression of dmvd. further studies should be performed to reveal the role of ht in inducing and accelerating spontaneous dmvd and to investigate if lowering serum ht concentration could alter the progression of dmvd. the objective of this prospective study was to evaluate the utility of cardiac troponin i (ctni) in differentiating between underlying etiologies of pericardial effusion in the canine patient. patients were prospectively recruited at time of diagnosis of novel pericardial effusion. serum samples were collected prior to pericardiocentesis. patients were evaluated by echocardiography and classified with the diagnosis of hemangiosarcoma (hsa), heart base tumor (hbt), or unknown etiology at the initial evaluation based on established characteristic echocardiographic findings. idiopathic pericardial effusion (ipe) was defined by histopathology, echonegative for a mass lesion with no recurrence of pericardial effusion months, or symptom free months from time of enrollment. patients were excluded from analysis if a diagnosis could not be established based on above criteria or concurrent moderate azotemia (creatinine . mg/dl) was present at time of sample collection. serum samples were frozen and analyzed in batches within days of collection by a ctni assay with a . ng/ml lower limit sensitivity. sixty-three patients were recruited over a one year period with patients excluded due to lack of diagnosis ( ) or azotemia ( ). median ctni levels of dogs with hsa (n ), hbt (n ), and ipe (n ) were . ng/dl (interquartile range (iqr) o . - . ), . ng/dl (iqr o . - . ), and o . ng/dl (iqr o . -o . ) respectively. concentrations of ctni differed significantly between dogs with hsa and hbt (p . ) and ipe (p . ). there was no difference between ctni concentrations between hbt and ipe dogs (p . ). receiver operating curve analysis to determine the optimal cutoff for differentiation of dogs affected with hsa and both hbt and ipe revealed a significant (p o . ) area under the curve ( . ). a cut-off point of ctni of . yielded a sensitivity of % ( % ci, - %) and specificity of % ( % ci, - %). utilizing a higher cut-off point of . yielded a lower sensitivity of % ( % ci, - %), but a higher specificity of % ( % ci, - %) which may have more clinical utility given the disparity in prognoses of the etiologies compared. in conclusion, this study supports the diagnostic utility of ctni concentrations to delineate between patients with hsa and other etiologies of pericardial effusion, but does not reliably differentiate between dogs with ipe and other neoplastic etiologies. the pathogenesis of degenerative mitral valve disease (dmvd) in dogs remains to be fully elucidated. the high sheer stress caused by mitral regurgitation damages the endothelial surface of the valve, and a previous study demonstrated increased transcription of intercellular adhesion molecule- (icam- ) and e-selectin in affected mitral leaflet tissue. we hypothesized that this may be responsible for platelet recruitment and adhesion, and initiation of a proliferative cascade, resulting in further myxomatous changes. the goal of this study was to compare plasma levels of icam- and e-selectin in healthy dogs and those with dmvd. the study population included dogs with echocardiographic evidence of dmvd and healthy control dogs year old with no heart murmur or known systemic diseases. dmvd dogs underwent d/color-flow doppler echocardiographic exam. blood samples were obtained for plasma icam- and e-selectin analysis using commercially available elisa kits. the study included dogs, of which had dmvd and were normal. the dmvd group had a median age of . yrs ) and included females and males. two ( %), ( %), ( %) and ( %) dogs were classified as isachc a, b, and a, respectively. of the control dogs, median age was . yrs [ - . ], with females and males. there was no statistical difference in plasma e-selectin between control dogs (median . [ . - . ]) and those with dmvd ( . [ . - . ]); p . . plasma icam- concentrations were higher in dmvd dogs ( . [ . - . ]) than controls (median . [ . - . ], but this difference did not reach statistical significance (p . ). linear regression analysis showed no significant correlation between icam- or e-selectin and serum serotonin level, nt-probnp or echocardiographic measures of dmvd severity (la:ao, lvidd:ao, lvids:ao). the results of this study demonstrate no significant difference in circulating adhesion molecules icam- and e-selectin in dogs with dmvd as compared with healthy controls. further studies investigating adhesion molecules within the mitral valve tissue itself are likely needed if icam- and e-selectin play a role in the pathophysiology of dmvd. the rate of glucose utilization in the heart is greater than in other tissues, and impaired glucose uptake may play a major role in the pathogenesis of heart failure (hf). glucose uptake across the sarcolemma is regulated by a family of membrane proteins called glucose transporters (gluts), which includes glut- , the major cardiac isoform, and glut- , a recently discovered isoform, the role of which is unknown in the heart. in addition, despite the wellknown regional differences in myocardial structure and function, potential regional patterns in glucose transport have not been investigated. thus, we hypothesized that glut- and - protein and gene expression would be chamber specific in healthy dogs and during chronic hf. using a canine model of tachypacing induced chronic hf, glut protein and messenger rna in both ventricles and atria were investigated by immunoblotting and real time pcr. in control dogs, glut- , but not glut- , protein expression were significantly higher in the atria compared to the ventricles, with the highest content in the right atrium (ra, p o . ). glut- and - mrna were homogeneously expressed in all the cardiac chambers. during chronic hf, glut - and - expression was highest in the left ventricle (lv, by . and . fold, respectively, p o . ), with a concomitant increase in glut- and - mrna (p o . ). glut - , but not glut- , was decreased in ra during chronic hf (p . ). our data suggest that glut- protein was differentially expressed across the cardiac chambers in the healthy heart. during chronic hf, lv was the primary site dependent on both glut and glut -mediated glucose transport, which was transcriptionally regulated. in addition, the paradoxical decrease in glut content in ra may induce perturbations in atrial energy production during chronic hf. some obese dogs are suspected to have cardiac disease because they have enlargement of the heart on thoracic radiograph. it has been reported in cats that the fat increases the cardiac silhouette, while echocardiograms revealed normal cardiac dimensions. the purpose of this study was to determine whether obesity overestimates cardiac dimension in radiographs compared to echocardiographic findings in dogs. twenty three obese dogs and controls were included based on a - body condition scoring (bcs). computerized radiography was obtained and vhs measurement was performed as previously described. echocardiographic measurements were interpreted based on reference values according to lean body weight regression equations. results for echo and vhs measurements were classified in scores as normal, mild, moderate or severe increase. student's t test was used for comparison of vhs between groups. mann-whitney rank sum test was used to assess echocardiographic scores between groups. spearman rank order correlation was used to assess relationships between any pairs of variables between echo and vhs scores, echo vs bcs and vhs vs bcs. groups were similar regarding age [obese ( ae ); control ( ae ); p . ], breeds and gender distribution. obese dogs had significantly higher vhs and echo scores compared with controls [vhs: ( . ae . ) vs ( . ae . ); p o . ; echo score: range ( - ) vs ( - ); p . ]. there were no relationships between any pair of variables analyzed. these results show that there are changes both in echo and radiographic appearance of the heart in obese dogs, but vhs overestimates cardiac silhouette compared to echo, probably related to pericardial fat accumulation. heart rate variability (hrv) is an indirect measurement of the autonomic modulation of heart rate (hr). reduced hrv measured from short-time electrocardiography is seen in dogs with heart failure (hf) secondary to myxomatous mitral valve disease (mmvd). however, hrv is suggested to increase with disease severity at early stages of mmvd. the aims of this study were ) to associate hr and hrv with severity of mmvd in cavalier king charles spaniels (ckcs) and ) to compare hr and hrv between ckcs and other dog breeds in a group of dogs in hf secondary to mmvd. one-hundred dogs were examined by echocardiography and hour electrocardiography. the dogs were divided into five groups: ) ckcs with no/minimal mitral regurgitation (mr) (mr jet % of the left atrial area using color doppler mapping) and no murmur, ) ckcs with mild mr ( % o jet %), ) ckcs with moderate/ severe mr (jet %) and no clinical signs of hf, ) ckcs in hf (hf defined as left atrium to aortic root ratio (la/ao) . , clinical signs of hf and furosemide responsiveness) and ) non-ckcs in hf. dogs in hf were allowed hf therapy. both hr and hrv were analyzed over a -hour period, while hrv were also analysed over a -hour nightly period. analyses of variance were performed with hr or hrv as response variables and the explanatory variables dog group and echocardiographic indices of mmvd were included separately. all p-values were bonferroni corrected. minimum-and mean hr were significantly higher in ckcs with moderate/severe mr and in hf compared to ckcs with no/ minimal and mild mr (all p o . ). seven out of hrv variables were significantly decreased in ckcs with moderate/ severe mr and in hf compared to ckcs with no/minimal and mild mr (all p o . ). another hrv variables showed the same groupwise differences (all p o . ), except that the difference between ckcs with mild mr and ckcs with moderate/severe mr did not reach statistical significance. mminimum hr, mean hr and the hrv variables ( and ) differing between dog groups, also consistently decreased with increasing mr, la/ao and the proximal isovelocity surface area in ckcs. non-ckcs in hf had a lower minimum hr compared to ckcs in hf (p . ) and a higher triangular index measured in both periods (all p o . ). in conclusion, hr increased and most hrv variables decreased with increasing severity of mmvd in ckcs, even prior to the development of hf. other breeds in hf secondary to mmvd had lower minimum hr, but higher triangular index compared to ckcs in hf. although the cells in the specialized conduction system in the heart are capable of initiating their own impulse, the rate in which those impulses are generated can be influenced by autonomic nervous system. different types of respiratory patterns can stimulate autonomic nervous system in different manners. thus, non-sedated rabbits were studied during forced respiration aiming to evaluate the influence of this breathing pattern on heart rate. twenty male, one-year-old healthy new zealand rabbits were enrolled in the study. animals were set in right lateral recumbency and maintained that way by physical contention. chemical sedation was not used. partial nasal obstruction by digital compression was applied to those rabbits for five seconds, eliciting a forced inhaling and exhaling against semi closed nostrils. heart rate was obtained by measurement of two consecutives rr intervals in the computerized electrocardiography, recorded continuously prior and during the maneuver. heart rate before the intervention was ae bpm (mean ae standard deviation). all rabbits submitted to the maneuver showed a dramatic reduction in this parameter. after nasal partial obstruction, heart rate was ae bpm. data was submitted to statistical analysis by paired student's t test and a significant difference between the heart rate before and after the maneuver was observed (p o . ). although the exactly mechanism involved in this response was not elucidated, the presented data support the applicability of this maneuver as an efficient method for non-pharmacological heart rate reduction in rabbits. obesity can affect cardiac function due to effects on cardiac rhythm, ventricular volume and blood pressure. the purpose of this study was to determine the effects of obesity and overweight on noninvasive systemic blood pressure and doppler echocardiographic parameters in cats without others causes of cardiac hypertrophy. the study groups comprised fifteen obese cats with mean body score index (bsi) of , , seven overweight cats (bsi , ) and seven cats with ideal bsi ( , ). the blood pressure was measured by doppler method and the doppler echocardiography was performed in conscious animals. the statistical analysis was performed by analysis of variance followed by tukey's test and pearson's correlation. the blood pressure values of the obese cats were superior ( , ae , mmhg, p o , ) than in overweight ( , ae , mmhg) and normal cats ( , ae , mmhg) and % of the obese cats had blood pressure higher than mmhg. there were observed differences on the ratio of early (e) and late (a) left ventricular filling velocity (p , ) of obese animals (e/a , ae , ) compared to overweight ( , ae , ) and normal cats ( , ae , ). seven obese cats ( %) had inversion of e/a compatible with diastolic dysfunction and there were negative correlation (r À , , p , ) between the e/a ratio and blood pressure values. other differences observed were increases in left ventricular septum in diastole (p , ) and in free wall in diastole (p , ) and systole (p , ) of the obese animals compared to overweight and normal cats. these results demonstrate the possibility of cardiovascular effects related to obesity in cats, such as systemic arterial hypertension and secondary diastolic dysfunction. diuretic therapy reduces preload, and relieves congestion secondary to cardiac dysfunction. torsemide (torasemide) is a loop diuretic with longer duration of action, less diuretic resistance, and adjunctive aldosterone antagonism as compared to furosemide. we hypothesized that torsemide was no less effective than furosemide at diuresis, control of clinical signs, and maintenance of quality of life in dogs with congestive heart failure. a double-blinded, randomized, crossover clinical trial was performed in dogs with stable heart failure receiving bid furosemide and adjunctive medications. dogs were randomized to their current furosemide dose or torsemide (calculated as / of the daily furosemide dose divided into bid dosing). crossover occurred at day and the study ended on day . clinical, laboratory, radiographic, and owner-perceived quality of life variables were evaluated on days , and . no dog developed recurrent heart failure during the study. average furosemide dose on day was . mg/kg/day (range, . - . ). following torsemide treatment, blood urea nitrogen (p . ), albumin (p . ), and albumin:globulin ratio (p . ) were significantly increased, and urine specific gravity (p . ) and chloride (p . ) were significantly decreased as compared to baseline and/or furosemide dosing (one-way anova with bonferroni correction). no differences in qol were found. results indicate that torsemide is equivalent to furosemide at controlling clinical heart failure in dogs, and might in fact, achieve greater diuresis vs. furosemide. larger clinical trials evaluating furosemide resistance and/or torsemide as a first-line loop diuretic for congestive heart failure in dogs with heart failure are warranted. the purpose of this study was to investigate the feasibility of speckle tracking echocardiography (ste) in healthy cats and to determine whether or not it can detect myocardial dysfunction in cats with diseased heart. radial and circumferential strain and strain rate were measured by ste using left ventricle short-axis view in clinically healthy cats. eighteen cats with hcm whose lv thickness at end-diastole with mm or more were evaluated with ste analysis, and compared with healthy cats. index of left ventricular synchrony (trs-sd) was also assessed in cats with hcm, and compared to healthy subjects. ste resulted in technically adequate images in % of the cats. fusions of early and late diastolic (e and a) wave in strain rate were seen in of cats. percent errors in analysis with or without simultaneous ecg monitoring were . - . % in all parameters. inter-and intraobserver variability of ste parameters in healthy cats was minimal ( . - . %) except for the systolic circumferential strain rate. sedation using buprenorphine and acepromazine did not affect any ste parameter. e wave in radial and circumferential strain rate of hcm cats was significantly decreased compared with healthy cats. no significant difference was seen in trs-sd. ste analysis was considered clinically feasible to assess cardiac function in cats, and could detect myocardial dysfunction in cats with hcm. further study is warranted to investigate to assess whether or not ste can differentiate the etiology of left ventricular concentric hypertrophy since it is clinically important. carvedilol, a rd generation non-selective beta-blocker with ancillary alpha -blocking and antioxidant properties may have therapeutic implications for multiple diseases in cats; however, pharmacokinetics and bioavailability of commercially prepared oral carvedilol has not been determined. hplc for carvedilol measurement in feline plasma was validated and standardization curves created. the pharmacokinetics (pk) of carvedilol was evaluated in apparently healthy male neutered adult cats (average weight of kg) following single dose intravenous (iv) of . mg/kg and single dose oral administration of . to . mg/kg. concentrations of the active parent compound, carvedilol, were detected in plasma using hplc analysis. lower limit of quantification was ng/ml. the mean peak concentration after iv administration of carvedilol was ng/ml (range, to ), elimination half-life was . hours (range, . to . ), and clearance was . l/hr/kg. the volume of distribution was . l/hr. after a single oral administration of carvedilol, the time to peak plasma concentration was minutes (range, to minutes) and the mean residual time was . hours. the half life was . hours. maximal concentration ng/ ml and the mean bioavailability was . % with a median of . % (range, . % to %). these data demonstrate a low bioavailability of oral carvedilol and a wide variation in cats. all cats tolerated the oral dose of carvedilol with no major adverse effects. also, a mean residual time of . hours would suggest that a more frequent dosing schedule may be required to maintain therapeutic plasma levels. pharmacodynamic studies investigating beta-adrenergic blockade duration may provide a more accurate dosing interval of carvedilol. abstract c- effects of sildenafil citrate on dogs with ei-senmenger's syndrome. k nakamura, m yamasaki, h ohta, m takiguchi. department of veterinary clinical sciences, graduate school of veterinary medicine, hokkaido university, sapporo, hokkaido, japan. sildenafil has shown to be effective for dogs with pulmonary hypertension; however, its efficacy for dogs with eisenmenger's syndrome (es) and secondary erythrocytosis has not yet been determined. the objective of this study is to determine the effect of sildenafil for dogs with eisenmeger's syndrome and secondary erythrocytosis. this was a prospective, single arm, open-label study. five clinical dogs with es and secondary erythrocytosis were included. new york heart association (nyha) functional class, pcv, and pulmonary artery acceleration time to ejection time ratio (pa at : et) were evaluated before and after sildenafil therapy ( . mg/kg, bid). nyha functional class was significantly improved after (median ; range - , p . ) and months (median ; range - , p . ) of sildenafil therapy, compared with the baseline (median , range - ). pcv was significantly decreased after month ( . ae . %, p . ) and months ( . ae . %, p . ) of therapy, compared with the baseline ( . ae . %). at : et was significantly increased after month of therapy ( . ae . , p . ) from the baseline ( . ae . ). sildenafil resolved the clinical signs and secondary erythrocytosis in dogs with es. sildenafil therapy could be the treatment of choice for dogs with es. sepsis is the number one cause of mortality in neonatal foals. the role of the raas and hpaa in systemic inflammation and response to stress is well documented in critically ill human neonates, but limited information exists in foals. we hypothesized that in septic foals the raas and hpaa will be activated by systemic inflammation and hypoperfusion and the degree of activation will be associated with severity of sepsis and mortality. blood samples were collected on admission from septic (sepsis score ), sick non-septic (sns), and healthy foals of o days of age. blood concentrations of corticotropin-releasing hormone (crh), adrenocorticotropin (acth), cortisol, aldosterone, angiotensin-ii (ang-ii), arginine vasopressin (avp) and plasma renin activity were determined by radioimmunoassays. acth, cortisol, aldosterone, ang-ii and avp concentrations were higher while crh was lower in septic and sns foals compared to healthy foals (p o . ). septic non-survivor foals had higher concentrations of aldosterone, cortisol, acth and avp and lower concentrations of ang-ii and crh than survivors. avp was associated with ang-ii in septic, and with acth in septic and sns foals (p o . ). there was no difference in renin activity and ang-ii concentrations among foal groups. septic foals had a higher acth:aldosterone ratio than healthy foals (p o . ). this study shows that in response to sepsis there is raas and hpaa activation in critically ill foals. we propose that in sick foals avp is more important than crh in regulating acth secretion. the increased acth:aldosterone ratio further supports relative adrenal insufficiency in septic foals. this prospective, cohort study aimed to characterize alterations in coagulation and blood-derived inflammatory biomarkers in adult horses that developed diarrhea during hospitalization. physical and hematological parameters were evaluated at times (onset of diarrhea), , , and h, then every h until resolution of diarrhea or death. each hematological analysis included a complete blood count (cbc), thromboelastography (teg), partial-thromboplastin-time (ptt), prothrombin-time (pt), plasma concentrations of lactate, tumor necrosis factor alpha (tnf-a), interleukin (il)- , il- , il- and nt-proc-type-natriuretic peptide (pcnp). horses were categorized into three groups based on the duration of diarrhea and evidence of systemic inflammation. group : diarrhea o h without systemic inflammation (si); group -diarrhea ! h without si; group -diarrhea with si. assessment of vital parameters and cbc established a diagnosis of si as previously described (levy, ) . descriptive and univariate outcome analyses were based on data normality. horses were enrolled, of which ( . %) survived to discharge. the mean age was . /- . years. eight horses ( . %) were categorized as group- , ( . %) as group- and ( . %) as group- . two horses developed thrombophlebitis. based on the results of teg, / ( . %) were normocoagulable, / ( . %) were hypocoagulable and / ( . %) were hypercoagulable, at one or more time points. of these, / ( . %) group- horses were coagulopathic. additionally, group- horses had a significantly lower ma than group- horses at baseline ( . ae . vs. . ae . ) and h ( . ae . vs. . ae . ). biomarker analyses are pending. in conclusion, si was associated with coagulation disorders in horses with hospital acquired diarrhea. clostridium difficile and clostridium perfringens are commonly associated with colitis and diarrhea in equines but asymptomatic carriers exist. reported carrier rates of toxigenic c. difficile and c. perfringens strains in feces range between - % and - % respectively. toxigenic c. difficile has also been isolated from the small intestine of diseased foals and is implicated as etiologic agent of duodenitis/proximal jejunitis in adult horses however scarce information is available on prevalence in gastrointestinal compartments other than feces in healthy horses, and it is unclear whether fecal samples are good predictors of the status of proximal intestinal sites. the objectives of this study were to investigate the presence of c. difficile and c. perfringens in various intestinal compartments of healthy adult horses and to molecularly characterize isolates. intestinal contents were collected from the stomach, duodenum jejunum, ileum, cecum, right dorsal and left ventral colon, small colon and rectum of euthanized horses free of apparent gastrointestinal disease. enrichment culture was performed for c. difficile and c. perfringens and c. difficile isolates were further characterized via toxin gene detection and ribotyping. c. difficile was isolated from / ( %) samples from / ( %) horses. between zero and three sites were positive per horse, and multiple sites were positive in three horses. isolates were recovered from duodenum (n ), right dorsal colon (n ), small colon (n ) and rectum (n ). in one horse, the rectal sample was negative but c. difficile was isolated from a proximal site, all other horses were positive on the rectal sample if a more proximal compartment was positive. in three horses multiple compartments were positive however different strains were always present within the same horse (n ). all isolates possessed genes encoding toxins a and b. five isolates were ribotype and also possessed genes encoding the binary toxin. the other isolates were ribotype and were negative for the genes encoding the binary toxin. despite using a method with a detection level as low as cfu/g of feces, no c. perfringens was recovered. rectal samples were a good predictor of overall c. difficile carrier status ( / horses), however rectal samples were not always representative for the ribotype carried in more proximal compartments. the presence of variable strains within the same horse suggests transient passage of the bacterium through the gastrointestinal system rather than actual colonization although further study testing multiple colonies per site is needed. the predominance of ribotype is consistent with recent emergence of this strain in this region, as earlier studies found other strains ( , ) to be more prevalent and a variety of ribotypes were typically recovered from horses. interestingly ribotype has recently emerged as a hypervirulent strain in humans in our area. clostridium difficile, clostridium perfringens and salmonella are important enteric pathogens in horses, however some healthy animals also harbour these pathogens. point prevalence studies have reported these carriage rates, but there are no data regarding longitudinal prevalence of these enteric bacteria, information that would be useful to better understand the epidemiology of these pathogens. additionally, antimicrobial resistance is a pressing concern. commensal e.coli is often used as an indicator organism to evaluate antimicrobial resistance of enteric bacteria, yet there are limited data from horses on farms. the objectives of this study were to longitudinally investigate the above enteric pathogens over the course of one year, molecularly characterize obtained isolates and determine the antibiotic susceptibility profile for e. coli. fecal samples were collected from adult horses from five farms on a monthly basis over the course of one year. selective cultures were performed for c. difficile, c. perfringens, salmonella and e. coli. c. difficile isolates were characterized via toxin gene pcr and ribotyping. broth microdilution was performed to assess antimicrobial susceptibility profiles of e. coli. clostridium difficile was isolated from / ( . %) samples from / ( %) horses. four horses were positive on more than one occasion, three were positive in two consecutive months. different ribotypes were found in two of the latter horses. most isolates were ribotype (n ) with ribotype (n ) and ribotype c (n ) also identified. ribotypes and c possessed genes encoding toxins a, b and binary toxin, while ribotype only possessed toxin a and b genes. despite a detection threshold of cfu/g feces, c. perfringens was not detected in any samples, nor was salmonella. e coli was isolated from / ( %) samples. resistance to ! antimicrobial was present in only / ( . %) isolates. multidrug resistance (! antibiotics) was present in / ( %). most commonly, isolates were resistant to sulfisoxazole ( / ) and trimethoprim sulfamethoxazole ( / ). the overall detection rate for toxigenic c. difficile in fecal samples of healthy horses was . % which is consistent with previous studies. the cumulative prevalence of % was striking but only one horse shed the same strain for more than one month, indicating c. difficile shedding is a transient and dynamic state. the predominant isolation of ribotype is consistent with the suspicion that this strain has emerged and become widely disseminated in this region in recent years. the low prevalence of c. perfringens and salmonella is in agreement with some other studies. the low prevalence of antibiotic resistance in commensal e. coli was encouraging and suggests that healthy horses on pleasure horse farms are not likely a major reservoir of resistance in enteric bacteria. type polysaccharide storage myopathy (pssm) in horses is associated with a dominant missense mutation (r h) in the skeletal muscle glycogen synthase gene (gys ). since disease severity varies between affected horses, we hypothesised that some clinical variability could be accounted for by the underlying genotype. belgian / percheron horses were genotyped using a validated restriction fragment length polymorphism assay enabling grouping of horses as homozygotes (hh), heterozygotes(hr) or normal (rr). subsequently, semimembranosis muscle samples were biopsied from each of six matched sedentary horses from each group; one sample was formalin-fixed and one fresh frozen. sections were stained using haematoxylin and eosin, periodic acid schiff /diastase. anti-dystrophin, nnos and myosin heavy chain immunohistochemistry was performed to examine sarcolemmal intregrity, there were significant differences in resting ck activity (p . ) (median hh u/l interquartile range(ir) - ; hr u/l ir - ; rr u/l ir - ) and ast activity (p o . ) (ast mean hh u/l sd ; hr u/l sd ; rr u/l sd ) and muscle pathology between the groups, with severity increasing rr o hr o hh. there were significantly more type a (p . ) and fewer type x fibres (p . ) in homozygotes ( a % sd . ; x % sd ) compared with the other groups (hr a % sd . , x % sd . ; rr: a . % sd . x % sd . ). more type a fibres contained polysaccharide inclusions in homozygotes ( % sd . ) than in heterozygotes ( . % sd . ) (p o . ). both dystrophin and nnos expression was normally localised to the sarcolemma in pathologically normal and vacuolated fibres from mutant horses. in conclusion, sedentary homozygotes have more severe skeletal muscle pathology and higher resting plasma ck and ast activities than heterozygotes, and pssm is associated with a fibre type shift towards type a. although subsarcolemmal vacuolation likely disrupts the contractile apparatus's attachment to the sarcolemma, the latter's integrity appeared intact. the recumbent horse presents a logistic, diagnostic, and therapeutic challenge to the equine practitioner. there is currently very little data available on the prognosis and outcome of horses that are recumbent. therefore, the purpose of this study was to investigate the outcome of hospitalized horses that had been recumbent in the field or in the hospital and the factors affecting their survival. records of horses admitted to the school of veterinary medicine, university of california davis from january of to december of with a history of recumbency or horses that became recumbent while hospitalized were evaluated. a horse was defined as recumbent if it was unable to stand on its own. the medical record was examined for the following criteria: history pertaining to the current illness including treatment by the rdvm, breed, age, weight, date of presentation, physical and neurological examination findings, cbc and biochemical profile results, initial drugs administered on arrival, time spent recumbent, time spent in a sling, diagnosis, and hospitalization costs. statistical analysis correlating factors associated with survival was performed using logistic regression. overall there were non survivors and survivors. factors that favored survival included early initiation of treatment in the field by the rdvm, horses that tolerated a sling and spent more time in a sling, increased duration and costs of hospitalization, horses that were recumbent post anesthesia, and those recumbent due to disease of the musculoskeletal system. factors that increased likelihood of non survival included horses that were ataxic on presentation, horses with increased bun, horses that spent more time recumbent, those that did not tolerate a sling, and horses diagnosed with botulism and spinal cord disease. in conclusion, this retrospective study demonstrated that both the cause of recumbency and the ability of horses to tolerate a sling had a direct effect on survival. abstract e- plasma peak and trough gentamicin concentra-tions in hospitalized horses receiving once daily gentamicin. jr read , pa wilkins , rd nolen-walston . university of pennsylvania, new bolton center, kennett square, pa. university of illinois, champaign-urbana, il. gentamicin is often used to provide gram negative antimicrobial coverage in horses at . mg/kg iv every hours. therapeutic drug monitoring in our hospital suggests larger doses are required in many clinical cases to achieve the desired concentration ( -  minimum inhibitory concentration) for common bacterial isolates (peak target range - mg/ml). the aim of this study was to determine the correlation between gentamicin dose and plasma concentration in hospitalized horses receiving gentamicin treatment in order to identify an optimum dose range for this population. review of records ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) identified horses ! months old receiving once-daily gentamicin with peak and trough assays performed (n sets). spearman rank correlation coefficient analysis revealed a weak (r . ) but statistically significant correlation (p . ) between gentamicin dose and peak plasma concentration. horses receiving . - . and . mg/kg gentamicin (groups and ) had higher median peaks ( mg/ml) than horses receiving . - . mg/kg (group ; . mg/ml). higher doses were more likely to result in peaks mg/ml ( and %, groups and respectively) than horses receiving . - . mg/kg (group ; %). all hour post-gentamicin administration trough values were o mg/ml. no correlation was found between dose and change in plasma creatinine during treatment, nor dose and trough level. these data suggest that gentamicin dosage in horses should be individually determined by therapeutic monitoring. additionally, these data support an initial dose of . - . mg/kg iv every hours in order to achieve desired peak concentration and an appropriately low trough concentration. heaves is a common respiratory inflammatory disease, characterized by a pulmonary neutrophilia. this disease is also characterized by an activation of circulating neutrophils after antigen challenge but their specific role in heaves is not well understood. also, there are anecdotal studies concerning heaves-affected horse to be more susceptible to infection. however, to our knowledge, the antibacterial host defense role mediated by circulating neutrophils was not investigated in heaves-affected horses. the objective of this study was to compare phagocytosis activity and bacterial killing by circulating blood neutrophils of heaves-affected and control horses. peripheral neutrophils were isolated from heaves-affected (n ) and control (n ) horses using a density gradient technique. the killing capacity was assessed by incubating neutrophils with streptococcus equi spp equi and spp zooepidemicus. after h of bacterianeutrophil coculture, total viable bacterial cells were measured by quantitative plating. the phagocytosis was evaluated by flow cytometry using fluorescent beads and gfp-transformed streptococcus suis suilysin-negative mutant strain. circulating neutrophils from heaves-affected horses showed a significant decrease in their killing capacity toward s. zooepidemicus (p . ). a reduced, although not significant (p . ), killing capacity of s. equi by these neutrophils was also observed. the phagocytosis activity was not different between groups. this impairment of blood neutrophil bactericidal activity in heaves-affected horses could contribute to an increase susceptibility to infection. obesity is a common disorder of the horse, with current prevalence estimated at %. in people, obesity is associated with dyslipidemia, insulin resistance, mitochondrial dysfunction and downregulation of lipid and glucose metabolic pathways. in the horse, obesity is similarly associated with insulin resistance and alterations in lipid profiles; however, metabolic regulatory gene expression profiles have not been fully characterized. we hypothesized that obese horses have decreased expression of metabolic regulatory genes and decreased mitochondrial content in skeletal muscle compared with non-obese horses. sixteen light breed horses, - years of age were included. body condition score (n ) and neck circumference (n ) were recorded. post-mortem biopsy samples of the semi-membranosus muscle were obtained. dna and rna were isolated. relative expression of the metabolic genes, peroxisome proliferator activated receptor g (pparg), pparg coactivator- a (pgc- a), fatty acid translocase (fat) and estrogen related receptor a (erra) was determined by quantitative polymerase chain reaction (qpcr). mitochondrial content was assessed by determining mitochondrial dna/nuclear dna ratio by qpcr, using nadh-dehydrogenase subunit and cytochrome oxidase subunit as mitochondrial genes and beta actin as the nuclear reference gene. non-normal data was log transformed for analysis and a pearson coefficient of correlation was calculated for relative gene expression, body condition score and neck circumference. a value of p o . was considered significant. body condition score was strongly correlated with neck circumference (n , r . , p . ). relative expression of erra and glut- increased with body condition score (erra: n , r . , p . ; glut- : n , r . , p . ). copy number of the mitochondrial genes (nadh-dh and cox- ) was not related to body condition score or metabolic gene expression. expression of glut- , erra, pparg, and fat were strongly correlated to each other, but not pgc- a. there was a strong trend towards correlation between pparg and pgc- a in horses with body condition score (n , r . , p . ). in this study, there was no change in mitochondrial content in obese horses. assessment of mitochondrial function in obese horses and horses with ems is under way. the strong correlation between pparg and pgc- a observed only in horses with high body condition scores suggests this pathway is activated with obesity. the role of pparg and pgc- a in equine obesity should be further investigated to determine their potential as therapeutic targets. upregulation of erra and glut- in horses with increasing body condition score is unexpected, and may indicate a compensatory response to dysfunction of a downstream pathway. further studies to better define the role of metabolic regulatory gene expression in obese horses and those with ems are ongoing. previously presented at the th annual harold hamm diabetes center research retreat, oklahoma city, ok. inflammatory bowel disease is a cause of weight loss, decreased performance, and colic in horses. this condition is difficult to diagnose and clinicians rely upon absorption tests to document malabsorption. the purpose of this study was to compare glucose and xylose absorption tests in normal horses and determine the repeatability of these procedures. eight horses received mg/kg dextrose or d-xylose powder mixed as a % solution in water or water alone via nasogastric intubation on three different occasions within the same week for three consecutive weeks ( tests/horse). a crossover design was employed and the order of treatments was randomized. blood samples were collected at time , , , , , , and min. data were analyzed by repeated measures anova and t-tests. results showed that the xylose response over time differed significantly from the glucose response over time (test  time; p o . ). mean time to maximum concentration differed (p o . ) between tests (glucose min; xylose min). within-horse area under the curve, maximum concentration, and time to maximum concentration values for dextrose and xylose did not differ significantly when tests were repeated. results indicate that glucose and xylose absorption tests are repeatable within the same horse, but plotted curves differ between tests, with peak concentrations occurring at a later time point for the glucose absorption test. we conclude that both tests provide repeatable measures of intestinal absorption, but glucose and xylose appear to differ in their rates of absorption and clearance. the purpose of this study was to examine the records of a population of thoroughbreds with cervical vertebral malformation (cvm) and to determine which factors have an effect on these horses achieving athletic function. this was a retrospective case study of thoroughbreds with cvm treated medically from to . forty-one were euthanized after diagnosis, while the remaining were discharged for treatment. racing records were reviewed to determine which horses raced after treatment. horses were separated into groups based on whether or not they raced. medical records were reviewed, and results of neurologic examination, radiographic and laboratory findings, treatments, and outcome were assessed and compared between groups. twenty-one of horses treated medically ( %) improved enough to race. median neurologic grade between groups was significantly different (p o . ), with a hind limb grade of . (range - ) for the raced group and . (range . - ) for the unraced. intravertebral sagittal ratios measured from standing lateral cervical radiographs were equivocal between groups. radiographs of all horses were examined for kyphosis, dorsal over-riding arch, caudal epiphysitis, degenerative joint disease, cystic bone lesions, and cranial stenosis of the vertebral canal. horses with kyphosis (p . ), degenerative joint disease (p . ), or cranial stenosis (p . ) at any site were less likely to return to racing. racing prognosis for horses with cvm treated conservatively is equivalent to that of those treated surgically as reported by rush moore et al (javma, ) . radiographic changes and neurologic grade may help serve as indicators for whether a horse will respond to conservative therapy. since pain assessment is vital for management of colic, a valid, reliable and feasible tool for assessing the severity of acute abdominal pain in horses is urgently needed. our aim was to construct and validate a behavior-based pain scale by methodology utilized in construction of pain scales in non-verbal humans. the project consisted of four stages. firstly, behaviors to include in a scale were empirically identified. thirty equine clinicians noted behaviors in each of random film clips of horses with colic using a checklist. nine behaviors (e.g. rolling, pawing, and flank watching) demonstrated good inter-observer agreement without bias (multi-rater kappas: . - . ). secondly, the clinical judgment of experts was utilized to identify and to weight behaviors. six expert clinicians independently expressed opinions as to which of behaviors to include and the severity of pain they indicate. two contending scales (equine acute abdominal pain scales (eaaps) & ) were constructed based on both the empirical and the judgmental approaches. each included identical behaviors with a - point score range; eaaps- with gradations to some of the behaviors and eaaps- without. in the third stage, blood cortisol and lactate levels and heart rate were shown to only approximate pain since they correlate poorly with degree of pain as assessed by visual analog scale (vas) in horses with colic and controls (spearman rho; lactate . ; cortisol . ; heart rate . ). finally, reliability and validity of the pain scales were evaluated including constructs of pain; face validity, convergent and discriminate validity and extreme groups. thirty of films of horses with colic were randomly presented to expert equine clinicians internationally who were randomly allocated into three groups to score pain; one group by both vas and numerical rating scale (nrs)), and two groups, each by one of the two eaaps scales. inter-observer reliability of both eaaps scales was excellent (intraclass correlation . ). intra-observer reliability based on scores given for identical films demonstrated; % and % agreement, kappa . and . , and spearman's rho . and . for eaaps- and , respectively. both scales varied by score between observations. face validity; each group reported their scale to be valid ( % & %). convergent validity; the scales compared favorably with vas/nrs scores (spearman's rho: . - . ). discriminate validity; correlation to heart rate, lactate and cortisol levels was predictably low (rho . - . ). extreme group validity; colic horses scored significantly higher than control horses; scores of . - . in controls versus . - . in cases. in conclusion, methodology established in human medicine but novel in veterinary medicine was used to construct and validate two clinically feasible equine abdominal pain severity scales that showed excellent reliability and validity. further refinement of the eaaps scale is advised prior to introduction into clinical practice. aortic valve prolapse (avp) is a common echocardiographic finding in horses, but when compared with mitral valve prolapse in dogs, little is known about the natural progression of this condition. previously published data has shown that echocardiographic identification of avp in horses is reliable, diagnostic criteria have been established and that development occurs with training. the aims of this study were to evaluate the different rna and protein expressions of smooth muscle actin (sma), transforming growth factor-b (tgf), nitric oxide synthase (nos) and the concentrations of elastin and collagen in normal, prolapsing and diseased cusps to evaluate what structural changes may predispose them to prolapse. valve cusps were harvested and processed from a group of horses at a commercial abattoir following disease classification using echocardiography. horses were aged . ae . years, weighing ae kg and with a median body condition score of / . cusps were collected in rnalater s and stored at À c prior to processing. cdna was produced from half a valve using a standard protocoland qrt-pcr performed to assess relative rna expression of sma, tgfb , endothelial (enos) and inducible nos (inos) and compared with the housekeeping gene s. a quarter of cusp was processed using an adapted commercial protocol to evaluate protein expression of sma, tgf b , enos and compared to vimentin. specific antibody binding was assessed with western blotting and protein expression evaluated using dot blots. the remaining quarter cusp was used to measure soluble collagen and elastin concentrations using commercial assays . statistical analyses included one way anova with post-hoc bonferoni, paired student's t-test, linear and logistic regression. there was no effect of gender or age on any of the measurements. valves from animals with avp had lower expression of sma and elastin compared to normal and diseased valves, increased expression of tgfb and enos, whereas inos expression was greater than normal valves (table ) . collagen content of valves from horses with avp was increased compared to normal but lower than horses with valve disease. prolapsing cusps appear to be a different phenotype from diseased cusps. further studies will help to elucidate the significance of these findings in vivo. a clear association between heart rate (hr) and body mass has been observed across a wide range of mammalian species. furthermore, it is well known that electrocardiographic (ecg) time intervals vary with heart rate and body mass. within the equine species, small breeds are generally thought to have higher heart rates than large breeds. however, despite the large differences in size among different equine breeds, there is little information about normal heart rates and normal ecg time intervals in horses and ponies of different body size. similarly, the relationship between hr and body mass in dogs of various breeds and sizes is still under debate. the goal of this study was to investigate the relationship between heart rate and ecg time intervals to body mass in apparently healthy horses and ponies and to calculate normal ranges for different weight groups. adult horses and ponies at an age of . ( - ) y [median (range)] and a body weight of ( - ) kg were included in the study. all animals were considered clinically healthy based on history and physical examination. a standard base-apex ecg was recorded at a speed of (n ) or mm/s (n ) using a multiparameter monitor (datascope passport). during the procedure, the horses were unsedated, standing quiet in a box stall. mean hr over sec was determined for each recording. the following ecg time intervals were measured in triplicate and averaged for further analyses: pq interval, qrs duration, qt interval, and difference between qt and qrs (qt-qrs duration). the relationship between hr, ecg time intervals, and body mass was assessed using linear regression analyses. normal ranges ( . % to . % percentile) were calculated for different weight groups. the level of significance was p . . heart rate was inversely related to body mass (p o . , r . ). the pq interval (p o . , r . ), qrs duration (p o . , r . ), qt interval (p o . , r . ), and qt-qrs duration (p o . , r . ) were directly related to body mass. normal ranges for hr, pq, qrs, and qt within the different weight groups were - bpm, - ms, - ms, - ms (o kg); - bpm, - ms, - ms, - ms ( - kg); - bpm, - ms, - ms, - ms ( - kg); - bpm, - ms, - ms, - ms ( - kg); and - bpm, - ms, - ms, - ms ( kg). we conclude that in healthy horses there is a significant but weak relationship between body mass and hr and between body mass and ecg time intervals, respectively. this study therefore supports the hypothesis that within the equine species, small breeds have faster heart rates and shorter ecg time intervals than large breeds. therefore, body mass has to be considered when comparing hr and ecg time intervals to normal ranges in horses. horses with pituitary pars intermedia dysfunction (ppid) often have elevated plasma acth concentrations. however, ppidaffected horses rarely have resting serum cortisol levels above the reference range or adrenal hyperplasia. we hypothesized that this apparent dissociation between plasma acth levels and adrenal response in horses with ppid is due to the secretion of acth that is less biologically active than that from normal horses. to test our hypothesis, a bioassay to evaluate acth activity was developed. adrenocortical explants were harvested aseptically from normal horses at euthanasia and stimulated with plasma from healthy (n ) and ppid-affected horses (n ). the assay was performed three times with explants obtained from different horses. cortisol secreted by the explants and plasma acth levels were measured with commercially available radioimmunoassays. cortisol secretion stimulated by each sample was standardized to the respective explant protein concentration. cortisol data was normalized for acth concentration in each plasma sample and expressed as a cortisol:protein:acth ratio. ratios from horses with ppid and normal horses were compared by unpaired t-test. horses with ppid had significantly lower cortisol:protein:acth ratios compared to normal horses. (assay : . ae . vs. . ae . , p o . ; assay : . ae . vs. . ae . , p o . ; assay : . ae . vs. . ae . , p o . ). these results suggest that plasma acth from ppid horses is less biologically active than plasma acth from normal horses. our findings give further insight into the pathophysiology of ppid and may aid in the development of novel diagnostic testing protocols. an online survey was conducted to determine perceived needs of potential employers of new acvim-laim diplomates. the survey was designed as the first step in determining what is needed for success in the various sectors of practice employing acvim-laim diplomates. demographic and background data were collected using questions and drop-down menus on the first page. the survey evaluated skills or concepts in areas of veterinary practice. participants answered questions about each skill or concept using drop-down ranked lists. those participants that had completed an acvim-laim training program were asked additional questions about whether they were taught the skill or concept during their own residency. data were collated and descriptive statistics calculated. the mean scores or frequencies of use for each skills or concepts were ranked to determine which of the skills or concepts were most important for an entry-level diplomate. eighty-eight individuals participated in the survey with respondents being acvim diplomates, respondent was not board-certified and respondent was an act diplomate. nineteen respondents were diplomates of acvim and an additional specialty. eighty-three respondents had completed an acvim residency. the majority of respondents were in academia ( %) with % being in private practice. equine specialists prevailed ( %) followed by mixed large animal ( %) and then food animal only specialists ( %). the distribution of years post-residency was slightly skewed toward younger diplomates, but overall there was a good distribution of diplomates across years of experience. most respondents stated that they did not make hiring decisions in their practice. competency in disciplines other than internal medicine was expected with ultrasonography and radiology being the most desirable followed by theriogenology and lameness. surgical skills, both equine abdominal ( %) and food animal general surgery ( %) were considered important by some respondents. thirty-six per cent of respondents thought that a new diplomate should expect to make o $ , per annum, while only % of respondents thought that a new diplomate should expect to make ! $ , per annum. not all respondents answered questions on all skills or concepts. the mean number of skills or concepts evaluated was (sd ) with only respondents answering all . all skills or concepts evaluated were found to be at least somewhat important, were estimated to be used at least occasionally, were recommended for inclusion in training programs as core or elective, and some level of knowledge was expected. at least some of the respondents were taught each of the skills or concepts during their residency, practiced the skill or concept at least occasionally during their residency, and some degree of competency was expected at the time of completion of their residency. these data will provide a framework for designing future laim residency programs. abstract this study evaluated pharmacokinetics and clinical safety of an oral paste formulation of a commercially available cox -sparing nsaid in clinically healthy pony foals in a randomized controlled clinical trial. values for complete blood count, serum chemistry profile, urinalysis, pharmacokinetic assay, and gastric endoscopy were evaluated in eighteen shetland pony foals treated with firocoxib ( . mg/kg, po, q h) or placebo for days. foals were divided into treatment groups. group and foals received firocoxib while a rd group was administered an oral placebo. gastric endoscopy was performed on group and foals prior to treatment and on days and to monitor for the presence of gastric ulcers. group and foals had blood and urine samples taken sequentially for pharmacokinetic analysis, cbc, serum chemistry evaluation, and urinalysis. physical examinations were performed prior to treatment and daily for days. data were analyzed using anova and paired t-tests (p o . ). none of the foals presented adverse clinical effects. there were no significant changes in cbc, biochemical profiles within groups, or differences between groups. pretreatment gastric endoscopy scores were not significantly different from evaluations at and days. firocoxib was quickly absorbed with an observed maximum concentration at hr, the first sampling interval, for the majority of animals. firocoxib plasma concentrations decreased in a log-linear manner after reaching the maximum concentration and steady state concentrations were achieved by the th dose. based on the sampling times after the final and th dose, an average half life of . days was estimated. administration of firocoxib did not cause any adverse effects on gastrointestinal, or hematological or serum biochemical variables, appears to have been well tolerated, and follows a predictable pharmacokinetic pattern in - week old foals. equine herpesvirus (ehv- ) is highly prevalent in most horse populations. horses are routinely vaccinated against ehv- , and neutralizing antibodies have helped to prevent disease. however, the usda has recently classified ehv- myeloencephalopathy (ehm) as an emerging disease, in response to the apparent increase in incidence, morbidity, and mortality of ehm that suggests a change in virulence of the virus. it has been reported that cellular immune mechanisms, in particular cytotoxic t-cells (ctls), are important in controlling ehv- viremia. interferon-alpha (ifn-a) has a key function in innate immune regulation by inducing the differentiation and maturation of ctls. here, we investigated the influence of abortogenic (racl , ny ) and neuropathogenic (ab ) ehv- virus strains on ifn-a, il- and il- secretion in equine pbmc. equine pbmc were infected with racl , ny or ab ehv- strains or kept in medium for hours. ifn-a, il- and il- secretion was detected in the supernatants by a fluorescent bead-based cytokine assay. the production of ifn-a increased with increasing viral doses and similarly for all three ehv- strains. the production of the antiinflammatory cytokine il- was significantly decreased after ab infection compared to racl and ny strains at viral infection doses of moi . - . at high doses (moi ), il- production was suppressed by all three ehv- strains. the results suggested that abortogenic and neuropathogenic ehv- strains equally induce antiviral ifn-a production in equine pbmc. they also illustrated the differences in the ability of ehv- strains to modulate anti-inflammatory il- . neuropathogenic ab strain had an increased potential to down-regulate il- production suggesting specific viral mechanisms that interfere with the control of inflammation in the host. the variations in innate il- secretion might influence the development of protective immunity and might offer an explanation why neuropathogenic ab induces more severe disease, including myeloencephalopathy, than abortogenic ehv- strains. previously presented at a conference of research workers in animal disease. rhodoccocus equi is the major cause of pneumonia in foals during the first six months and control measures are frequently ineffective. treatment protocols are long, expensive and do not always produce good results. rhodococcosis prevention through immunization of foals using a safe and efficient vaccine is still a challenge. recent studies are based on the use of the virulence associated protein a (vapa) which has been described as an important inducer of immunity against r. equi. the present study evaluated the clinical and immune response of foals vaccinated with an attenuated strain of s. enterica typhimurium expressing vapa antigen (test group) or s. enterica typhimurium without the vapa gene (control group), previous to and following experimental challenge. two experimental phases were established according to the immunization route: intranasal or oral vaccination up to hrs following birth and at days of age. the experimental and control groups were challenged on day with a virulent stain of r. equi. clinical examination, cbc and image complementary exams were used to evaluate the development of clinical signs. immune response patterns were evaluated though immunoglobulin dosage, cytokine expression, lymphocyte proliferation assays, isolation of r. equi and cytological profiles of tbw. clinical manifestation was less intense in the test group during the second experimental phase, and death occurred only in the control group ( / ) and was due to r. equi pneumonia. the test group produced a more intense iggb response when compared to controls however no statistical difference was observed. lymphoproliferation and th cytokine expression were higher in the test group. in contrast, controls produced an il- response. local iga was significantly higher in animals immunized with salmonella carrying vapa. immunization protocols produced no severe toxic effects. the vaccination of neonatal foals with s. enterica typhimurium expressing vapa was considered safe, produced efficient modulation of the immune response and is apparently able to protect against experimental r.equi infection. this study was conducted to test the hypothesis that the kd protein, myristolated alanine-rich c-kinase substrate (marcks), is involved in equine neutrophil migration and adhesion. in other species, marcks phosphorylation and dephosphorylation causes the protein to cycle between the cell membrane and cytosol, respectively. to investigate marcks phosphorylation in horses, neutrophils were isolated from whole blood and stimulated with platelet activating factor (paf), leukotriene b (ltb ) or phorbol myristate acetate (pma). western blot was performed using specific phospho-marcks and total marcks primary antibodies. these results determined marcks phosphorylation is maximal seconds following stimulation and that dephosphorylation occurs within minutes. to investigate the requirement for marcks in equine neutrophil chemotaxis, isolated neutrophils were pre-treated with mans (a cell permeant peptide identical to the n-terminal amino acids of marcks), rns (a control peptide) or vehicle control (vc) prior to a migration assay toward known neutrophil chemoattractants (ltb or paf). pre-treatment of equine neutrophils with mans significantly inhibited migration while rns pre-treatment had no effect. to investigate marcks requirement in equine neutrophil adhesion, mans, rns or vc treated cells were stimulated to adhere to immulon plates coated with % fbs. pre-treatment of equine neutrophils with mans significantly inhibited adhesion while rns pre-treatment had no effect. inhibition of marcks using a cell permeant peptide identical to the protein's n-terminus significantly inhibited equine neutrophil adhesion and migration. these results indicate that marcks is an important regulator of equine neutrophil chemotaxis and represents a potential target for anti-inflammatory therapy. amongst other tests, a thorough neurologic examination of horses may include walking with the head elevated and during blindfolding, in order to help differentiate normal from abnormal and to help with neuroanatomically localising any lesion(s) i.e. in the ataxic horse. consensus amongst equine neurologists suggests that gait abnormalities associated with these specific tests are often exacerbated in horses with underlying proprioceptive deficits however the effect of these tests on temporal gait characteristics in normal horses has not previously been assessed quantitatively. we hypothesized that head elevation or blindfolding, in comparison with walking in a straight line would result in a compensatory decrease in lateral (left front-on to left hind-on and right front-on to right hind-on) and diagonal coupling intervals (left front-on to right hind-on and right front-on to left hind-on) in normal horses. four thoroughbreds without any history or clinical signs suggestive of neurological disease (age range to years) were included in the study. retroreflective markers were applied to the withers, to the sacrum and to left and right tuber coxae; for each limb, lateral fetlock markers and dorsal and lateral hoof wall markers were used. a minimum of trials each with - walk strides for each task were analysed as horses walked across an -force-plate runway i surrounded by a -camera kinematic system. ii force-plate data were processed with semi-automated custom written matlab iii scripts. data were analysed with a mixed model using the statistical software r. there was a significant fixed effect of normal walk on a straight line and head elevation on left and right lateral coupling intervals (p o . ) and of the left and right diagonal coupling intervals (p o . ). there was no significant effect of blindfolding on neither lateral nor diagonal coupling intervals. the random effect of horse had no influence on the coupling intervals. the decrease of the lateral coupling intervals indicates a tendency towards a pacing gait during head elevation. we conclude that there is a significant change in temporal gait characteristics of non-neurologic horses when the head is elevated but not during blindfolding compared to normal walking. current results suggest that pacing and increased variation in foot-placement during head elevation should be interpreted with caution however further work is required to determine whether the change differs between horses with neurological disease and non-neurologic disease. hereditary equine regional dermal asthenia (herda) is an autosomal recessive connective tissue disorder associated with a mutation in cyclophillin b that leads to impaired collagen folding, aberrant wound repair, and corneal abnormalities. it affects young quarter horses, appaloosa, and paints. herda shows similarities to the human hereditary connective tissue syndrome ehlers danlos (eds). many eds patients suffer from joint pain and osteoarthritis (oa) as adults. the similarity between eds and herda raises the question whether horses suffering from herda develop oa. in oa, excess production of inflammatory mediators such as prostaglandin e (pge ) activate enzymes that degrade cartilage as well as impede wound healing. the present study examined articular cartilage from yearling horses afflicted with herda. we hypothesized that chondrocytes from these horses are continually activated to produce inflammatory mediators. to test this hypothesis, articular cartilage from carpal and tarsal joints of herda horses were evaluated using histology. pge production by chondrocyte cultures was measured by elisa and analyzed by one-way anova, tukey post-hoc test, p o . significance. we also determined the antiinflammatory effects of an avocado/soybean unsaponifiables (asu), glucosamine (glu), and chondroitin sulfate (cs) mixture (ingredients found in cosequin s asu) and phenylbutazone (pbz) on chondrocytes. cosequin s asu and pbz are used alone or in combination for the management of oa. chondrocyte cultures were incubated for hrs with control media alone, a clinically relevant concentration of pbz ( mg/ml), or the combination of asu (nmx s , . mg/ml) glu (fchg s , mg/ml) cs (trh s , mg/ml). articular cartilage from joints of five herda-afflicted horses showed gross and histologic evidence of osteoarthritic lesions. chondrocyte cultures from cartilage of horses suffering from herda spontaneously produced greater pge than chondrocytes from normal horses ( -fold). pbz significantly decreased pge production by $ % (p o . ). the combination of asu -glu cs also significantly reduced pge production by $ % (p o . ). the present study supports anecdotal findings that horses suffering from herda are likely to develop oa. the inhibition of pge synthesis by asu glu cs suggests that this combination may be beneficial for the management of oa in herda. research supported by nutramax laboratories, inc. equine inflammatory airway disease (iad) is a common condition often treated empirically with corticosteroids. gene expression analysis in the bronchoalveolar lavage fluid (balf) may help understand the effects of corticosteroids in iad. the first part of the study aimed at identifying reference genes in the balf of iad horses treated with corticosteroids. the second part of the study investigated the effects of dexamethasone and fluticasone propionate treatments on the mrna expression of il- b, il- , il- and il- . the expression stability of seven candidate reference genes was determined in balf taken pre-and post-treatment with dexamethasone and fluticasone propionate in horses with iad. primers' efficiencies were calculated using linregpcr. normfinder, genorm and qbaseplus softwares were used to rank the genes according to their stability. gapdh was the most stably expressed gene whereas b m was the least stable gene. in addition, genorm analysis revealed that the number of genes required for optimal normalization was four (gapdh, sdha, hprt, rpl ). in the second part of the study the mrna expression of il- b, il- , il- and il- was measured in balf samples from seven iad horses treated in a randomized cross-over design with dexamethasone ( . mg/kg sid, days) or inhaled fluticasone propionate ( mcg bid with aerohippus, days). the balf samples were taken at baseline and after each treatment period. there was no significant effect of the corticosteroids treatment on the mrna expression of il- b, il- and il- in the balf. the mrna expression of il- was suppressed by dexamethasone and fluticasone propionate treatments. pneumonia is observed in horses after long distance transportation in association with confinement of horses' head position leading to a reduction in tracheal mucociliary clearance time (tmct). we hypothesize that clenbuterol, a beta- agonist shown to increase tmct in the horse, will ameliorate the affect of a fixed head position on large airway contamination and inflammation in a long-distance shipping model. six adult horses were enrolled in a cross-over design prospective study. horses were housed with their heads in a fixed position for hours to simulate long distance transport, and treated with clenbuterol ( . ug/kg po q h) or a placebo starting hours before simulated shipping. tmct was measured using a charcoal clearance technique. data were collected at baseline and hours, and included tmct, tracheal wash cytology and quantitative culture, rectal temperature, cbc, fibrinogen, and serum tnfa, il- and il- levels. there was a -week washout between study arms, and each horse served as its own control. the data was analyzed using regression analysis and wilcoxon rank-sum tests. no statistically significant difference was seen between treatment and placebo groups for any of the variables investigated. tmct did not differ after treatment ( . ae . cm/min) versus placebo ( . ae . cm/ min; p . ), and intratracheal bacterial counts were similar for treatment (  ae  cfu; p . ) and placebo (  ae  cfu) groups. a reduction of tracheal b hemolytic streptococcus. spp. after clenbuterol versus placebo was also nonsignificant ( % versus %; p . ). in conclusion, treatment with clenbuterol does not appear to combat the deleterious effects of this long-term shipping model. breathing cold air during strenuous exercise is associated with airway inflammation. under these conditions, warming and humidification of inspired air occurs in the lower respiratory tract resulting in mucosal cooling, desiccation, and hyperosmolarity. the purpose of this research was to test the hypothesis that airway hypertonicity causes inflammatory cell migration and alterations in cytokine expression associated with exercise induced airway inflammation. horses (n ) were examined in a randomized crossover design after exposure to hypertonic aerosols ( minute nebulization with solutions of either isotonic or hypertonic mannitol). airway leukocytes were harvested and hours post aerosol challenge via bronchoaveolar lavage, and were used to determine total and differential nucleated cell count and expression of cytokinespecific mrna. hypertonic aerosol challenge resulted in an increase in total number of cells hr after challenge, characterized by increased macrophage (p . ) and neutrophil (p . ) concentrations, but there was no effect on airway leukocyte concentrations hours after nebulization. no significant changes in the relative quantity of mrna for airway cytokines were noted at either time point. these data demonstrate that transient airway hypertonicity can cause airway leukocyte influx and may be responsible for the airway inflammation commonly found in athletes that exercise in cold conditions. however, our data do not support the hypothesis that hypertonicity is the sole initiating cause of changes in cytokine expression secondary to cold weather exercise. it is likely that factors such as airway temperature, shear stress or epithelial damage also play a role in this phenomenon. we studied the importance of abdominal sonograms in neonatal foals suffering from gastrointestinal conditions. we hypothesized that there would be a subgroup of neonates with sonographically detectable pneumatosis intestinalis (pi) as a reflection of a necrotizing component of the disease. records of foals days of age hospitalized between and with signs of gastrointestinal disease were evaluated (n ). the association of sonographic, clinical, pathological and clinicopathological signs with outcome and severity of disease was determined. pneumatosis intestinalis was imaged in foals. twenty-seven foals were classified as having necrotizing gastrointestinal disease based on the presence of gastrointestinal signs (colic, diarrhea, gastric reflux or abdominal distension) and pi detected sonographically ( ), surgical ( ) or pathological ( ) evidence of gastrointestinal necrosis. there was a difference between overall survival rate ( %) and survival rate in foals with necrotizing disease ( %, p . ) or foals with pi detected sonographically ( %, p . ). pneumatosis intestinalis was the only sonographic finding associated with outcome. sonographic abnormalities in peritoneal fluid, stomach, duodenum, jejunum, cecum, umbilicus or the presence of meconium were associated (p o . ) with surrogates of severity of disease (hospitalization cost or days of hospitalization). hypoproteinemia was associated with pi (p . ). the presence of blood in the feces, reflux and abdominal distension was associated with necrotizing gastrointestinal disease (p o . ). abdominal sonograms have prognostic value in neonatal gastrointestinal disease. pneumatosis intestinalis was a common sonographic sign that worsened the prognosis. the therapeutic implications of detecting a necrotizing component of the gastrointestinal disease deserve further study. the interaction of insulin and the microvascular endothelial insulin receptor (irc) plays an important role in the normal and insulin resistant (ir) individual. while endothelial irc signaling is normally vasodilatory, this effect is well-documented to reverse in the ir individual, resulting in vasoconstriction. although vascular dysfunction has been reported in sepsis-associated equine laminitis, the role of the laminar microvasculature in endocrinopathic laminitis remains poorly characterized. the purpose of this study was to characterize the pattern of irc expression in digital laminae in ponies subjected to a dietary carbohydrate challenge that mimicked abrupt exposure to pasture rich in nonstructural carbohydrates (nsc). mixed-breed ponies (body weight . /- . kg) received a diet of hay chop (nsc $ % on a dm basis) for weeks prior to initiation of the experimental feeding protocol. following conditioning, ponies either remained on the control diet (n ) or received the same diet supplemented with sweet feed and oligofructose (total diet $ % nsc; n ) for a period of days. serum insulin concentrations were measured prior to and after completion of the feeding protocol. at the end of the feeding protocol, sections of numerous tissues, including dorsal digital laminae, were collected immediately following euthanasia. the samples were formalin-fixed for hours, transferred to % ethanol, and paraffin-embedded. laminar sections were stained immunohistochemically for irc using a commercially-available antibody (abcam); the number of irc ( ) cells was quantified in x light microscopy fields (n ) for each section. the total number of irc ( ) cells was greater in the laminae of challenged ponies than control ponies (p . ), and there was a significant correlation between the change in serum basal insulin concentration and number of laminar irc ( ) endothelial cells (r . ; p o . ). while the number of irc ( ) endothelial cells was significantly greater in the dermal laminae of challenged ponies (p . ), there was no difference in the number of interstitial irc ( ) cells (p . ). no epithelial irc ( ) cells were observed in any laminar section, and irc ( ) cells were conspicuously absent from the deep dermal tissue (including vessels). up-regulation of irc expression in the laminar vasculature occurs acutely in response to dietary carbohydrate challenge and accompanies hyperinsulinemia in ponies. the dramatic increase in endothelial irc expression in the laminar microvasculature in nutritionally challenged ponies, with no apparent epithelial irc present, suggests that hyperinsulinemia associated with exposure to increased dietary nsc may induce laminar injury by causing a similar vasoconstriction in ir equids as described in the microvasculature of ir humans. glucose transport from the blood stream into cells, the limiting step in whole-body glucose utilization, is regulated by a family of glucose transporter (glut) proteins in insulin-sensitive (i.e., muscle and adipose) tissues. we previously demonstrated that glut , the major isoform, is a key factor in the pathogenesis of equine insulin resistance (ir). while it has been recently demonstrated that glut (a newly discovered isoform) increases insulin-stimulated glucose transport in human muscle, its role in other tissues, particularly in the setting of ir, is not well characterized in any species. in addition, as has recently emerged as a key downstream signaling molecule regulating translocation of glut to the cell surface, the rate-limiting step in glucose uptake. we hypothesized that glut content would be differentially expressed across tissues and that ir would induce alteration in glucose transport by affecting active cell surface glut . biopsies of skeletal muscle, and subcutaneous and visceral adipose tissue were collected from light-breed horses, characterized as either insulin sensitive or compensated ir, based on the results of an insulin-modified frequently-sampled intravenous glucose tolerance test (n /group). we specifically quantified active cell-surface glut in these biopsies, using an innovative exofacial bismannose photolabeled assay, which has not been previously applied to glut . total glut protein expression was measured by western blots, as well as total and phosphorylated (indicating activation of) as . glut was expressed in all the depots with a significant regional effect. total glut protein content was increased (p o . ) in visceral (omental and mesenteric) compared to subcutaneous (nuchal ligament and tailhead) adipose tissue and skeletal muscle of healthy horses. ir did not induce alterations in active cell-surface and total glut content nor in total and phosphorylated as in any of the tissues evaluated. our data suggests that glut is abundant in visceral adipose tissue and is therefore likely to play a substantial role in the regulation of glucose transport. however, neither glut translocation nor as activation are impaired in insulin-sensitive tissues of ir horses. it is concluded that, in contrast with glut , glut does not appear to contribute to glucose transport alterations during naturally-occurring equine ir. insulin resistance (ir), characterized by exaggerated glycemic or insulinemic responses to glucose challenge, is a key metabolic disturbance in horses that develop obesity-associated laminitis. in addition to obesity, diet and age have been demonstrated to affect tissue sensitivity to insulin in other species but these factors have received limited investigation in horses. we hypothesized that there would be greater glycemic and insulinemic responses to a sweet feed meal in aged horses, as compared to adult horses, as well as in horses adapted to a forage-only diet. three diets, grass hay only, grass hay plus sweet feed (starch and sugar-rich, ss), and grass hay plus a fat and fiber (ff) feed, were fed to mares, adult ( - yr) and aged ( yr), for a -week adaptation period in a randomized design. glycemic and insulinemic responses to a standardized meal of sweet feed ( g/kg bw offered for hour) were determined for hours from the onset of feeding. peak glucose and insulin concentrations and areas under the glucose or insulin vs. time curves (auc-g, mg/ dl/ min, and auc-i, mu/ml/ min) were determined and data were analyzed by one-and two-factor repeated measures anova. there were no differences between age groups in glycemic responses to any of the diets. however, in aged horses peak glucose concentration (p o . ) and auc-g (p o . ) were greater after adaptation to the forage-only diet, as compared to the other two diets. in contrast, aged horses had a greater peak insulin concentration (p o . ) and auc-i (p o . ) than adult horses on all diets but no differences in peak insulin concentration or auc-i was found between diets within age groups. as hypothesized, the insulin response, but not the glycemic response, to a sweet feed meal was greater in aged horses, regardless of background diet. further, the glycemic response was greatest after adaptation to a forage-only diet, but this finding was only significant in aged horses. morbidity, mortality, and economic loss to the equine industry. in obese humans and rodent models of nutritional obesity, systemic insulin resistance and hyperinsulinemia are followed temporally in a majority of individuals by decreased glucose tolerance, pancreatic bcell failure, and type ii diabetes mellitus. in stark contrast to humans, obese horses and ponies chronically remain in what is termed a ''prediabetic'' state in human ir, characterized by hyperinsulinemic euglycemia. few data exist describing the biology of the equine endocrine pancreas in the chronically ir animal that may both: ) explain this unique equine endocrine physiology and ) characterize the animal at-risk for hyperinsulinemia-associated laminitis. the purpose of the study reported here was to characterize the morphology and physiology of the equine endocrine pancreas in response to a dietary carbohydrate challenge. twenty-two mixedbreed ponies (body weight . ae . kg) were conditioned to a diet of chopped hay (nsc $ % on dm basis) for weeks; following conditioning, ponies either remained on the control diet (n ), or received the same hay supplemented with sweet feed and oligofructose (total diet $ % nsc; n ) for days. serum insulin concentrations were measured prior to and after completion of the feeding protocol. at the end of the feeding protocol, sections of numerous tissues, including pancreas, were collected immediately following euthanasia. the samples were formalin-fixed for hours, transferred to % ethanol, and paraffin-embedded. immunohistochemistry was performed on pancreas sections using a commerciallyavailable anti-insulin antibody (abcam), and measurements of islet surface area and b-cell surface area were performed (n islets per tissue section) using a commercially-available computer software program (image j). there was a trend for greater total islet surface area in pancreatic tissue from ponies fed the high nsc diet when compared to the ponies on the hay diet (p . ); however, no difference was noted in b-cell surface area between diet treatments (p . ). the change in serum insulin concentration was significantly greater in the high nsc-fed ponies than in controls ( . /- . miu/l vs. . /À . miu/l; p . ); however, this variable was not correlated with total islet surface area (r . ; p . ) or b-cell surface area (r . ; p . ). due to the relatively modest changes in pancreatic islet surface area that accompany marked increases in serum insulin concentrations in ponies fed a high nsc diet, it is important to assess both b-cell function and insulin clearance mechanisms in future studies to delineate the mechanism(s) of hyperinsulinemia in this model. humans that suffer from obesity show exaggerated inflammatory responses and this may be relevant to the association between increased adiposity and laminitis in horses with equine metabolic syndrome (ems). this study was performed to test the hypothesis that inflammatory responses to endotoxemia differ between healthy horses and those affected by ems. six healthy adult mares and horses with ems received an intravenous infusion of lipopolysaccharide (lps; ng/kg in ml sterile saline) or saline alone. a crossover design was employed with a -day washout period. physical examinations were performed hourly for h and whole blood was collected at , , , , , and min for assessment of inflammatory cytokine gene expression. a liver biopsy was performed between and min postinfusion. data were analyzed using mixed model anova. mean rectal temperature, heart rate, and respiratory rate increased following lps infusion (treatment  time; p o . ), with higher heart (group  treatment; p . ) and respiratory rates (group; p . ) detected in ems horses. lipopolysaccharide infusion significantly increased whole blood gene expression of tumor necrosis factor a (tnfa), interleukin (il)- b (p o . ), il- (p o . ), il- (p o . ), and il- (p . ), and hepatic gene expression of il- (p o . ), il- (p o . ), and il- (p . ). inflammatory gene expression did not differ significantly between groups, so our hypothesis was not supported. heart rates tended to be higher when lps was administered to horses with ems. elevated serum concentration of cardiac troponin i (ctni) is a biomarker for myocardial damage in horses. preferred times to test blood for ctni levels following athletic performance or other events that may cause myocardial injury are not yet established and would be affected by time of release from the myocytes, location of release within the myocytes, duration of release and half-life of ctni in the horse. this information would be necessary to more accurately and reliably test horses for myocardial injury. the aim of this study was to determine the elimination half-life (t / ) of equine ctni. to establish the t / of equine ctni in horses, ctni was recombinantly expressed in e.coli. two healthy ponies received intravenous injections of recombinant equine ctni and plasma ctni concentrations were measured with a point-of-care ctni analyzer at multiple time points after injection. standard pharmacokinetic analysis was performed to establish the elimination half-life of ctni. for comparative purposes, data were subjected to pharmacokinetic models describing a single versus biphasic elimination profile. elimination of recombinant equine ctni following intravenous administration exhibits a short half-life. establishing the t / of troponin provides critical information in understanding the clinical application of this cardiac biomarker in clinical practice. this study describes a true biological ctni t / , which has not been documented in any species thus far. stall-side assessment of this cardiac biomarker in horses should enhance the ability of clinicians to detect myocardial damage and aid in the management and treatment of horses with cardiac disease. the objective of the study was to evaluate the between-pony, within-pony, between-analyser and within-analyser variation of flow-mediated vasodilation (fmd) measurement in healthy ponies, to investigate the hypothesis that fmd occurs in healthy ponies. six healthy, native breed, unrelated pony mares of varying weight ( - kg), body condition score ( / - / ) and age ( - years) were used. the median artery was occluded for minutes. twodimensional ( d) ultrasonographic images of the artery were recorded for seconds prior to and for minutes after occlusion. the peak luminal diameter was compared to baseline diameter to calculate the relative percentage increase in luminal size (fmd). images were obtained from six ponies on one occasion and from one pony on six occasions. analysis of images was performed by two independent analysers and by one analyser twice. the mean (sd) fmd in ponies was . % ( . %) and in pony ( occasions) was . % ( . %). coefficients of variation were . % and . % respectively. agreement between analysers was fair (icc . ) and within analyser was poor (icc . ). fmd is used to assess endothelial function in humans and has recently been assessed for its use in canine subjects. fmd occurs and measurement is feasible in ponies. fmd could be used to assess endothelial function, in the context of laminitis or other cardiovascular diseases. current state-of-the-art technique for measuring blood pressure (bp) in the horse is invasive and involves cannulation of the facial artery. indirect techniques, such as oscillometry, have proven useful in the anaesthetised horse, but have not become routine in the standing horse. monitoring bp can be indicated for the diagnosis and treatment of the hypotensive patient (ie. caused by endotoxemia, hypovolemia, systemic inflammatory response syndrome and cardiac failure) or the hypertensive patient (ie. due to equine metabolic syndrome or pain). the objective of this study was therefore to a) describe the methodology for application of oscillometric bp using a cuff applied to the tail in the standing horse and b) and to determine accuracy and precision of this method applied to the normotensive standing horse. the oscillometric method is simple to apply in a clinical setting. a pneumatic cuff is snugly applied to the unclipped tail-base with the cuff bladder centered over the middle coccygeal artery. the tail circumference must match the manufacturers description of the cuffs diameter range. the oscillometric apparatus inflates the cuff and obtain systolic, diastolic and mean arterial bp (sap, dap and map). at least consecutive measurements must be obtained. a correction of . mmhg/cm vertical distance between cuff and heart level is added to the measurement to correct for hydrostatic pressure difference. for determination of accuracy and precision of indirect sap, dap and map, eight healthy horses (age to years), was equipped with an intra-arterial catheter ii in the facial artery and a commercial tail-cuff oscillometric apparatus. i measurements were recorded every minutes for minutes. the data were analysed with the statistical software r using a mixed model with repeated measurements and a bland-altman analysis corrected for repeated measurements. oscillometric bp was accurate and precise for map (mean bias, lower confidence level, upper confidence level, variation in difference, all mmhg) (À . , À . , . , . , respectively) in the conscious horse but not for sap (À . , À . , . , . , respectively) and dap ( . , . , . , , respectively) . there was no significant contribution to the statistical model of either horse or measurement number. all horses tolerated the tail-cuff well and the method was simple to apply. only map could be measured with acceptable accuracy and precision in the normotensive standing horse using the described oscillometric method. reference intervals for thyroid hormone (th) concentrations have not been established for donkeys. therefore, clinicians must use reference ranges from horses, potentially leading to misdiagnosis of thyroid diseases. we hypothesized that th concentrations are different between donkeys and horses. the purposes of this study were: a) to compare th concentrations between donkeys and horses and, b) to determine whether the age may influence th concentrations. thirty-eight healthy donkeys ( . ae . years), mixed breeds, and healthy andalusian horses ( . ae . years) were used. donkeys were divided into three groups: o years (n ), - years (n ), and years (n ). serum concentrations of total triiodothyronine (tt ), free triiodothyronine (ft ), total thyroxine (tt ), free thyroxine (ft ), reverse triiodothyronine (rt ) and thyroid-stimulating hormone (tsh) were quantified by radioimmunoassay. all blood samples were collected the same day. neither horses nor donkeys had received any treatment for days before sampling and both farms had similar production conditions. total t , ft , ft and tt concentrations were higher (p o . ) in donkeys than horses. in contrast, no statistical differences were found for rt and tsh concentrations. young donkeys ( o years) had higher ft , tt and rt concentrations compared to other donkey groups (p o . ). old donkeys ( years) had lower tt and ft concentrations than both younger donkeys groups (p o . ). this study shows that there are differences in th concentrations between donkeys and horses, raising awareness on the possibility of misdiagnosis of thyroid gland dysfunction when using values from horses, being necessary to determine exclusive reference intervals for donkeys. ovariectomy is associated with alterations of responses to many hormones, not just those associated with reproductive function. in humans and rats, ovariectomy leads to insulin resistance, increased adiposity and altered fat mobilization. the effects of ovariectomy on energy metabolism have not been reported in horses. ovariectomized mares have been shown to respond normally to an acth stimulation test, but the response to suppression of the hypothalamo-pituitary-adrenal axis has not been previously described. the aim of this study was to evaluate the effect of ovariectomy on insulin response in mares and to determine if mares exhibit alterations in response to dexamethasone administration after ovariectomy. six healthy mares underwent an intravenous glucose tolerance test (ivgtt), an insulin sensitivity test (ist) and a dexamethasone suppression test (dst) before and weeks after bilateral ovariectomy. body weight, cortisol values at baseline, and hours after dexamethasone injection and acth values at baseline, and hours after dexamethasone injection, basal insulin/glucose ratio, time to reach a % decrease in blood glucose in the ist, time to reach baseline glucose concentration in the ivgtt and area under the curves plotting blood glucose and time to injection of glucose or insulin were compared before and after ovariectomy using a paired t-test or an anova for repeated measures. significance level was p o . . average body weight was decreased after surgery ( kg ). the injection of dexamethasone resulted in a serum cortisol concentration of less than mg/dl in all mares before ovariectomy, whereas after ovariectomy, dexamethasone injection resulted in a serum cortisol concentration of less than mg/dl in out of mares. in all cases, acth concentration was within the reference range ( - pg/ml) before and after ovariectomy. however, acth concentrations at t and at t were significantly higher after ovariectomy. each mare had a normal ivgtt, both before and after ovariectomy. additionally, no significant differences were observed in basal blood glucose ( ae mg/dl before and ae mg/dl after) or in the time to reach glucose baseline ( ae min before and ae min after). serum basal insulin concentration and insulin/glucose ratio was not significantly different before or after ovariectomy ( . ae . miu/ml and . ae . miu/ml and . ae . and . ae . , respectively), nor was the average time to reach a % decrease in blood glucose after insulin injection ( ae min and ae min, respectively). these findings suggest that, as reported in other species, the shortterm effect of ovariectomy may modify dexamethasone response in mares and that, contrary to other species, it may not modify insulin response. equine gastric ulcer syndrome (egus) is a common medical problem in horses. the high prevalence of gastric ulcers, vague clinical signs and negative effect on performance make it a significant clinical and economic problem within the horse industry. current pharmaceutical treatments are expensive and alter the acidic environment of the stomach. berries and pulp from the seabuckthorn plant (hippophae rhamnoides) are a rich source of vitamins, trace minerals, amino acids, antioxidants, and other bioactive substances and have been used successfully to treat stomach ulcers in man and rats. the purpose of this study was to evaluate the efficacy of a commercially sold, liquid extract of seabuckthorn berries (seabuck tm sbt gastro-plus) for treatment and prevention of gastric ulcers in horses. eight thoroughbred and thoroughbred-cross horses ( - years of age, geldings & mares, - kg) were used in a blinded two-period cross-over study. treatments consisted of control (untreated) and treatment (seabuck tm sbt gastro-plus) twice daily mixed with the grain meal. horses were treated for weeks followed by a week alternating feed-deprivation period to induce or worsen existing ulcers. gastroscopies were performed on all horses on day , week , and week (at the end of the alternating feed-deprivation period). gastric juice was aspirated and ph was measured. during gastroscopy, gastric ulcer scores were assigned to each stomach based on lesion number and severity. horses acted as their own controls, and between each treatment period the horses had a -week washout period. data was analyzed by anova for repeated measures via the glm procedure (sas inst. inc., cary, nc). when significant differences (p o . ) were observed, a post-hoc tukey's test was used to determine differences. non-glandular gastric ulcer scores significantly increased in all control and sbt-treated horses from week to week , after the feed-deprivation phase of the study. there was no significant difference in the non-glandular gastric number (p . ) and nonglandular gastric severity (p . ) scores in sbt-treated horses compared to non-treated controls. glandular ulcer number (p . ) and glandular ulcer severity (p . ) was significantly lower in the sbt-treated horses compared to the control horses. there was no significant difference in the ph (p . ) in sbt-treated horses compared to non-treated controls. thus, seabuck tm sbt gastro-plus, mixed in the feed twice daily, may be efficacious in controlling the severity of glandular ulcers in horses during stress, without increasing stomach ph. the availability of rapid and accurate quantitative fibrinogen measurements may be useful for evaluation of hospitalized equine patients. the abaxis vspro analyzer was evaluated for precision using two levels of human fibrinogen controls ( mg/dl and mg/dl), four different vspro machines, and two different lots of cartridges, assessed over subsequent days. the coefficients of variation of the assay ranged from % ( mg/dl) to % ( mg/ dl). we subsequently evaluated the abaxis vspro fibrinogen assay compared to fibrinogen concentration measured using the beckman coulter acl- in equine samples of varying fibrinogen concentrations obtained from horses with gastrointestinal disease. all samples were measured in citrated plasma. fibrinogen samples measured on the acl- ranged from to mg/dl (median mg/dl). vspro samples were run in duplicate, and the mean compared to the acl values. pearson correlation coefficient analysis generated an r value of . (p o . ). duplicate measurements on the vspro were strongly correlated to each other with an r value of . (p o . ). bland-altman analysis of these samples for the vspro compared to the acl- noted a bias of À ae mg/dl the results of this study indicate that the vspro benchtop fibrinogen analyzer provides accurate and precise fibrinogen data compared to the acl- reference analyzer. the immune response of foals to r. equi is incompletely understood and believed to be responsible for clinical disease caused by this pulmonary pathogen. in a recent study foals receiving a large inoculum exhibited th skewing with pneumonia and a small inoculum exhibited th skewing without clinical disease. we hypothesized that cytokine/chemokine production by pulmonary alveolar macrophages, in vitro, would increase with the infective dose and that the magnitude of the response would differ between foals and adults. alveolar macrophages were obtained by bronchoalevolar lavage from healthy mares and their -week-old foals. macrophage cultures were infected with r. equi ( or -) at a multiplicity of infection (moi) of or . total rna was harvested and hours post-infection, reverse transcribed and used as template for quantita-tive pcr. the ddct method was used to calculate relative gene transcripts for il- , il- p , tnfa and cxcl . cellular infections at moi resulted in significantly higher expression of il- , il- p and tnfa mrna transcripts compared to moi . however, the dose-effect was reversed for cxcl with significantly lower expression at the higher moi. there was no difference in magnitude of cytokine/chemokine responses by the alveolar macrophages between adults and foals. dose-dependent responses of alveolar macrophages may represent a novel mechanism by which r. equi could modulate immune responses and therefore disease. significant down-regulation of cxcl mrna transcripts associated with a higher dose is of particular interest as this chemokine plays a role in development of protective th responses. the intent of this study was to develop likelihood ratios (lrs) for infection attributable to corynebacterium pseudotuberculosis in horses based on synergistic hemolysis inhibition (shi) test titers. medical records for horses presented to the uc davis veterinary teaching hospital with serum submitted for shi titer determination were evaluated and cases met study inclusion criteria. these cases were grouped based on evidence of internal and/or external infection attributable to c. pseudotuberculosis and likelihood ratios with % confidence intervals determined. results showed increasing lrs indicating increasing odds for any form of active disease as titer increased with all cases considered. lrs for internal infection were for titers ! overall and for titers with external abscess cases excluded. no difference from (and therefore no significant change in pre-test to post-test odds) was seen in any lrs for internal disease when only cases with external disease were examined (external and internal disease vs. external only). overall, the shi test results showed usefulness in determining internal c. pseudotuberculosis infection in horses with no evidence of external abscessation. overall, however, higher titers were more indicative of active external or internal disease than internal disease specifically in contrast to previous reports. the shi test was unable to distinguish internal infection when external abscesses were present. salmonella enterica is a zoonotic pathogen that has tremendous impact on many different animal production and management systems. rapid detection of s. enterica in fecal samples may facilitate effective infection control practices. current detection methods require - hours (polymerase chain reaction or pcr) or - hours (enriched aerobic culture) to obtain results. alternatives have been developed, lateral flow antigen detection systems (lfads), which are currently marketed for salmonella detection related to food safety microbiology. the objective of this study was to evaluate two commercially available rapid salmonella detection systems in equine feces. fecal samples collected from repeatedly culture-negative horses were inoculated with known concentrations of salmonella enterica serotype typhimurium (five uninoculated control samples, and samples of each -fold dilution [ .  - .  cfu/gram of feces]). all samples were aerobically cultured using a standard enrichment technique. in a blinded fashion, samples were tested using two different lfads as well as plated on agar media for confirmatory testing. at hours of incubation, using bacterial culture as the reference method, test was correctly identify % of samples ( bacterial contamination of stalls with salmonella sp. is a serious problem in equine hospitals. salmonella sp. exposure to horses in the facility can result in nosocomial infections which results in temporary facility closure, until the organism is eradicated. hospital closure can result in loss of revenue, damage to reputation and interference with patient care. the purpose of this study was to evaluate three stall cleaning methods on eradication of salmonella sp. at an equine veterinary teaching hospital (vth). horses admitted to the vth were assigned to salmonella sp.negative stalls within areas of the vth during the study period (september -january . when the horses were discharged stalls were randomly assigned to one of three cleaning methods (pressure-washing only [pw] , pressure washing and hand scrubbing [pws] , or hand scrubbing only [s]) in a single period, non cross-over design. all stalls were stripped of bedding and surfaces sprayed with tap water and cleaned with a disinfectant quaternary-ammonia solution (super hdq neutral, spartan chemical co., inc, maumee, oh). the pressure-washing system (psc cleaning systems, inc., toronto, canada) used, provided a pressure of psi and a temperature range of - f. following cleaning, each stall was allowed to air dry and within hours, stall surfaces were sampled using three  sponges moistened with sterile saline. the person collecting the samples was masked to the method of cleaning. sponges were submitted to the louisiana animal disease diagnostic laboratory (laddl) for culture of salmonella sp. a chi-squared analysis was used to determine significant differences (limit p o . ) between cleaning methods and salmonella sp. isolation. during the study period, stalls (pw [n ]; pws [n ]; s [n ] were included. all stalls had negative environmental salmonella sp. cultures prior to beginning the study. for pw cleaned stalls, / ( . %) were salmonella sp.-positive, for pws cleaned stalls, / ( %) were salmonella sp.-positive, and for s cleaned stalls, / ( . %) were salmonella sp.-positive. although, there were fewer salmonella sp.-positive stalls ( . %) in the handscrubbed stalls, cleaning method did not significantly (p . ) affect the isolation of salmonella sp. from the stall environment. in conclusion, power washing alone, power washing and hand scrubbing, and hand scrubbing alone, using a quaternary-ammonia solution did not significantly affect environmental isolation of salmonella sp. from stalls surfaces in the vth during this study. the objectives of this study were to determine the plasma and pulmonary disposition of gamithromycin in foals and to investigate the in vitro activity of the drug against streptococcus equi subsp. zooepidemicus (s. zooepidemicus) and rhodococcus equi isolates. a single dose of gamithromycin ( mg/kg of body weight) was administered intramuscularly. concentrations of gamithromycin in plasma, pulmonary epithelial lining fluid (pelf), bronchoalveolar lavage (bal) cells, and blood neutrophils were determined using hplc with tandem mass spectrometry detection. the minimum inhibitory concentration of gamithromycin required to inhibit growth of % of r. equi and s. zooepidemicus isolates (mic ) was determined. additionally, the activity of gamithromycin against intracellular r. equi was measured. mean peak gamithromycin concentrations were significantly higher in blood neutrophils ( . ae . g/ml) and bal cells ( . ae . g/ml) compared to pelf ( . ae . g/ml) and plasma ( . ae . g/ml). mean terminal half-lives in neutrophils ( . h), bal cells ( . h), and pelf ( . h) were significantly longer than that of plasma ( . h). the mic of s. zooepidemicus isolates was . g/ml. the mic of gamithromycin for macrolide-resistant r. equi isolates ( g/ml) was significantly higher than that of macrolide-susceptible isolates ( . g/ ml). the activity of gamithromycin against intracellular r. equi was similar to that of azithromycin and erythromycin. intramuscular administration of gamithromycin at a dosage of mg/kg would maintain pelf concentrations above the mic for s. zooepidemicus and phagocytic cell concentrations above the mic for r. equi for approximately days. eight western stock yearling horses were infected with ehv- (ab ) by nasopharyngeal instillation. venous blood samples for collection of plasma were collected in na-citrate tubes on the day prior to infection (d - ) and on d through d . in addition, clinical data, nasal swabs and peripheral blood mononuclear cells (pbmc) for detection of viremia were collected on the day before infection (d - ) and on d through d post-infection. d-dimer concentrations were determined in citrated plasma samples using a latex agglutination test (minutex d-dimer, biopool, ireland). viral load in pbmc was determined using quantitative pcr. all horses showed bi-phasic fevers typical for ehv- infections. one horse developed acute ehm on d and was euthanized after samples were collected. in all horses d-dimers were undetectable on d - and on d , and . in contrast, all horses had increased ddimer concentrations for to consecutive days starting on day post-infection. d-dimer concentrations in horses increased to ug/ml and one of these horses was the horse with acute ehm. interestingly, mean increased d-dimer concentrations showed timely overlap with the mean fever curve and, delayed by day, with the mean viremia curve. because plasma samples for d-dimer measurements were not collected during the first days post-infection, which are typically associated with a primary fever, conclusion on the association of d-dimers with fever of viremia await analysis of a second study currently conducted in our laboratory. in conclusion our data indicates that during ehv- infection with neuropathogenic strains activation of the coagulation cascade and production of cross-linked fibrin is wide-spread; not limited to horses with clinical signs of ehm, and can be expected between days and post-infection. lawsonia intracellularis is an emerging pathogen in horses and the causative agent in equine proliferative enteropathy (epe). the goal of this study was to evaluate the exposure of pre-weanling foals and broodmares to lawsonia intracelluaris on several farms in louisiana with a history of epe and compare the results to several farms with no known clinical cases of epe in foals. an additional goal of the study was to identify whether a relationship exists between lawsonia intracelluaris and other gastrointestinal pathogens in foals. whole blood and fecal samples were collected from mares and foals from four breeding farms in louisiana. farms a and b had no known clinical cases of epe, while farms c and d had previous know cases of epe in . serum samples were examined for the presence of antibodies against lawsonia intracellularis using an immunoperoxidase monolayer assay (ipma). dna was extracted from fecal samples using a commercial dna kit and molecular detection of lawsonia intracelluaris was assayed using real-time pcr. fecal ova were determined using quantitative sucrose floatation. the presence of fecal clostridium difficile toxin was measured using a commercial enzyme linked immunosorbent assay (elisa). three of the farms examined had foals and mares with exposure to l. intracellularis as evidenced by serum antibodies against the organism. of the total population sampled, foals ( . %) and mares ( . %) had evidence of antibodies to l. intracellularis based on serology. three foals ( . %) tested positive for l. intracellularis organism by fecal pcr, and all of these foals were located on farm c. of these, one of the foals was seronegative, while the other two were seropositive. farm c also had the highest percentage of mares ( . %) serologically positive for l. intracellularis, while farm a had the highest percentage of foals ( . %) with antibody titers against l intracellaris. farm c also had the only pairs (n ) of serologically positive mares with seropositive foals. while farm a and b had seropositive mares and/or foals, none of the foals were positive for l. intracellularis fecal shedding by pcr. all serum and fecal samples were negative for evidence of l. intracellaris on farm d. ten foals ( %) had fecal egg counts greater than egg per gram and foals ( %) were positive for c. difficile toxin. this study demonstrated evidence of natural exposure to l intracellularis on farms both with and without a history of epe in louisiana. further, this study failed to establish a relationship between l intracellularis and other gastrointestinal pathogens. the objective of this study was to examine the clinical, hematological, biochemical, and outcome data from equids infected with anaplasma phagocytophilum presented to a primary care field setting in southeastern pennsylvania. computerized medical records from febrile equids with confirmed anaplasma phagocytophilum infection were reviewed. confirmation of anaplasma phagocytophilum was defined by the presence of granular inclusion bodies seen within leukocytes or eosinophils on microscopic blood smear evaluation and/or a positive polymerase chain reaction (pcr) for anaplasma phagocytophilum. horses and donkey presented with a mean fever of . f and mean fever duration of hours. the mean age at presentation was . years and the mean pack cell volume was . %. / cases were diagnosed in the months of may to december. equids ages to years had significantly lower platelet counts. / cases were positive on blood smear for inclusion bodies and / cases were positive for anaplasma phagocytophilum on pcr. treatments included intravenous oxytetracycline, oral doxycycline, or both. mean treatment duration was . days and mean treatment cost was $ . / cases were normothermic within hours. the treatment used in the two remaining cases was changed from oral doxycycline to intravenous oxtetracycline and was successful. this is the first case series of equine granulocytic anaplasmosis in the mid-atlantic states. all cases were examined and treated in the field. in order to make a definitive diagnosis, some cases required pcr. treatment failures were documented with the use of oral doxycycline alone. % of the cases survived. a high incidence of clinical and possibly genetic abnormalities has been reported amongst friesian horses including dwarfism, hydrocephalus, dissecting aortic aneurism and esophageal dysfunction. the purpose of the current study was to develop a new electromyography (emg) method to assess neurophysiological function of the esophagus especially for friesian horses. five friesian horses with esophageal dysfunction were included (ranging in age from . - years and comprising mares and a stallion) and two friesian control horses (a -and -year-old gelding). all five horses with esophageal dysfunction had a history of recurrent esophageal obstruction and were examined histopathologically post-mortem. barium contrast radiography was used as the gold standard to distinguish the diseased from the control horses. an endoscopically-guided percutaneous needle emg procedure (viking quest r ; software version . ) was performed just caudal to the larynx and just cranial to the thoracic inlet (to monitor striated and smooth muscle, respectively) to visualize esophageal motility. esophageal contractility in both control horses was predominantly reflected by interference patterns associated with longer duration and lower amplitude in smooth muscle compared to striated muscle. mean (ae sd) values were . ae . ms and . ae . mv (n readings) and . ae . ms and . ae . mv (n readings), respectively. in diseased horses, aperistalsis in smooth muscle was the most remarkable finding suggesting a loss of inhibitory neurogenic input resulting in aperistalsis and thus esophageal dysfunction. preliminary findings suggest that endoscopically-guided percutaneous needle emg might become a valuable method in elucidating the pathophysiology of dysfunction of esophageal motility especially in friesian horses. lymphoma affects horses of all ages. unlike in humans, no etiologic agent has been discovered. a year old thoroughbred/warmblood cross mare presented with signs of upper and lower respiratory disease and was subsequently diagnosed with lymphoma and equine multinodular pulmonary fibrosis (empf) and was positive for equine herpes virus (ehv- ) in both the pulmonary tissue and the lymph nodes. retrospective polymerase chain reaction (pcr) testing of six lymphoma cases found that of of the cases were positive on pcr for ehv- ( . %, p . , rr . ). electron microscopy was performed on one sample and herpes virus particles were identified. of the samples in which immunohistochemistry was performed ( of ), only t-cell rich b-cell lymphoma was identified. samples of mesenteric or submandibular lymph nodes from clinically healthy horses were submitted for ehv- pcr analysis; % were positive. gamma herpesviruses in humans have been associated with lymphoproliferative diseases such as kaposi's sarcoma and burkitt's lymphoma. equine herpesvirus , also a gamma herpesvirus, is found in association with equine lymphoma; although the exact role this virus plays in the initiation or perpetuation of lymphoproliferative neoplasia remains unknown. pathologic events reported to occur in the digital laminae in early stages of sepsis-related equine laminitis include leukocyte extravasa-tion into the laminar interstitium, pro-inflammatory cytokine expression, and epithelial stress. while these events have been documented early in the disease process at both a developmental stage and at the onset of obel grade (og ) lameness in the carbohydrate overload (cho) model of laminitis, the later events occurring at the onset of obel grade lameness(og , time point at which structural failure of the laminae usually occurs) have not been determined. we hypothesized that the inflammatory events described above are sustained through og lameness, likely playing an injurious role culminating in laminar failure. our objectives were to determine pro-inflammatory gene expression, leukocyte extravasation, and epithelial stress at og induced using the cho model. archived laminar tissue samples (snap frozen and paraffin embedded sections) were used from a previous cho study at louisiana state university (control group [n , water], cho group [n , corn starch]. calprotectin (cp) immunohistochemistry (ihc) was used to assess both laminar myeloid leukocyte numbers and epithelial stress; rt-qpcr was used to assess inflammatory gene expression. minimal inflammatory changes were present at og compared to published values at og stage in the cho lameness model including decreased mrna concentrations of cytokines (i.e. -fold increase in il- at og vs. -fold increase at og , no increase in il- b at og vs. -fold increase at og ), chemokines (no change in mcp- at og vs. fold increase at og , -fold increase in il- at og vs. fold increase at og ) and adhesion molecules (no change in e-selectin at og vs. -fold increase at og ). laminar leukocyte emigration was also decreased at the onset of og lameness compared to previously reported leukocyte infiltration at og . interestingly cox- , underwent a greater increase at og (approx. -fold) compared to that reported at og lameness ( -fold). finally, epithelial stress at og evidenced by cp ihc did not follow the uniform widespread distribution reported at og lameness, but instead was present in focal areas in which secondary epidermal laminae on either side of a common primary dermal vascular supply demonstrated increased cp signal. overall, laminar inflammation appears to be subsiding at og lameness, with epithelial stress possibly more dependent on vascular dysregulation instead of inflammatory events. the sustained increase in cox- , central to the induced production of vasoactive prostanoids in disease processes, may play a role in vascular dysregulation. this study was conducted to characterize clinical, laboratory and postmortem findings associated with oleander toxicosis in equids and to determine factors predictive of survival in these cases. retrospective analysis of medical records from our veterinary medical teaching hospital from january , to july , was completed. records of equids demonstrating detectable oleandrin in serum, plasma, urine or gastrointestinal fluid samples or detectable serum digoxin in the absence of pharmaceutical cardiac glycoside administration were included. descriptive statistics were used to evaluate the history, physical examination, and laboratory and postmortem data of affected individuals. logistic regression analysis was used to detect physical examination and laboratory factors significantly associated with survival. thirty equids met inclusion criteria of the study. three of subjects were dead on arrival or died immediately upon arrival ( %). of the remaining equids, % presented with gastrointestinal abnormalities, % were azotemic and % had cardiac arrhythmias. mortality was % for all subjects and % for those treated. the predominant cause for non-survival was cardiac dysfunction. factors significantly associated with survival included relatively decreased hematocrit and serum glucose, relatively increased serum chloride, absence of cardiac arrhythmias, and increased duration of hospitalization. equids with oleander toxicosis frequently present with gastrointestinal upset and may develop cardiac and renal disturbances. patients with cardiac arrhythmias and relatively increased hematocrit and serum glucose and decreased serum chloride are significantly less likely to survive. oleander intoxication is a differential diagnosis for colic in endemic areas, particularly with concurrent azotemia or cardiac dysrhythmia. the quantitative physicochemical approach emphasizes the importance of strong ions (na, k, cl, lactate), pco , and the plasma protein concentrations in determining plasma ph. serum concentrations of strong ions, proteins, and total co are reported on modern biochemical profiles. we hypothesized that the results of serum biochemical analysis can be used for acid-base interpretation in horses. the objective was to determine whether blood ph, anion gap, and strong ion gap could be quantitatively estimated and clinically used based on the results of serum or plasma biochemical analysis. horses ( adults and foals) presented to the isolation unit of our veterinary teaching hospital for suspected infectious diseases were prospectively enrolled. a venous serum sample was analyzed using a hitachi or copas c automated machine. measured parameters included strong ion difference (sid {na k}-{cl lac-tate}), total protein concentration (tp), and total co (tco ), with lactate being measured by blood gas analyzer. a second venous blood sample was collected into a na-heparin blood gas syringe and analyzed for ph (ph m ), pco and concentrations of na, k, cl, and lactate using a radiometer flex blood gas analyzer; sid was calculated from the measured values, and total solids (ts) were estimated using refractometry. serum/ plasma ph (ph calc ) was calculated using stewart's factor equation from the results of serum or plasma biochemical analysis, assuming pco mmhg for serum and pco accurate for plasma. anion gap (ag) was calculated as: ag (na k)-(cl tco ). strong ion gap (sig) was calculated as: sig . x[total protein, g/l]/ ( {pka-ph} )-ag. linear regression analysis was used to compare ph calc to ph m, as well as ag and sig to blood lactate concentrations. measured ph ranged from . to . ( . ae . ). measured sid from serum biochemistry (sid sb ) ranged from . to . meq/l ( . ae . meq/l) and sid from blood gas analyzer (sid bg ) from . to . meq/l ( . ae . meq/l; r . ; sid bg .  sid sb ). sid sb and sid bg showed small variability in measurements. tp ranged from to g/l ( . ae . g/l) and ts from - ( . ae . g/l; r . ; ts .  tp). using sid sb and tco values with constant pco , ph calc was poorly associated with ph m (r . ; ph calc . . ). in contrast, using sid bg with accurate pco , ph calc was closely associated with ph m (r . ; phcalc . . ) and the equation was not different from the line of identity. anion gap and sig (meq/l) calculated were significantly linearly correlated with lactate concentrations (mmol/l); ag .  [lactate] . (r . ), and sig À .  [lactate] . (r . ). we conclude that ph calc using sid sb , tco and constant pco values is not accurate. however, variability of measured biochemical parameters between machines was small, permitting use of serum biochemistry for clinical metabolic acid-base abnormalities interpretations of patients. these results reemphasize the importance of strong electrolytes and proteins in acid-base balance. metalloproteinases (mmps) are critically important in remodeling processes and in wound healing. however, excessive activation of mmps by pro-inflammatory mediators including cytokines, prostaglandin e , and nitric oxide lead to tissue breakdown. this is observed in osteoarthritis (oa) which is characterized by erosive lesions in articular cartilage. in hereditary equine regional dermal asthenia (herda), afflicted horses exhibit collagen abnormalities and can have associated chronic inflammation and aberrant wound repair. herda affects horses with quarter horse bloodlines and is similar to the human hereditary connective tissue syndrome ehlers danlos (eds). many adult eds patients suffer from joint pain and oa. we hypothesized that chondrocytes from articular cartilage of herda horses have increased activity of mmps. to test this hypothesis, chondrocytes were retrieved from articular cartilage of homozygous herda carpal and hock joints. chondrocytes from normal horses were also obtained for comparison. chondrocytes were seeded at x /ml into -well plates and incubated at c, % co for up to seven days. activity of secreted mmps was determined by zymography using equal amounts of proteins for loading. secreted mmps were analyzed by western blot. zymography showed that normal chondrocytes secreted two major bands with gelatinolytic activity observed at and kda suggestive of the latent form of mmp- and mmp- , respectively. less intense bands of gelatinolytic activity were observed at about and kda suggestive of the active form of mmp- and mmp- . another band of activity was also seen at kda which is suggestive of a dimer of mmp- that has been reported when mmps are in excess of tissue inhibitors of metalloproteinases (timps). chondrocyte cultures from homozygous herda cartilage showed a similar profile but with decreased activity by % at kda and - % increased activity at kda compared to normal chondrocytes. western blot analysis detected mmp- and mmp- immunoreactivity in chondrocyte culture media of herda-afflicted and normal horses. the present study demonstrates for the first time that horses suffering from herda have increased mmp activity which may predispose them to the development of lesions in articular cartilage. research supported by nutramax laboratories, inc. equine polysaccharide storage myopathy (pssm) type is a dominantly inherited glycogenosis caused by a mutation in the gene coding for skeletal muscle glycogen synthase type (gys- ). the disease has been reported to affect the haflinger breed but so far its prevalence is unknown. aim of this preliminary study was to estimate the occurrence of the gys- mutation in austrian haflingers and establish which of the seven haflinger sire lines appear mostly affected. gys- genotyping of randomly chosen haflingers was performed with a validated restriction fragment length polymorphism assay. resting and post-exercise muscle enzyme activities (creatine kinase (ck), aspartate aminotransferase (ast), lacate dehydrogenase (ldh)) and blood lactate concentrations were compared between horses with and without the mutation. among the horses were heterozygous (hr) carrier of the mutation. no homozygotes (hh) were identified. all horses with the gys- mutation were descendents of the a-or w-sire lines. the estimated hr prevalence was % ( % ci: . - . %). ck activity after exercise (p . ) was significantly higher in hr horses compared with horses not carrying the mutation (rr). ast activity was significantly higher in the hr group at rest and after exercise (p o . ). there was no statistically significant difference in resting ck, resting and post exercise ldh activity or blood lactate between hr and rr. results suggest that the prevalence of hr in the austrian haflinger population is higher than in the overall quarter horse population and might be as high as %, similar to some draft horse breeds. further research is needed to establish the prevalence within the different breeding lines. hereditary equine regional dermal asthenia (herda) is an autosomal recessive connective tissue disorder affecting quarter horse lineages. although a mutation in the gene encoding cyclophilin b has been genetically linked to herda, its causal association with the disease is not yet documented. previously, we demonstrated reductions in ultimate tensile strength (uts), modulus of elasticity, and energy to failure (toughness) of skin from many corporal regions of herda animals. given the presumed relationship between her-da and abnormal collagen structure, and the predominance of type i collagen in skin, we hypothesized that altered biomechanical properties would be detected in tendons which are rich in type i collagen. to evaluate this hypothesis we compared the uts, modulus of elasticity, and energy to failure of forelimb deep digital flexor tendons (dft) from six herda horses to six age-matched controls. isolated dft was secured and pulled to failure on an instron s universal testing instrument using purpose-built cryogenic clamps. analysis of variance was executed using sas . proc glimmix program (sas institute, ). p-values . were identified as significant. uts and modulus of elasticity were significantly lower in herda dft when compared with controls (p o . ); energy to failure did not differ between groups. these findings document abnormal biomechanics in herda tendon, leading us to postulate that lower uts and modulus of elasticity associated with the herda defect could convey a competitive advantage in the athletic disciplines in which this defect has segregated. (references on request). a proprietary herbal biocontamination product (bios) approved for cosmetic use in france, inhibits proliferation of medically relevant bacteria, mold, and viruses. these properties make bios potentially useful as a topical wound medication, prompting us to compare bios to silver sulfadiazine (ssd) in a distal extremity wound healing model in horses. using general anesthesia, two . cm wounds were aseptically created on the dorsomedial aspect of all limbs. for the duration of the study, two contralateral limbs were randomly chosen to be bandaged; the other two limbs were un-bandaged -with one limb of each group being treated with % bios and the other with ssd. for each limb the most proximal wound served as an untreated control. every hours wounds were evaluated, digitally photographed, and perimeter and area determined using morphometric software (imagej, nih). analysis of variance did not identify significant differences between ssd or bios treatment for wound perimeter (p . ) or area (p . ). at individual time points the effect of bandaging was significant when area was evaluated (p . ) and trended toward significance for perimeter (p . ) comparisons, substantiating published reports that bandaging modifies wound healing. difference in perimeter and area between control and treatment were highly significant (p o . ), substantiating the importance of topical treatment. over the study duration, effects of bandaging (p o . ) and topical treatment (perimeter p o . ; area p . ) continued to be highly significant. bios performance in the equine distal extremity wound model was equivalent to ssd. both bandaging and topical treatment significantly impacted wound healing. this effect was compounded when both variables were evaluated over time. radiolabeled leukocytes are the only scintigraphic method currently available for identifying sites of infection and/or inflammation in horses; however the clinical applicability of this technique is limited by expense and poor efficacy. this pilot study compares the accumulation of m tc-labeled igg, peg-liposomes and leukocytes in an equine muscle abscess model. three mixed breed adult horses had  cfu s. equi zooepidemicus inoculated into the right semitendonosis to create an abscess. peg-liposomes were prepared via the film hydration method and labeled using mci m tc-hexamethyl-propylene-amine-oxime ( m tc-hmpao). autologous leukocytes were obtained from ml whole blood and labelled using mci m tc-hmpao. commercial equine polyclonal igg was conjugated with the chelator hydrazinonicotinamide (hynic) and labelled with mci m tc. radiopharmaceutical administration was initiated hours after inoculation. horses and received mg m tc-igg, . mmol/kg m tc-liposomes and m tc-leukocytes, with a hour interval between each radiopharmaceutical. horse received only m tc-leukocytes. scintigraphic examinations were performed at and hours post injection (p.i.) with each radiopharmaceutical. after the final study, horses were euthanized and tissue samples collected. the percentage of injected dose per kilogram of tissue (%id/kg) was calculated for the region of the abscess, normal muscle and multiple organs. scintigraphic examinations demonstrated increased radiopharmaceutical in the region of the abscess with all three techniques at both time-points. at hours p.i. abscess-to-background ratio was highest using m tc-igg ( . ae . ). at hours p.i. abscess to background ratio was highest using m tc-liposomes ( . ae ). tissue biodistribution data revealed abscess to muscle ratios of ( m tc-igg), ( m tc-liposomes), and . ( m tc-leukocytes). this preliminary data demonstrates that m tc-liposomes, m tc-igg and m tc-leukocytes exhibit long circulating characteristics and accumulate at inflammatory/infectious foci after intravenous injection in horses. m tc-igg and m tc-liposomes appear to be superior to m tc-labelled leukocytes in this model. due to its low cost and ease of preparation, m tc-igg has great potential for clinical use where identification of infectious or inflammatory foci is necessary. digital hypothermia is used clinically to decrease the incidence of sepsis-related equine laminitis, a disease causing structural failure of digital laminae resulting in crippling lameness. due to the fact that hypothermia was recently reported to effectively decrease laminar expression of inflammatory molecules including pro-inflammatory cytokines, chemokines and cox- in equine laminitis, our laboratory is investigating the effect of hypothermia on central upstream signaling cascades which may induce expression of these diverse inflammatory molecules. the p mapk pathway has recently been reported to be a central component of inflammatory signaling in multiple diseases including human sepsis, and is currently being assessed as a therapeutic target. we thus hypothesized that ) p mapk is upregulated and activated in affected laminae in equine laminitis and ) digital hypothermia inhibits inflammatory mediator expression by blocking p mapk phosphorylation (indicator of p mapk activation). western hybridizations using both a total p mapk and a phospho-p mapk antibody were performed on archived pooled laminar samples from black walnut extract (bwe) model ( control, developmental (dev) groups [ . h & h post bwe administration] and the onset of obel grade lameness (og ) [n each]) and carbohydrate overload (cho) models (con [n ], dev [n ], og [n ]) of laminitis, and individual laminar samples from two groups of horses from a digital hypothermia (dh) study. in the dh study, one forelimb of each horse was kept at approximately c in ice water and the other at ambient temperature following administration of g/kg oligofructose (of). dorsal laminae were harvested for snap freezing at either hours after of administration (dev, n ) or at the onset of lameness (og , n ) using protein extracted from treated and untreated digital laminae of each horse. increased laminar concentrations of phospho-p mapk were present in the developmental periods ( . h and h) in the bwe model, and in both the dev and og periods in the cho laminitis models. however, digital hypothermia had no effect on laminar phospho-p mapk concentrations. thus, p mapk is activated in affected laminae in multiple models of laminitis, but does not appear to be the central signaling cascade through which hypothermia works to block the expression of inflammatory molecules. therefore, p mapk is not likely to be a viable therapeutic target as a sole source for blocking the multiple inflammatory signaling mechanisms inhibited by local hypothermia. abstract e- does cefquinome penetrate the blood brain barrier in the normal horse? hollis ar duggan ve and corley ktt . scott dunn's equine clinic, berkshire, uk; university college dublin, dublin, ireland; anglesey lodge equine hospital, the curragh, ireland. meningitis is a rare but serious condition that occurs in both foals and adult horses. there is currently a restricted choice of antimicrobials that are both safe to use in horses and penetrate the blood brain barrier. cefquinome is a fourth generation cephalosporin that has activity against streptococcus, the most commonly reported causative organism in adult horse meningitis. therefore, if cefquinome were to achieve therapeutic concentrations in cerebrospinal fluid following routine administration, this would be an exciting advance for the treatment of meningitis in the horse. mature, healthy horses were used on separate occasions, seven days apart, in a crossover design. each horse was administered either cefquinome ( mg/kg) or saline (equivalent volume). cerebrospinal fluid was collected via atlanto-occipital puncture under general anaesthesia and hours after administration of cefquinome or saline placebo. blood samples were collected prior to, and and hours after administration of cefquinome or placebo. all samples were analysed for the presence of cefquinome by a laboratory masked to treatments administered. cefquinome was detectable in the cerebrospinal fluid in all horses hours after intravenous administration, and in horses hour after administration. cefquinome penetrates the blood-brain barrier and it is therefore a potential treatment for equine meningitis. further investigation of the pharmacokinetics and pharmacodynamics of cefquinome in the cerebrospinal fluid is warranted to establish the optimum intravenous dose. the purpose of this study was to determine if enrofloxacin alters the pharmacokinetics of firocoxib in the horse. firocoxib is a coxibclass nonsteroidal anti-inflammatory drug (nsaid) approved for use in horses to control pain and inflammation associated with osteoarthritis. dosages of firocoxib are species dependent, with the recommended dose for horses being . mg/kg as an oral paste every h. the main elimination pathway of firocoxib is hepatic; however the effects of concurrent administration of drugs that may inhibit its metabolism have not been evaluated. enrofloxacin is a synthetic antibacterial agent from the flouroquinolone group developed for veterinary use. it is primarily used for gastrointestinal, urogenital, skin and respiratory tract infections in various animals. a well acknowledged problem associated with flouroquinolone usage is their effect on the metabolism of other drugs. co-administration of multiple drugs can result in unpredictable therapeutic outcomes. often it is either diminished therapeutic efficacy or increased toxicity of one or more of the administered drugs. various pharmacokinetic interactions between antimicrobials and nsaids have been described. six healthy, adult mares were administered . mg/kg of firocoxib orally. samples were collected by direct venipuncture of the jugular vein at (control), , , and min, , , , , , , , , and h after administration. after a day washout period the six horses were pretreated days with enrofloxacin mg/kg intravenously every h then on the fourth day given . mg/kg of firocoxib orally. samples were collected at (control), , , and min, , , , , , , , , and h after administration. all samples were stored at À c until analysis using a validated hplc method. the t / , c max , t max , auc - and auc -f after firocoxib administration were . angiotensin converting enzyme (ace) inhibitors improve survival and quality of life in humans and small animals with cardiovascular and renal disease. there is limited information regarding their effects in horses. the purpose of this study was to determine the pharmacokinetics of quinapril and its effects on ace inhibition in horses. six healthy horses were administered quinapril at mg iv, mg po or mg po in a -way crossover design. blood was collected at predetermined times for measurement of quinapril and quinaprilat concentrations using high pressure liquid chromatography, as well as ace concentrations using a radioenzymatic assay. normally distributed data were analyzed with one way repeated measures analysis of variance (rm-anova) and non-normally distributed data were analyzed using friedman rm_anova on ranks. significance was set at p o . . no adverse effects were observed during the study period. plasma quinapril concentrations were low and rapidly declined after iv administration. quinaprilat concentrations were below the limit of quantification ( . mg/ml). ace activity was significantly decreased from baseline at . and hour after iv dosing and at all timepoints after oral dosing. maximum % ace inhibition was , and % with the iv, high and low oral doses, respectively. these results suggest that, despite low plasma concentrations, quinapril has sufficient oral absorption and results in inhibition of ace in healthy horses. controlled studies in clinically affected horses are indicated. this study determined the pharmacokinetic profile of firocoxib in healthy neonatal foals. foals are more sensitive to the side effects of nsaid, primarily due to immature renal clearance mechanisms and ulcerogenic effects on gastric mucosa. firocoxib, a novel, second generation nsaid, is reported to have reduced side effects due to cox- selectivity. the pharmacokinetic profile of firocoxib in neonates has not been established. we hypothesized that firocoxib given po to neonatal foals would achieve therapeutic concentrations in plasma. seven healthy foals of mixed gender were administered . mg/kg firocoxib po q h for nine consecutive days, commencing at h old. blood was collected for firocoxib analysis at (dose # only), . , . , , , , , and h after doses # , and . for all other doses ( , , , , and ) blood was collected immediately prior to the next dose ( h trough). elimination samples were collected after dose # . plasma was stored at À c until analysis. physical examinations were performed on foals daily and body weight obtained every two days during the sampling period. analysis of plasma samples by liquid chromatography-mass spectrometry revealed firocoxib was rapidly absorbed. after the initial dose, a maximum plasma concentration was reached in min, minimal accumulation after repeat dosing occurred and steady state was obtained after approximately four doses. after the final dose, plasma drug concentration decreased in a linear manner with an estimated terminal t / of h. seventy-two hours after the final dose, firocoxib was not detectable (o ng/ml). erythrocytosis is reportedly a rare finding associated with hepatocellular carcinoma in horses. the purpose of this study was to determine the relative frequency of erythrocytosis and the clinicopathologic abnormalities and hepatic histopathology associated with erythrocytosis in horses with liver disease. ninety-seven horses aged ! year with clinicopathologic or clinical signs of liver disease, a complete blood count (cbc), and hepatic histopathology were included. information on cbc, biochemical variables, and hepatic histopathology was collected from records. data from horses with erythrocytosis (packed cell volume %) were compared to those without using the mann-whitney rank sum test with significance set at p o . . there were no differences between groups in white blood cell count, gamma-glutamyl transferase, sorbitol dehydrogenase, aspartate aminotransferase, and alkaline phosphatase activities, total protein, albumin, globulin, blood urea nitrogen, or glucose concentrations. fibrosis ( %), biliary hyperplasia ( %), inflammatory infiltrate ( %), megalocytosis ( %), degeneration ( %), necrosis ( %), cholestasis ( %), anisocytosis and anisokaryosis ( %), and lipidosis ( %) were observed in livers of horses with erythrocytosis. neoplasia ( %) was observed rarely. this study reports a high frequency of erythrocytosis in horses with liver disease. erythrocytosis is associated with higher total bilirubin and serum bile acids concentrations. common histopathologic changes include fibrosis, biliary hyperplasia, and inflammatory infiltrate. hepatic neoplasia was rare. this study was performed to determine if horses diagnosed with equine proliferative enteropathy (epe) from lawsonia intracellularis (li) infection had long term effects from disease based on their sale price as yearlings and race earnings. a retrospective review of medical records of thoroughbred horses that were treated for lawsonia intracellularis infection between january , and january , at hagyard equine medical institute in lexington, kentucky was performed. three criteria were used for inclusion in this study. first, each horse had presumptively been diagnosed with li based on physical examination findings such as ventral edema, diarrhea, lethargy, or poor body condition. second, horses had hypoalbuminemia of less than . mg/dl (normal reference range: . - . mg/dl). third, each horse had a positive fecal polymerase chain reaction (pcr) for li, a positive serum immunoperoxidase monolayer assay (ipma), or both. an ipma titer greater than or equal to was considered positive for disease. horses met the initial criteria. of the horses sold at public auction as yearlings. the sale price of these horses was compared to the average sale price of all yearlings by the same sire as the affected horse (control group). of the horses raced in the united states. their monetary earnings from racing were compared to the average monetary earnings of all progeny by the same sire as the affected horse (control group). earnings of horses that were between and years of age ( / horses) at the conclusion of the study were compared to the lifetime average earnings of the stallion's progeny. earnings from horses that were two years of age ( / ) at the end of the study were compared to the two year old average earnings of the stallion's progeny. monetary earnings from all races prior to december , were included in the study. horses both sold at public auction and raced. as well as being included in the total number of horses that sold and raced, their sale records and monetary earnings were compared to the averages from their respective sire as a separate group. this retrospective study indicated that yearling horses previously infected with li do not sell for as much at public auction as their herdmates, but their monetary earnings from racing are not significantly different from other horses. these results should assist practitioners in guiding owners in determing if treatment of horses with epe is appropriate and it may aid in reassuring owners that despite the poor condition of the horse during and shortly after the course of disease, horse may still have future athletic potential. this abstract was presented at the aaep in december . bronchopneumonia caused by streptococccus equi subsp. zooepidemicus (s. zooepidemicus) is one of the most important causes of morbidity in weanling foals. ceftiofur crystalline free acid (ccfa) is a long acting third-generation cephalosporin antimicrobial recently approved for the treatment of bronchopneumonia associated with s. zooepidemicus in adult horses. the objective of the present study was to determine the disposition of ccfa in plasma and pulmonary epithelial lining fluid (pelf) of weanling foals. six healthy -to month-old weanling foals were administered a single intramuscular injection of ccfa at a dose of . mg/kg of body weight. concentrations of desfuroylceftiofur acetamide (dca) and related metabolites were measured by use of ultra-high performance liquid chromatography and tandem mass spectrometry. following im administration, median time to maximum plasma and pelf concentrations was h ( - h) . mean (ae sd) peak dca concentration in plasma ( . ae . mg/ml) was significantly higher than that in pelf ( . ae . mg/ml). terminal half-life of dca in plasma ( . ae . h) was not significantly different from that of pelf ( . ae . h). time above the therapeutic target of . mg/ml was significantly longer in plasma ( ae h) than in pelf ( ae h). based on the results of the present study, intramuscular administration of ccfa at a dose of . mg/kg would be appropriate for the treatment of bronchopneumonia caused by s. zooepidemicus and other susceptible pathogens in weanling foals. fgf- is secreted by osteocytes and osteoblasts in response to hyperphosphatemia. fgf- enhances phosphaturia and is postulated to have a central role in the development of secondary renal hyperparathyroidism. hyperthyroid cats have elevated plasma phosphate and parathyroid hormone concentrations, which may in part be associated with underlying chronic kidney disease (ckd). the aim of this study was to determine if plasma fgf- concentrations were associated with the presence of underlying ckd in hyperthyroid cats, and to investigate the changes in plasma fgf- concentrations that occur following treatment of hth. hyperthyroid cats were recruited from two london-based first opinion practices between and . cats that were azotemic at diagnosis were excluded. hth was treated with anti-thyroid medication alone or in combination with thyroidectomy. cats were included in the study if they had a plasma total thyroxine concentration o nmol/l documented for a six month period following commencement of treatment. cats were classified as having azotemic ckd if they developed renal azotemia within six months of establishment of euthyroidism. otherwise cats were deemed to have normal renal function. stored edta plasma samples were assayed for fgf- using a recently validated elisa. the mann-whitney u test and the wilcoxon signed rank test were used to compare between the groups and assess the response to treatment respectively. results are reported as median [ th , th percentiles]. correlations were made using spearman's correlation coefficient. thirty one cats with hth ( azotemic and non-azotemic) were included in the study. plasma phosphate concentrations decreased following treatment in cats that did not develop azotemia ( . [ . , . ] mg/dl vs. . [ . , . ] mg/dl; n , p . ) whereas plasma phosphate concentrations did not change significantly following treatment in cats that did develop azotemia ( . [ . , . ] mg/ dl vs. . [ . , . ] mg/dl; n , p . ). plasma fgf- concentrations were significantly higher in cats that developed azotemia than cats that did not at both pre treatment ( . [ . , . ] pg/ml vs. . [ . , . ] pg/ml; p . ) and post treatment ( . [ . , . ] pg/ml vs. . [ . , . ] pg/ml; p . ) timepoints. plasma fgf- concentrations increased following treatment in both azotemic (p . ) and non-azotemic groups (p . ). plasma fgf- concentrations and plasma phosphate concentrations were not correlated at baseline (r s . , p . ) or following treatment (r s . , p . ). plasma fgf- concentrations were higher in pre-azotemic cats than non-azotemic cats and increased following treatment of hth. the reason that fgf- concentrations increased following treatment, particularly in the face of decreasing plasma phosphate concentrations in cats that remain non-azotemic, is unclear but may be related to the decline in glomerular filtration rate. hyperthyroidism is a disorder resulting from the excessive production and secretion of t and t by the thyroid gland. although the disorder and its pathological lesions have been well studied and described the cause remains illusive. whole blood and solid tissue samples from non-diseased, severe disease and mild disease cats based on t levels and thyroid histology were used in this study. whole blood samples from non-disease cats, severe disease cats and mild disease cats as well as solid thyroid tissue samples from non-disease cats, severe disease cats and mild disease cats were collected and processed. the resulting total rna samples were used for genechip analysis using our custom feline gene chip designed by affymetrix. data analysis was performed using the partek s gs software for gene expression data. the robust multichip average algorithm was used for background adjustment, normalization, and probe-level summarization of the raw data. anova analysis was performed to find significant differentially expressed genes with a minimal false discovery rate control of . and a fold change of . in each direction. during the mild disease state, pathways associated with dna damage and apoptosis are most prominent. at later stages when the histopathological disease is more severe in addition to the aforementioned pathways others associated with tgf-beta signaling, cell adhesion and extracellular matrix remodeling take more prominence. the analysis of this unique data set generated from the use of our proprietary genechip revealed molecular mechanisms that are associated with the transition from non-disease, to mild disease to severe disease, in the thyroid tissue as well as the blood. these mechanisms could provide insights into the causes of the disease and identify potential new therapeutic and diagnostic targets. although it is well established that concurrent chronic kidney disease (ckd) develops in about % of hyperthyroid cats, no one has reported the use of the iris staging system for ckd before and after treatment of these hyperthyroid cats. the purpose of this study was to compare the effects of treatment in hyperthyroid cats with known stage and ckd in order to determine the effects of restoring euthyroidism or inducing hypothyroidism has on the iris stage in these cats. we evaluated hyperthyroid cats (median age, years) in this study. one day prior to treatment, serum t concentration, serum chemistry analysis, complete urinalysis, and urine protein-to-creatinine ratio (upc) were measured. all cats were again evaluated with the same parameters again months after treatment with i. prior to treatment, ( %) of the cats had no evidence of azotemia (serum creatinine o . mg/dl), whereas cats ( %) had stage ckd (serum creatinine, . - . mg/dl). in the cats, iris staging revealed proteinuria in cats ( %), with borderline proteinuria (upc, . - . ) and with overt proteinuria (upc . ). hyperthyroidism was cured in all cats (median post-t , . mg/dl). all cats had a good response to treatment; there were no signs of ckd except for polyuria and polydipsia in some cats. a significant (p o . ) increase in median values for both serum urea nitrogen ( mg/dl to mg/dl)) and creatinine ( . to . mg/dl) occurred after treatment. nine of the cats ( . %) classified as nonazotemic or iris stage prior to i progressed to stage ckd after i. all cats with stage ckd before treatment remained azotemic after i, with cats remaining in stage ckd, and cats progressing to stage ckd (serum creatinine, . - . mg/dl). there was a significant inverse relationship (p . ) between pretreatment urine specific gravity (usg) and post-treatment serum creatinine in the cats. of the cats with post-treatment serum creatinine values . mg/dl (stage to ckd), ( %) had pretreatment usg of o . . in contrast, in the cats with post-treatment serum creatinine values o . mg/dl, only ( %) had pretreatment usg of o . . a significant (p o . ) decrease in median upc from . to . occurred after treatment, but there was no relationship between degree of proteinuria and iris stage in these cats. two cats developed iatrogenic hypothyroidism after i, diagnosed by finding low serum t and high ctsh concentrations. both hypothyroid cats had progressed from stage before treatment to stage and ckd, respectively, after i; after thyroxine replacement, serum creatinine decreased to near pretreatment concentrations in both cats. conclusions: ) iris stage ckd is common in untreated hyperthyroid cats. ) progression to next higher iris stage is common after treatment, but most cats with remain relatively asymptomatic for ckd. ) usg may be helpful in predicting which cat's iris stage will progress after i. ) iatrogenic hypothyroidism worsens azotemia, an effect that appears reversible with replacement therapy. home blood glucose monitoring (hbgm) of diabetic pets is likely to result in superior glycaemic control, minimizing episodes and impact of dangerous hypoglycaemia and reducing costs. nevertheless, it has proven difficult to objectively establish a clear benefit of hbgm using biological parameters (clinical signs, blood glucose, fructosamine). the current study aimed to assess the impact of hbgm on owner perceived quality of life (qol) aspects of diabetes mellitus (dm) treatment, using the recently validated psychometric tool diaqol-pet. owners of insulin treated diabetic cats were recruited to complete the -item tool, evaluating areas affecting the cat's and owner's qol, including: worry about pet's dm, hypoglycaemia, costs, owner's desire for autonomous control over the pet's dm, etc. item-weighted-impact-scores (iwis), reflecting frequency and importance ratings of each item, were calculated, as well as averageweighted-impact-scores (awis; average iwis of all items), as an overall measure of diabetes dependent qol. frequencies, iwis and awis were compared between owners practising hbgm and those who did not using mann whitney u test (significance p o . ). two hundred and eleven owners of insulin treated diabetic cats completed the diaqol-pet; owners practised hbgm, whereas the remaining did not practise any form of home monitoring (including urine glucose). iwis for 'excessive drinking' and 'owner wanting more control' were significantly different between the hbgm-group (mean /-standard deviation: À . /À . and À . /À . ) and the non-hbgm-group (À . /À . and À . /À . ). there was no significant difference between the groups with regards to the iwis for other items, including 'worry about hypoglycaemia' or 'worry about pet's dm'. polydipsia was reported significantly more frequently in the non-hbgm-group and this was the reason for the difference between groups in this item's iwis as it was considered of equal importance. frequency and iwis of reported occurrence of hypoglycaemia signs were not significantly different. awis for both groups was not significantly different (hbgm: À . /À . ; non-hbgm: À . /À . ). the current study suggests that hbgm is predominantly practised by owners who desire more autonomous control over their cat's dm. the frequency of polydipsia was lower in the hbgm-group perhaps suggesting superior control. however, hbgm did not detectably affect the impact of the majority of qol-items, nor the frequency of hypoglycaemic episodes. overall diabetes dependent qol of diabetic cat and owner, as measured per diaqol-pet, was unaffected by hbgm. these data argue for the use of hbgm in selected pet-owner combinations rather than as part of a practice's standard dm management protocol, although further studies are indicated. insulin resistance is associated with impaired activation of the insulin signaling pathway in peripheral tissues such as skeletal muscle, visceral and subcutaneous (sc) adipose tissue. high plasma glucose, fatty acid and endotoxin levels are three major causes of insulin resistance in feline and human obesity and in type diabetes mellitus. however, the mechanisms by which these factors influence insulin action are still unclear. therefore, our aim was to investigate the tissue-specific expression of crucial mediators of insulin action such as the insulin-receptor substrate (irs ), the serine/threonine protein kinase b (pkb/akt) and of the principal insulin-dependent glucose transporter protein (glut ) in feline models of hyperglycemia, hyperlipidemia and subacute endotoxemia. healthy cats were infused through the jugular vein with glucose (n ), lipids (n ) or lipopolysaccharide (lps; n ) for days to clamp their blood concentrations at the approximate level found in untreated feline diabetes (glucose: - mmol/l; triglycerides: - mmol/l) or to induce a systemic low-grade inflammation (lps; rectal temperature: . - . c), respectively. healthy control cats were infused with saline (n ). on day , specimens were collected from skeletal muscles, visceral and sc fat and processed for irs mrna expression, total and phosphorylated pkb/akt and glut protein expression. gene transcripts of irs were not different between the groups. compared to controls, skeletal muscle pkb/akt phosphorylation was % lower in cats infused with glucose (p o . ); lipid-infused cats showed a trend for a decrease in pkb/akt phosphorylation ( % lower than saline) and had decreased glut expression (p o . ) in muscle. total (p o . ) and phosphorylated (p o . ) pkb/akt protein expression were decreased in the sc adipose tissue of lps-infused cats compared to controls. in these cats, phosphorylation of pkb/akt protein was also decreased in visceral fat (p o . ). sustained hyperglycemia and, to a lesser extent, hyperlipidemia impaired insulin signaling and glucose transport pathways primarily in skeletal muscle; endotoxemia reduced insulin sensitivity mainly in adipose tissues. thus, the development of insulin resistance in response to hyperglycemia, hyperlipidemia or endotoxemia might be affected by tissue-specific mechanisms in cats. separately used, single photon emission computed tomography (spect) and computed tomography (ct) both lack sensitivity and are additionally hampered by a poor anatomical location capacity and a lack of specificity, respectively. these drawbacks suggest an interest in the fusion of images obtained by the techniques. the aim of this study is to test spect/ct fusion performance in dogs with insulinoma. inclusion criteria were: / a biological diagnosis of insulinoma; / an examination by high resolution ct scan and in-pentetreotide spect followed by spect/ct fusion; / a surgical or post mortem examination completed by histopathological analysis. spect examination showing abnormal foci and ct scan showing pancreas, lymph nodes (ln) or liver abnormalities were considered positive. in case of double positivity, presence (imp ) or absence (imp-) of superimposition of abnormal images was noted. ten dogs were included. in / dogs, superimposition of abnormalities couldn't be tested. ct scan detected abnormal images [ pancreatic nodules (pn), enlarged ln (eln)] while spect failed to show any abnormal uptake. both dogs became euglycemic after removal of pn and ln designed by ct scan. in / , all abnormal images were classified as imp [ pn, eln and diffuse hepatic infiltration (dhi)]. surgery performed on / resulted in euglycemia in ; dog remained hypoglycemic after partial removal of pn. pn localization and dhi were confirmed after necropsy in the th dog. in / dogs imp and imp-images were both recorded. in dog, a dhi was classified as imp but pn localization was imp-: localized in the left lobe by ct scan and in the corpus by spect, the latest localization being confirmed after necropsy. in the other dog pn localization was imp but a diffuse spect signal superimposing to the liver considered as normal on ct scan was noted. hepatic biopsy confirmed spect results. this study confirms an imperfect sensitivity of both ct scan and spect. it confirms that ct scan can be associated with unspecific abnormal images. subject to a confirmation on a larger cohort of dogs, it indicates that imp images provide specific detection and accurate localization of canine insulinomas' primary lesions and metastasis. the majority of dogs with primary hypoadrenocorticism (ph) reveal clinical and laboratory abnormalities of gluco-and mineralocorticoid deficiency. in some of them sodium and potassium levels are normal, a phenomenon currently called atypical addison's. it has been postulated that in those cases adrenal destruction is confined to the zona fasciculata/reticularis, resulting in isolated glucocorticoid deficiency. however, there are no histological studies confirming a normal zona glomerulosa and in most reported cases diagnosis was based solely on low post-acth cortisol levels. the aim of the study was to evaluate aldosterone (aldo) levels in dogs with ph with and without electrolyte abnormalities. seventy dogs with newly diagnosed ph were included. aldo concentrations (ria, coat-a-count s , siemens) were measured before and min after administration of mg synthetic acth (synacthen s , novartis) iv. results were compared to those of healthy dogs and dogs with diseases mimicking ph. to confirm that peak concentrations were not missed aldo was additionally measured , and min after acth in dogs ( with ph, with ph mimicking diseases). results were analysed by means of non-parametric statistical methods (p o . ). post-acth aldo was significantly lower in dogs with ph ( - pg/ml, median pg/ml) than in healthy dogs ( - pg/ml, median pg/ml) and in dogs with ph mimicking diseases ( - pg/ml, median pg/ml). low post-acth aldo was found in / dogs with ph, in / of them levels were below the detection limit of the assay. normal sodium and potassium levels were found in / dogs ( %), / dogs ( %) had hyponatremia and normal potassium, / dogs ( %) had hyponatremia and hyperkalemia. electrolyte abnormalities ranged from mild to severe. there was no correlation between post-acth aldo and sodium and a weak correlation between post-acth aldo and potassium (r À . ). aldo concentrations were not different , and min after acth. the results demonstrate that aldo levels are low in most dogs with ph independent of the degree of electrolyte abnormalities. this implicates that all three zones of the adrenal cortex are compromised and that there are mechanisms which allow maintenance of a normal electrolyte balance without aldo. definitive diagnosis of canine hypoadrenocorticism (ha) is based on inadequate cortisol secretion following adrenocorticotropic hormone (acth) administration. an abnormal serum sodium to potassium (na:k) ratio can be used to determine whether an acth stimulation test is warranted. the aim of this study was to examine the utility of combining the na:k ratio with white blood cell counts to determine whether an acth stimulation test is warranted. a retrospective review of medical records of dogs examined between and was performed. dogs diagnosed with ha and control dogs, in which a diagnosis of ha was excluded during the study period, were included. inclusion criteria for all dogs were hospitalization with intravenous fluid therapy, a complete blood count, and serum na and k measurements at the time of initial examination. dogs were included in the ha group if they also had pre and post acth stimulation serum cortisol concentrations . mg/dl. dogs were included in the control group if they had resting or post acth stimulation serum cortisol concentration . mg/dl. exclusion criteria were recent administration of glucocorticoids, prior treatment of hyperadrenocorticism, or serum cortisol concentration . mg/dl but . mg/dl. continuous variables were compared between groups using the mann-whitney u test. receiver operating characteristic (roc) curves were produced to assess the sensitivity and specificity of detecting ha with various cutoffs for each variable. data is presented with % confidence intervals (ci) and statistical significance was defined as p o . . the na:k ratio, neutrophil count and neutrophil:lymphocyte ratio were significantly lower in dogs with ha than in dogs without ha (p o . for each). lymphocyte and eosinophil counts were significantly higher in dogs with ha compared to dogs without ha (p o . for each). the areas under the curve by roc analysis were largest for na:k ratio ( . , ci: . - . ) and lymphocyte count ( . , ci: . - . ). a na:k ratio . was % sensitive (ci: - %) but only % specific (ci: - %) for detecting ha. a lymphocyte count ! . x cells/ml was % sensitive (ci: - %) and % specific (ci: - %). conversely a na:k ratio . was % sensitive (ci: - %) but % specific (ci: - %) and a lymphocyte count ! .  cells/ml was % sensitive (ci: - %) but % specific (ci: - %). a na:k ratio . was % sensitive (ci: - %) and % specific (ci: - %) for detection of ha and a lymphocyte count ! .  cells/ml was % sensitive (ci: - %) and % specific (ci: - %) for detection of ha. a combination of this na:k ratio ( . ) and lymphocyte count (! .  cells/ml) was % sensitive (ci: - %) and % specific (ci: - %) for detection of ha. these results indicate that the combination of lymphocyte count and na:k ratio results in a better screening test for ha than the use of the na:k ratio alone. pheochromocytoma is a malignant, catecholamine-producing, adrenomedullary tumor. clinical signs resulting from excessive catecholamine secretion are typically non-specific, making differentiation from other adrenal tumors a challenge. elevated plasma concentrations of the catecholamine breakdown products metanephrine (mn) and normetanephrine (nmn) are used to identify pheochromocytoma in humans. this study tested the hypothesis that plasma metanephrine concentrations are greater in dogs with pheochromocytoma than in dogs with other adrenal neoplasms, healthy dogs and dogs with non-adrenal illness. edta plasma was collected from healthy dogs and unwell, hospitalized dogs with non-adrenal illness, pheochromocytoma and cortical tumors between april and october . samples were stored at À c before measurement of free mn and nmn concentrations using high pressure liquid chromatography at the central laboratory for clinical chemistry at the university of groningen ( samples) or the mayo clinic, rochester, minnesota ( samples). kruskal-wallis tests followed by dunn's multiple comparison analysis were used to compare results between groups. significance was set at p o . . results are reported as median [range] . eight dogs with pheochromocytoma, healthy dogs, dogs with non-adrenal illness and dogs with cortical tumors were sampled. pheochromocytoma was diagnosed histologically ( dogs) or cytologically ( dog). cortical tumors were diagnosed histologically ( dogs) or by response to trilostane treatment after obtaining consistent endocrine test results ( dogs occult hyperadrenocorticism (hac) has been theorized to exist in which excess adrenal sex hormone secretion induces the clinical signs and laboratory changes associated with classic hac. however, the ability of sex hormones to cause such alterations has never been closely evaluated. if sex hormones can cause a syndrome similar to classic hac, they should be able to induce expression of classic glucocorticoid-induced genes. the purpose of the study was to determine if in vitro expression of the gene for corticosteroid-induced alp (cialp) could be induced by clinically relevant concentrations of cortisol and sex hormones believed to cause occult hac. canine hepatocytes were purchased from a commercial source (cellzdirect or invitro) in -well plates. upon arrival ( - plates per shipment), the cells were allowed to recover in general media per supplier recommendations. after hrs, media was changed to william's e media (-l-glutamine) containing concentrations of cortisol or sex hormones that have been documented in the literature in dogs with hac or with purported occult hac. each plate was treated with a different hormone (cortisol, -hydroxyprogesterone [ ohp], progesterone, estradiol or androstenedione), and each well contained a different concentration (starting with no hormone added as a negative control) to evaluate a dose response. media was changed daily. after days of hormone exposure, rna was extracted. reverse transcription was performed and the product used for quantitative pcr for cialp and beta-actin (roche lightcycler) using a gene-specific fluorescent probe for detection. standard curves were created for each gene. all samples and standards were run in duplicate. using the lightcycler software (vers . ), cialp expression was normalized to that of beta-actin. fold change in expression was determined relative to the negative control. each sex hormone was used to treat plates; one plate in each shipment was treated with cortisol as a positive control. for cortisol, a dose response was seen in expression of the cialp gene. compared to no cortisol, , , , , and nmol cortisol increased expression . , . , . . . , . and . fold, respectively. a -fold increase is considered significant (j.vandesompele et al genome biol ). expression of cialp was not significantly induced in response to any concentration of ohp ( nm maximum), progesterone ( nm maximum), estradiol (max pm maximum) or androstenedione ( nm maximum). we conclude that in vitro these sex hormones do not induce expression of the cialp gene which is classically induced by cortisol in vivo; indeed, elevated serum cialp activity is a hallmark of classic hac. thus, the ability of the sex hormones to induce the gene in vivo must be questioned and evaluated. measurement of sex hormones has been advocated as an adjunct means for diagnosing typical hyperadrenocorticism (hac), i.e. disease due to excess cortisol secretion, as well as for diagnosis of atypical hac, i.e. disease due to excess adrenal sex hormone secretion. however, measurements in either setting have not been widely studied. therefore, our objectives were: . to determine the sensitivity of -hydroxy-progesterone ( ohp) and estradiol concentrations pre-and post-acth for diagnosis of typical hac. . to determine the specificity of ohp and estradiol concentrations preand post-acth for diagnosis of occult hac. dogs that had pdh (n ), dogs that were suspected to have hac but proven not to (had non-adrenal illness [nai, n ]) or dogs that were healthy (n , used to establish reference ranges [rr]) were enrolled. acth stimulation tests were performed ( mcg/kg cosyntropin iv); blood samples were drawn pre and min post; ohp and estradiol were measured by previously validated radioimmunoassays. a kruskal-wallis rank sum test was used to compare values between the groups. significance was set at p o . . for basal and acth-stimulated ohp concentrations, the rr were determined to be . - . ng/ml (mean ae s.d.; range . - . ) and . - . ng/ml (range . - . ), respectively. in pdh dogs, and had basal and post-acth ohp concentrations above the rr, respectively; in the nai group, and dogs had concentrations above the rr, respectively. thus, the sensitivity of basal and post-acth ohp measurement for diagnosis of hac is % and %, respectively. specificity of diagnosis is % and %, respectively. post-acth ohp concentration was significantly different between groups. for basal and stimulated estradiol concentrations, the rr were determined to be - pg/ml (range - ) and - pg/ml (range - ), respectively. for both basal and stimulated estradiol, pdh dogs (n ) had concentrations above the rr; for those with nai (n ), and had concentrations above the rr, respectively. thus, the sensitivity of estradiol measurement for diagnosis of hac is % for both pre-and post-acth. specificity of estradiol for diagnosis for hac is % and % for pre-and post-acth, respectively. overall, dogs with nai had at least one elevated estradiol concentration (total specificity %). post-acth estradiol concentration was not significantly different between groups. we conclude that use of ohp and estradiol concentrations for diagnosis of hac can be problematic. sensitivity and specificity are relatively low, potentially leading to misdiagnoses. diabetes mellitus is one of the most common feline endocrinopathies and is considered to have a similar pathophysiological basis to human type diabetes. several studies have identified risk factors for development of diabetes mellitus in cats, which include age, obesity, inappropriate diet and physical inactivity. however, to date, no specific genetic risk factors have been identified. genome-wide association studies in humans have identified several genes that predispose to obesity and/or diabetes mellitus, one of which is the melanocortin receptor (mc r) gene. the aim of the current study was to identify polymorphisms (snps) in the feline mc r gene and to use these to perform a case:control study to determine whether these candidate gene snps were associated with diabetes mellitus in cats. genomic dna from cats ( domestic short hair [dsh], burmese) was initially analysed by pcr and direct sequencing using felmc r-specific primers, which identified a missense mutation (mc r:c. c t) in the region encoding the extracellular domain of the receptor protein in dsh cats only. one hundred and nineteen dsh cats were subsequently recruited into the case:control study. fifty nine cats were obese diabetic ( male, female), mean age . years (range - y); mean weight . kg (range . - kg). sixty lean cats were used as controls ( male, female), mean age . years (range - y), mean weight . kg (range . - . kg). the t to c base change alters a restriction site in the sequence recognized by the enzyme bstoi, such that dna from cats with the mutant (c) allele can be cut, whereas that from the wild-type (t) allele cannot. primers were designed that flanked the mutation to allow pcr amplification of this region of mc r from genomic dna obtained from edta blood. the pcr products were purified and subject to restriction fragment length polymorphism (rflp) analysis. bstoi digestion products were then analysed by agarose gel electrophoresis. of the diabetic cats, ( %) were homozygous for the mutation (cc), compared to ( %) of control cats. statistical analysis (two tailed fisher's square test) revealed that this difference between groups was statistically significant (p . ). in conclusion, this pilot study has identified a missense mutation in the coding sequence of mc r. this could be an important predisposing factor for development of diabetes and/or obesity in dsh cats. polymorphisms in a similar region of human mc r predispose to obesity, which in turn is a major risk factor for type diabetes. hyperadrenocorticism (hac) is one of the most common endocrine disorders of dogs. the two most effective medical treatments are trilostane (vetoryl s ) and mitotane (lysodren s ). previous studies evaluating the effect of treatment on aldosterone secretion measured the hormone at min post-acth administration. however, the optimal sampling time would be at the time of maximal secretion, which occurs minutes after the mg/kg dose commonly used for the test (carlson et al, jvim, ). thus, the true effect of either medication on aldosterone secretory capacity is unknown. our objectives were: ) to assess and compare the effect of treatment with trilostane and mitotane in dogs with pituitarydependent hac (pdh) on aldosterone secretory reserve at min post-acth stimulation and ) to determine if changes in aldosterone concentration at that time correlate with changes in serum sodium and potassium concentrations. forty-six dogs being treated for pdh with either mitotane (n ) or trilostane (n ) have been enrolled. the dogs could be treated for any length of time. all had acth stimulation tests performed ( mcg/kg cosyntropin iv); blood samples were drawn before and at and min post-acth for monitoring of cortisol and aldosterone concentration using previously validated radioimmunoassays. ten historical normal controls were also included. serum sodium and potassium concentrations were measured in the basal samples. a kruskal-wallis rank sum test was used to compare values between normal dogs and those treated with mitotane or trilostane. linear regression analysis was used to determine if a correlation existed between electrolyte and aldosterone concentrations or between cortisol and aldosterone concentrations. significance was set at the p o . level. acth-stimulated aldosterone concentrations in mitotane-treated but not trilostane-treated dogs were significantly lower than that in normal dogs at both the and min time points. no difference was detected between aldosterone concentrations at and min after acth injection in either treatment group. a positive correlation existed between the -min cortisol and -min aldosterone concentrations in the trilostane-treated group (r . ), i.e. the peak post-acth concentration for each hormone, but not in dogs treated with mitotane. basal serum sodium and potassium concentrations were not correlated with the basal aldosterone concentration in either treatment group. in conclusion, treatment with mitotane resulted in decreased aldosterone secretory reserve, but this did not correlate with hyperkalemia or hyponatremia. measurement of aldosterone concentrations is not predictive of electrolyte concentrations. previously presented at the auburn university phi zeta research emphasis day, november , . antioxidant depletion is documented in humans with hyperthyroidism, and is reversible with treatment. in addition, antioxidant depletion has been shown to increase the risk of methimazole toxicity in rats. the primary aim of this study was to determine whether deficiencies in glutathione (gsh), ascorbate (aa), or vitamin e, along with increases in urinary -isoprostanes, were present in hyperthyroid cats, and were reversible after radioiodine treatment. a secondary aim was to determine whether antioxidant abnormalities were associated with a prior history of methimazole toxicity. ongoing prospective, controlled, observational study. otherwise healthy client-owned hyperthyroid cats presenting for radioiodine therapy (n to date) and healthy age-matched controls (n to date) were recruited. all cats were screened with cbc, biochemical panel, urinalysis, and t , as well as red blood cell (rbc) gsh, plasma aa, plasma vitamin e, and urinary -isoprostanes. hyperthyroid cats were re-evaluated months after radioiodine treatment. unlike in humans, median blood antioxidants were not significantly different in hyperthyroid cats (gsh . mm; aa . mm, and vitamin e, g/ml) compared to controls (gsh . mm; aa . mm, and vitamin e, g/ml). results for urinary isoprostanes are pending, and associations with methimazole toxicity will be investigated after full recruitment. rbc gsh concentrations did increase significantly (to . mm; p . ) after radioiodine treatment. however, this modest change is unlikely to be clinically significant. preliminary data do not indicate clinically significant blood gsh, ascorbate, or vitamin e deficiencies in hyperthyroid cats. with appropriate insulin therapy and a low carbohydrate diet, up to % of newly diagnosed diabetic cats are eventually able to maintain euglycemia without insulin administration, and these cats are considered to have achieved remission. there are currently no published data reporting the glucose tolerance status of cats classified as being in remission, and it is unknown whether these cats are truly in diabetic remission, or should be classified as non-insulin dependent diabetics, or having impaired glucose tolerance, and/or impaired fasting blood glucose. the aim of this study was to determine fasting blood glucose concentrations and glucose tolerance status of cats in remission. the study was a prospective study in a feline-only clinic. for inclusion, diabetic cats had to have achieved remission through insulin therapy, and insulin withheld for a minimum of two weeks. five diabetic cats in remission and five matched non-diabetic cats were enrolled in the study. blood samples were obtained via the ear vein but where the cat's temperament precluded this, from the jugular.glucose concentration was measured using a meter calibrated for feline blood (abbott alphatrak). a simplified glucose tolerance test was performed after food was withheld for hours. a g catheter was placed in a cephalic vein three hours before the gtt was commenced, to minimize the effects of stress on blood glucose concentration. blood glucose concentration was measured at time and then a g/kg dose of glucose was administered slowly via the intravenous catheter. further blood glucose measurements were made at hours and then hourly until glucose had returned to o mg/dl (o . mmol/l). in the control group, all cats had a fasting blood glucose below mg/dl, and following glucose administration, glucose had returned to o mg/dl by hours. fasting blood glucose in the remission group was o mg/dl ( mmol/l) in all cats except one, which had fasting blood glucose of mg/dl ( . mmol/l). following glucose administration, all five cats in remission had blood glucose above mg/dl ( . mmol/l) at three hours, four were o mg/dl at four hours, and one returned to o mg/dl at five hours. the cat with impaired fasting glucose subsequently became diabetic after steroid administration. the results of this study show that these cats, while no longer diabetic, have mildly impaired glucose tolerance compared to nondiabetic cats, and a minority have impaired fasting glucose. the objective of this study was to determine the role of iodine restriction in the nutritional management of cats with naturally occurring hyperthyroidism. five domestic shorthair cats ranging in age from - years were confirmed to have hyperthyroidism based on persistently increased serum total thyroxine concentrations (tt ), palpable thyroid nodule and weight loss. serum tt concentrations ranged from - nmol/l (reference range - nmol/l). the cats were then fed a low iodine containing food ( . ppm iodine dmb, as measured by epiboron neutron atomic activation). serum tt concentrations were measured every weeks. biochemistry parameters were also evaluated at weeks , and . at weeks, serum tt concentrations had decreased in all cats with of cats ( %) being euthyroid (mean nmol/l; range - nmol/l). the remaining hyperthyroid cat had an initial serum tt of nmol/l, which decreased to nmol/l after being fed the iodine-restricted food. mean decrease in tt for all cats was nmol/l (range - nmol/l). renal parameters remained stable in all cats. these cats along with additional newly diagnosed hyperthyroid cats were transitioned to a similar food that contained less iodine ( . ppm dmb). baseline serum tt concentrations in the new cats ranged from - nmol/l. serum tt and other biochemical parameters were monitored every weeks for weeks, and then every weeks for an additional weeks. with the . ppm iodine food the four new cats became euthyroid with a mean tt concentration of nmol/ (range - nmol/l). the euthyroid cats from the earlier feeding study had a further decrease in tt concentration (mean tt nmol/l, range - nmol/l). the single non-euthyroid cat from the first study had a serum tt concentration of nmol/l, a decrease from the baseline concentration of nmol/l. the average decrease in serum tt for all cats was nmol/l (range - nmol/l). finally, of the cats were fed a third iodine-restricted food ( . ppm dmb) along with one other newly diagnosed hyperthyroid cat ( nmol/l serum tt ) and evaluated every weeks. all cats in this evaluation were euthyroid (mean tt nmol/l; range - nmol/ l). this result included the cat whose serum tt remained in the hyperthyroid range in the first two evaluations. the average decrease in tt was nmol/l (range - nmol/l). biochemical features of renal function remained stable and no other biochemical abnormalities were observed. in summary, the results of these three feeding studies demonstrate that feline hyperthyroidism can be managed effectively with dietary iodine restriction. we have shown previously that restriction of dietary iodine (i) is a safe and effective method for decreasing serum thyroxine concentrations (tt ) in cats with hyperthyroidism. the objective of this study was to determine the maximum level of iodine in a nutritionally balanced feline mature adult food required to maintain normal serum tt concentrations in hyperthyroid cats currently being controlled on a food containing . ppm i (dmb) as measured by epiboron neutron atomic activation. all cats were previously diagnosed at least months prior to the start of the study and their tt concentrations were maintained in the normal range by dietary iodine restriction for a minimum of months (range months- years). serum tt concentrations ranged from - nmol/l (reference range - nmol/l) at the beginning of the study. the cats were divided into two groups each containing cats. groups were similar in age and gender distribution (mean age . years, range - years). one group (group a) was placed on a food that was formulated for mature adult cats containing . ppm i (dmb). the other group (group b) was placed on a similar food that differed only in that it contained . ppm i (dmb). blood was collected from all cats every three weeks and analyzed for serum tt concentration. biochemistry parameters were also evaluated at weeks , and . all group a cats exhibited increases in serum tt concentration (mean increase of nmol/l above baseline, range - nmol/l). seven of the cats remained in the euthyroid range (mean serum tt nmol/l, range- - nmol/l). two cats exceeded the upper limit of the reference range ( and nmol/l respectively). the cats in group b also exhibited increases in serum tt concentration but to a greater degree than the cats in group a (mean increase nmol/l, range - nmol/l). four cats remained in the euthyroid range (mean serum tt , range - nmol/l). the five remaining cats all exceeded the upper limit of the reference range (mean serum tt nmol/l, range- - nmol/l). all cats returned to a euthyroid state within month of being returned to a diet containing . ppm i (dmb). it was determined that serum tt concentrations are not ideally controlled in the normal range in hyperthyroid cats fed a food containing ! . ppm i (dmb). hyperthyroidism is a common disease in old cats. excessive production of thyroid hormones is the hallmark of the disease. three main treatments for feline hyperthyroidism include radioactive iodine, thyroidectomy, and antithyroid drugs such as methimazole. previously we have shown that limiting dietary iodine to or below . ppm induces euthyroidism in cats with hyperthyroidism compared with a similar diet containing . ppm iodine. the objective of this study was to test whether dietary iodine at . ppm would induce euthyroidism in cats with naturally occurring hyperthyroidism. fourteen cats with hyperthyroidism confirmed by serum tt and ft measurements were stratified into two groups based on gender and age. one group (control: males and females, age ranged from to years) was given a positive control dry cat food ( . ppm iodine) while the other group (test: males and females, age ranged from to years) was fed a commercial dry cat food ( . ppm iodine) for at least weeks before the study. afterwards (week ), the control cats continued to receive the same food while cats in the test group were given a test food ( . ppm iodine) for additional weeks. all cats had free access to their food and deionized water during the study. blood samples were collected during weeks , , , and of the study. the control cats maintained euthyroidism during the study. the test food significantly reduced serum tt ( ae , ae à , ae à , ae à nmol/l in weeks , , and , respectively; à : p o . compared with week , dunnett's t test). it also significantly reduced ft at the end of the study ( ae vs. ae pmol, week vs. week ; dunnett's t test, p o . ). serum ft was within the reference range ( - pmol/l) in cats in both groups. serum tt , ft , and tsh were not affected by the test food and were within the reference ranges (tt : . - . nmol/l, ft : . - pmol/l, and tsh: - mu/l) in cats of both groups during the study. this study demonstrates that dietary iodine at or below . ppm provides an effective and inexpensive therapy for cats with naturally occurring hyperthyroidism. radioactive iodine ( i) is a widely used treatment for feline hyperthyroidism. prior to i administration, many cats receive methimazole therapy. it has been suggested that recent withdrawal of methimazole prior to i may increase the risk of hypothyroidism, inhibit the response to therapy, or have no effect. to further address this question, a retrospective medical records search was performed to identify hyperthyroid cats that received i therapy after methimazole treatment. inclusion criteria included documentation of the time interval between discontinuation of methimazole and i administration, and measurement of thyroxine (t ) at - days after i. cats were divided into groups: those receiving i within day of stopping methimazole, and those receiving i treatment or more days after stopping methimazole. sixty cats met the inclusion criteria. forty received i within day of stopping methimazole. of those, ( %) had a low t (o . mcg/dl), ( . %) had a normal t ( . - . mcg/dl), and ( . %) had an elevated t ( . mcg/dl) at - days after i therapy. fourteen cats received i or more days after stopping methimazole: ( %) had a low t , ( %) had a normal t , and ( %) had an elevated t at - days after i therapy. the results were compared with a fisher's exact test and there was no difference between the groups (p . ). these findings indicate that stopping methimazole therapy within day of i therapy does not inhibit the response to therapy. pharmacokinetic studies evaluating synthetic insulin analogs such as glargine necessitate the ability to measure the blood concentrations of glargine without cross-reactivity to endogenous insulin. although the cross-reactivity between endogenous human insulin assays and synthetic analogs is often known for commerciallyavailable assays, the degree of cross-reactivity of human insulin assays with feline insulin is not. the purpose of this study was to evaluate the cross-reactivity of feline insulin with a commerciallyavailable human insulin elisa with known cross reactivity to several synthetic analogs. pre-and post-prandial blood samples were collected from four healthy cats immediately prior to and approximately minutes following a meal, for a total of samples. dextrose was added to the meals given to two of the cats. blood samples were immediately centrifuged and the serum was collected, aliquoted, and stored at À c until analysis. serum insulin levels were determined in parallel with commercially-available feline insulin and human insulin elisas. the elisas were run in duplicate and according to the manufacturer's instructions. concentrations of serum insulin measured by the feline insulin elisa ranged from . ng/l to ng/l. despite the wide range of concentrations of feline insulin, all samples evaluated with the human insulin elisa yielded absorbance readings equal to or lower than the absorbance of the negative control, indicating no crossreactivity between the evaluated human insulin assay and feline insulin. since this assay is reported to cross-react significantly with glargine, it is a great candidate for determination of serum glargine concentrations in cats. the aim of this prospective, controlled study was to compare the efficacy of two trilostane protocols for treatment of canine pituitary-dependent hyperadrenocorticism (pdh). among the client-owned dogs diagnosed with pdh, only the dogs weighing o kg were selected (n ). group a (n ; low-dose treatment group) and group b (n ; high-dose treatment group) received . ae . mg of trilostane/kg orally every hours and mg of trilostane/ body orally every hours, respectively. all of the dogs were reassessed at , , , and weeks after the initiation of treatment. the improvement in post-acth stimulation serum cortisol concentration, as well as clinical signs in group a, required more time than group b; however, of dogs in group b had clinical signs and abnormal laboratory findings consistent with hypoadrenocorticism after treatment for weeks. twenty-four weeks later, all of the dogs of both groups improved the abnormal clinical findings. the present study suggests that twice daily, low-dose administration of trilostane is effective in the management of canine pdh and may be safe without the potential adverse effects of once daily, high-dose treatment. however, because this study involved only a small number of dogs, a population-based control study will be needed to clarify the efficacy of low-compared to high-dose trilostane treatment. cobalamin is essential for a variety of metabolic processes in many tissues and organs, and has effects on cell growth and peripheral and central nervous system function. chronic distal small intestinal disease in humans, cats, and dogs has been shown to cause cobalamin deficiency. an immunoassay for the measurement of serum cobalamin concentration in these species is being used in routine practice for the diagnosis of cobalamin deficiency. in pigs, the role of cobalamin has not yet been extensively investigated. thus, the aim of this study was to analytically validate an immunoassay, labeled for use in humans, for the measurement of cobalamin in porcine serum samples and secondly to determine serum cobalamin concentrations in weaned pigs. for the analytical validation of the assay, serum cobalamin concentrations were measured using the commercially available immulite s cobalamin immunoassay (siemens healthcare diagnostics ltd., deerfield, il, usa) in surplus porcine serum samples from a variety of studies. validation of the assay consisted of determination of dilutional parallelism, spiking recovery, and intra-and inter-assay variability. additional surplus serum samples from piglets from four litters at a texas a&m university farm were obtained. each piglet had been bled twice, the first at weaning ( days of age) and the second one days later. to investigate results in comparison between age groups, serum cobalamin concentrations were compared using a wilcoxon matched pairs test. significance was set at p o . . observed to expected ratios (o/e) for serial dilutions ranged from . to . % (mean ae sd: . ae . %) for four different serum samples at dilutions of : , : , and : , and from . to . % (mean ae sd: . ae . %) for one serum sample at dilutions of : , : , and : . o/e for spiking recovery ranged from . to . % (mean ae sd: . ae . %) for five different porcine serum samples that had been spiked with each other in a : dilution. intraassay coefficients of variation (%cv) for five different serum samples were . , . , . , . , and . %. inter-assay %cvs for five different serum samples were . , . , . , . , and . %. serum cobalamin concentration was significantly lower in piglets post weaning (median: ng/l) compared to those at the time of weaning (median: ng/l; p . ). the immulite s cobalamin immunoassay labeled for use in humans is linear, accurate, precise, and reproducible for measurement of serum cobalamin concentrations in pigs. this study also showed that piglets that differ in age by only days have significantly different serum cobalamin concentrations. further investigations of cobalamin concentrations in both sows and piglets at different stages of weaning are warranted. primigravid dairy heifers can be infected with mastitis pathogens during the periparturient period. the prevalence of intramammary infection (imi) ranges from - % of quarters pre-partum and - % at parturition. some pre-partum infections self-cure before parturition, however a number of these imis persist into early lactation. these imis may impact milk production and quality and may serve as a reservoir for contagious pathogens. no study has specifically investigated the risk of an imi persisting from the prepartum period into early lactation. the objectives of this study were to describe the prevalence of mastitis pathogens in heifers on a grazing dairy before and after parturition and calculate the relative risk (rr) and attributable fraction of population (afp) for the association between a post-partum and pre-partum imi. two-hundred-ninety-four heifers were systematically assigned to of groups: g ) pre-partum secretions from all mammary quarters (n ), g ) no pre-partum secretions collected (n ) and g ) pre-partum secretions from two diagonal quarters (n ). group assignments were designed to assess whether pre-partum sampling increased the likelihood of imi at calving. mammary quarter secretions were collected for bacterial culture approximately weeks prior to expected calving date. quarter milk samples were collected for bacterial culture once weekly during the st -weeks of lactation. bacterial isolates were classified as staphylococci, non-agalactiae streptococci and gram-negatives. mammary quarter samples yielding different bacteria were classified as mixed infections and those yielding ! bacterial types were classified as contaminated. bacterial isolates were speciated using gene sequencing methods and strain-typed using pulse-field-gel-electrophorysis to evaluate the relatedness of bacteria isolated from pre-and post-partum samples from the same mammary quarter. relative risk and afp were calculated using  tables. forty-five percent of mammary quarters had a pre-partum imi. during the st weeks of lactation the mean prevalence of imi was . % of quarters. staphylococci were most frequently isolated bacteria from pre-partum secretions and milk with s. chromogenes and s. aureus being the most common species. using data from mammary quarters, the rr and afp for the association between a post-partum and pre-partum imi were and %, and %, and and % for all staphylococci, s. aureus only and cns only imis, respectively. mammary quarters sampled pre-partum were no more likely to have a post-partum imi than those not sampled (chisquare, p ! . ). these data demonstrate that pre-partum imis persist into early lactation and that pre-partum secretion cultures may be a useful, not only in predicting imi at calving, but also in assessing risk of introducing new contagious mastitis pathogens, e.g., s. aureus, into the lactating herd. despite concerns about antimicrobial resistance and clostridium difficile in food animals, there has been little study of the prevalence or mechanisms of resistance. this study evaluated the impact of tetracycline treatment on c. difficile shedding in veal calves and the impact on resistance. calves arriving on veal farm received oral oxytetracycline for days as per farm protocols. calves were sampled at arrival and days later. selective culture for c. difficile was performed. isolates were ribotyped, and tested for tetracycline susceptibility and the presence of tetracycline resistance genes. multivariable logistic regression models were used to determine the relationship between tetracycline resistance and the presence of tetracycline resistance genes. clostridium difficile was isolated from % ( / ) and % ( / ) calves, at the first and second samples, respectively. the percentage of tetracycline resistant isolates increased from % to %. isolates from the second sample were times more likely to be tetracycline resistant (p . ) and times more likely to possess tet(m) (p . ). tet(m) was detected in % ( / ) and % ( / ), tet(o) in % ( / ) and % ( / ) and tet(w) in % ( / ) and % ( / ) of isolates from first and second samples, respectively. tet(l), tet(k) and tet(s) were not detected. resistant isolates were not carrying any of the genes investigated. routine tetracycline use may have had an impact on both the prevalence of c. difficile, as well as the strain distribution and resistance patterns. this is the first report of presence of tet ( the objectives of this study were to ) estimate the prevalence of antimicrobial resistance in the study population and ) to investigate the associations between exposures to antimicrobial drugs and antimicrobial resistance in fecal non-type specific e. coli (ntsec) recovered from individual feedlot cattle. two-stage random sampling was used to identify cattle for enrollment at western canadian feedlots. a fecal sample was collected per rectum from each individual at arrival and in the middle of the feeding period when cattle were rehandled as part of standard feedlot protocol. from samples collected at this second time point, a total of , ntsec isolates were tested for susceptibility to antimicrobial drugs by disk diffusion. parenteral and in-feed exposures to antimicrobial drugs were recorded for each individual enrolled in the study. the least square means estimates and % confidence intervals for the prevalence of resistance at each time point were modeled using poisson regression. multivariable logistic regression was used to investigate associations between antimicrobial resistance and exposure to antimicrobial drugs. regression models were adjusted for clustering of observations among individuals and pens. the most common resistances identified in arrival samples were sulfisoxazole ( . %; %ci: . - . ), streptomycin ( . %; %ci: . - . ) and tetracycline ( . %; %ci: . - . ). at the second sampling point, resistance prevalence was . % ( %ci: . - . ) for sulfisoxazole, . % ( %ci: . - . ) for streptomycin, and . % ( %ci: . - . ) for tetracycline. logistic regression modeling identified weak associations of exposures to tetracycline and macrolide classes of drugs with antimicrobial resistance at the second time point. abstract fa- premature/dysmature syndrome in cria: a ret-rospective study of cases ( ) ( ) ( ) ( ) ( ) ( ) ( ) . c. gerspach, d. anderson. the ohio state university, columbus oh. prematurity is widely acknowledged as risk factor for subsequent morbidity and mortality in llama and alpaca cria. a review of medical records for premature cria alive at the time of admission to the veterinary teaching hospital between and was performed to determine risk factors of prematurity and to report the outcome and related conditions or diseases in affected cria. medical records for premature or dysmature cria were included in this study. of these cria, were alpaca and llama, were female and were male. reasons for referral were prematurity, failure of passive immunity, dyspnoea, weakness and failure to gain weight. cria were presented at a mean age of . days and were premature by a mean estimated time of . days. overall survival rate was . %, with all llama cria surviving. a multivariate logistic regression model was used to identify risk factors associated with not surviving. cria receiving camelid colostrum had a significant better outcome than cria receiving no colostrum or colostrum from different species. dyspnea and tachypnea was associated with a poor outcome. all cria that were able to nurse, without assistance prior to referral, survived. clinical pathology parameters most commonly associated with death were hyperphosphatemia and acidosis. enrofloxacin is approved for the treatment of swine respiratory disease, however there are no published studies describing the pharmacokinetics of enrofloxacin at the approved dose and route in pigs ( . mg/kg subcutaneously). furthermore no studies have assessed the unbound concentrations of enrofloxacin at its site of action, the extracellular tissue fluid. therefore the objective of this study was to use an in-vivo ultrafiltration method to measure the active fraction of enrofloxacin, and the metabolite ciprofloxacin, at tissue sites relevant to pigs, and to compare these concentrations with plasma concentrations collected at similar time points. six healthy pigs were used in this study. pigs were recently weaned and weighed an average . kg. on the day before the experiment, pigs were anesthetized for the placement of jugular vein sampling catheters and interstitial fluid collection probes. three ultrafiltration probes were placed in each pig in a subcutaneous site near the right shoulder, an intramuscular site along the epaxial muscles, and in the pleural space of the chest cavity. each pig received an injection of enrofloxacin (baytril , bayer animal health) at a dose of . mg/ kg subcutaneously behind the left ear. plasma and interstitial fluid samples were collected at pre-determined time points, and enrofloxacin and ciprofloxacin concentrations were measured using hplc with fluorescence detection. protein binding was determined with a microcentrifugation system. pharmacokinetic data was analyzed using a one compartment model. the analysis of plasma and isf showed that only a small fraction of ciprofloxacin was produced in these pigs, therefore ciprofloxacin concentrations were not used in pharmacokinetic measurements. the plasma half-life (t / ), volume of distribution, clearance, and peak concentration (c max ) for enrofloxacin was . hr (ae . ), . l/kg (ae . ), . l/kg/hr (ae . ), and . mg/ml (ae . ), respectively. the concentrations from each of three tissues were not different in each pig. when pharmacokinetic values from all tissues were combined for the isf, the t / was . hr (ae . ) and the c max was . mg/ml (ae . ). the enrofloxacin plasma protein binding was . % (ae . ) and . % (ae . ) at a high and low concentration, respectively. this study has demonstrated that the concentration of biologically active enrofloxacin in tissues exceeds the concentration predicted by the unbound fraction of enrofloxacin in pig plasma. the half-life of enrofloxacin is longer in tissues and plasma than has been reported in previous studies. the high tissue concentrations and long half-life produce an auc/mic ratio sufficient for the pathogens that cause respiratory infections in pigs. ceftiofur crystalline free acid (ccfa), a long-acting ceftiofur formulation labeled for use in cattle, pigs, and horses for treatment of respiratory disease has been used for treatment of ovine respiratory infections in clinical practice. pharmacokinetic data, however, do not exist for ccfa administered subcutaneously in sheep. the present pharmacokinetic study evaluated the single dose subcutaneous administration of ccfa in sheep (n ) at . mg/kg body weight. concentrations of ceftiofur free acid equivalents (cfae) in plasma were measured by high performance liquid chromatography for days following drug administration. pharmacokinetics of subcutaneous ccfa in sheep were best described using a single compartment model with the following average (ae sd) parameters: area under the concentration time curve ! ( . hÃug/ml ae . ), observed maximum plasma concentration ( . ug/ ml ae . ), and observed time of maximum plasma concentration ( . h ae . ). no significant adverse drug reactions were observed. adequate cfae plasma concentrations were attained to effectively treat respiratory tract pathogens associated with pneumonia in sheep. the purpose of this study was to assess, using thoracic ultrasonography, the prevalence of lung lesions in pre-weaned dairy calves. subsequent aims were to describe ultrasonographic changes within the lung, clinical respiratory score, and treatment of respiratory disease. a longitudinal study was performed using female dairy calves from commercial dairy farms in new york state. calves were enrolled based on age. thoracic ultrasound and clinical respiratory scoring were performed on each calf at time points. a standard mhz linear ultrasound probe was utilized to evaluate intercostal spaces through of each hemi-thorax with the calf in lateral recumbency (us ) or standing (us ). lesion appearance, size, and location were recorded. respiratory score (rs) was assigned based on a previously published protocol incorporating fever, nasal discharge, cough, ocular discharge and ear droop, with a higher numerical score corresponding to more severe disease. abnormal lung on ultrasound was defined as one or more areas of ! cm width or depth of non-aerated lung. farm records were evaluated to identify treated calves. calves were treated for respiratory disease at the farm manager's discretion, not based upon ultrasound findings or rs. non-parametric methods were used to evaluate the data. ninety-one calves were enrolled into the study, with lost to follow-up. an average of minutes was spent performing the rs and ultrasound on each calf. the median ages at first (us ) and second (us ) examination were (interquartile range - ) and (interquartile range - ) days, respectively. the majority of calves had a low rs (o ) and only . % of calves had a rs high enough to warrant treatment based on previous recommendations (rs! ). the prevalence of calves that had abnormal lungs on ultrasound but a low rs (o ) was . % (us ) and . % (us ). the prevalence of calves that had abnormal lungs on ultrasound and a high rs (! ) was % (us ) and . % (us ). of the calves that had abnormal lungs on ultrasound but a low rs, % were treated with antimicrobials within days of examination. none of the calves with high rs and abnormal lungs on ultrasound were treated with antibiotics within days of examination. this study demonstrates a high prevalence of abnormal lungs, as detected by thoracic ultrasonography, without significant clinical signs in pre-weaned dairy calves. the relatively low treatment rate in these calves may suggest an area of opportunity for improvement in calf health, welfare, and herd longevity. further studies and follow up are needed to elucidate the significance of these findings and whether or not treatment is indicated. literature regarding diseases causing lameness in beef cattle is limited. this retrospective study was undertaken to examine beef cattle presented for lameness. medical records of beef cattle having a lameness examination done during the period to were reviewed and descriptive statistics generated. lameness was classified based on clinical diagnosis. the medical records of beef cattle were reviewed of which . % were male and . % were female. beef cattle presented for lameness most often during the summer months ( %) and least during autumn ( %). causes of lameness were categorized as infectious ( . %) or non-infectious ( . %) and infectious lameness subcategorized as either a primary disorder or a secondary infection. all cases of a primary infectious disorder were interdigital phlegmon. secondary infections diseases included sole abscess ( . %), septic arthritis ( . %), tenosynovitis ( . %), and pedal osteitis ( . %). non-infectious lameness included proximal limb lameness ( . %), foot trauma ( . %), hoof horn cracks ( . %), hoof defects ( . %), interdigital fibromas ( . %), overgrown hooves ( . %), sole bruise ( . %), subclinical laminitis ( . %), white line disease ( . %), osteoarthritis ( . %), heel erosion ( . %), sole ulcers ( . %), and sole hemorrhage ( . %). the most frequently affected claw was the lateral digit of the hind limb ( . %), followed by the medial digit of the front limb ( . %), lateral digit of the front limb ( . %), and the medial digit of the hind limb ( . %). the findings of this study suggest significant differences in the frequency of disease causing lameness in beef cattle compared to published reports for dairy cattle. in people, endoscopic ultrasound (eus) has become the technique of choice for assessing pancreatic disease and eus-guided fineneedle aspiration (eus fna) has proven a useful and safe modality for characterizing pancreatic lesions. reported complications include infections, bleeding and acute pancreatitis. in dogs, laparoscopic-assisted pancreatic biopsy has been suggested to be a safe procedure, however eus and eus fna have not been evaluated in dogs so far. thus the aim of the present study was to assess the practicability and safety of eus examination of the abdominal cavity as well as pancreatic eus fna in healthy dogs. this study was approved by the cantonal committee for the authorization of animal experimentation, zurich, switzerland. the study population consisted of healthy beagle dogs with a median bodyweight of . kg ( . - . ). eus was performed with an olympus gf-uc p-echoendoscope and fna were performed using g needles (cook echotipultra). after completion of the eus-examination of the abdominal cavity from the stomach (liver, gallbladder, bile ducts, kidneys, adrenals, pancreas), the scope was advanced into the duodenum and eus fna of the pancreas was performed. fna tissue acquisition was made applying negative pressure and to needle passes were made. all dogs received mg/kg metimazole im after eus fna and were re-checked ultrasonographically minutes post eus fna. postoperative activity was assessed using a standardized scoring system. a cbc, serum biochemistry, urinalysis and spec cpl s were measured before, as well as and h after eus fna. the eus examination was complete in / dogs, the pancreas could not be visualized in dog. the pancreas was hypo-( / ) to isoechoic ( / ) to the surrounding mesenterium in all cases. in / dogs parts of the pancreas presented hyperechoic. the mean measured thickness was . cm. the pancreas was aspirated in dogs using a transgastric approach ( ) or transduodenal approach ( ). duodenal transmural puncture was not accomplished in dog where a re-sterilized needle was used. a minimal amount of peripancreatic fluid was observed in / dogs after eus fna. all dogs recovered uneventfully and required no further analgesia. all laboratory results including the spec cpl s measurements were within reference ranges on all three time points. cytologically, conglomerates of exocrine pancreatic cells were seen in / cases, duodenal villous epithelial cells were seen in / cases. in dog the aspirated pancreatic material was sufficient for a histological assessment. the aspirates with exocrine pancreatic cells on cytology were obtained by transgastric ( ) and transduodenal ( ) aspirations. in conclusion, ( ) eus examination of the abdomen is feasible in medium-sized dogs, ( ) the healthy canine pancreas can be difficult to visualize completely, and ( ) eus-guided pancreatic fna using a g needle is a safe procedure in healthy dogs. studies evaluating its use in dogs with pancreatic disease are warranted to assess its clinical utility. miniature schnauzers have a high prevalence of idiopathic hyperlipidemia, which is characterized by an increased serum triglyceride (tg) concentration, with or without an increased serum cholesterol (chol) concentration. a common initial therapeutic approach for the management of hyperlipidemia is the use of a low-fat diet. also, it is believed that low-fat diets may be beneficial in the treatment of pancreatitis in dogs. however, the efficacy of this approach has not been evaluated for either condition. the aim of the present study was to evaluate the effect of a commercially available low-fat diet on serum concentrations of tg, chol, and canine pancreatic lipase immunoreactivity (cpli; measured as spec cpl s ) in apparently healthy miniature schnauzers with hypertriglyceridemia. blood samples were collected from apparently healthy miniature schnauzers with hypertriglyceridemia (serum triglyceride concentrations mg/dl). common causes of secondary hyperlipidemia were excluded based on historical information, physical examination findings, and the measurement of serum glucose, total t , and free t (by ed) concentrations. the owners of the dogs were asked to switch their dog to the study diet (royal canin gastrointestinal low fat s ; fat content: . g/ , kcal) and have a second blood sample collected weeks after their dog had been on the new diet. all blood samples were collected after food had been withheld for hours. serum tg, chol, and spec cpl concentrations were measured both before and after the diet change. results were compared between the two time-points using the wilcoxon signed rank and fisher's exact tests. serum tg concentrations were significantly higher before (median: mg/dl) than after the diet change (median: mg/dl; p . ). the proportion of dogs with hypertriglyceridemia was significantly higher before ( / ) than after the diet change ( / ; p . ). also, the proportion of dogs with serum tg mg/dl was significantly higher before ( / ) than after the diet change ( / ; p . ). serum chol concentrations were significantly higher before (median: mg/dl) than after the diet change (median: mg/dl; p . ). the proportion of dogs with hypercholesterolemia was significantly higher before ( / ) than after the diet change ( / ; p . ). finally, the difference in serum spec cpl concentrations before (median: mg/l) and after the diet change (median: mg/l) approached but did not reach significance (p . ). also, the proportion of dogs with high serum spec cpl concentrations before ( / ) and after the diet change ( / ) was different, but this difference was not significant (p . ). in summary, a commercially available low-fat diet was effective in reducing serum tg and chol concentrations in miniature schnauzers with hypertriglyceridemia. toll-like receptor (tlr ) is an extracellular pattern recognition receptor which recognizes flagellin present in motile bacteria. we have previously demonstrated a significant association between three non-synonymous single nucleotide polymorphisms (snps) in the tlr gene (g a, c t and t c) and inflammatory bowel disease (ibd) in german shepherd dogs (gsds). recently, we have confirmed that two of these tlr snps (c t and t c) are significantly associated with ibd in other canine breeds. to further substantiate the role of tlr in canine ibd functional analysis of these polymorphisms would be needed. therefore the aim of this study was to determine the functional significance of the tlr snps by transfecting wild-type and mutant receptors in to human embryonic kidney cells (hek) and carrying out nuclear factorkappa b (nf-kb) luciferase assay and il- elisa. the tlr gene containing the risk haplotype for ibd (acc) and wild-type haplotype (gtt) as determined by the case-control analysis in gsds with ibd were cloned into plasmids expressing yellow-fluorescent protein (yfp). these were then stably transfected into hek cells. nf-kb activity was measured by transiently transfecting the cells with nf-kb firefly and hsv-thymidine kinase promoter (prl-tk) renilla plasmids. the cells were then stimulated with various ligands ( . mg/ml flagellin, . mg/ml flagellin, mg/ml lps, mg/ml pam csk and media control). firefly and renilla luciferase activities were measured using the dual-glo luciferase assay system (promega, uk) according to the manufacturer's recommendations. the supernatants were harvested and used in an il- elisa (r&d systems). human tlr transfected hek cells (invivogen) served as positive controls in all experiments. independent t-test was used to determine the significance of relative luciferase activity and il- concentration between wild-type and mutated tlr cells. although there was no significant difference between the wild-type and mutated receptor when they were stimulated with . mg/ml of flagellin (p . ), there was a significant increase when the cells with mutated tlr were stimulated with . mg/ml of flagellin compared to the cells expressing wild-type tlr (p . ). similarly, there was a significant increase in il- concentration in the supernatants in the cells with the mutated tlr receptor when stimulated with . mg/ml flagellin compared to the wild-type (p . -one-tailed, . -two-tailed) but not with . mg/ml flagellin (p . ). we show for the first time that polymorphisms associated with ibd are functionally hyper-responsive to flagellin compared to the wild-type receptor. this suggests that tlr may play a role in canine ibd and that blocking the hyper-responsive receptor found in susceptible dogs with ibd may alleviate the inappropriate inflammation seen in this disease. however, further in-vivo functional analysis of tlr , especially at the intestinal mucosal level would be needed to confirm these findings and predict the usefulness of any future therapeutic interventions. tlr has been shown to play a role in the inappropriate inflammation seen in human inflammatory bowel disease (ibd). similarly, we have recently demonstrated a significant association between three non-synonymous single nucleotide polymorphisms (snps) in the canine tlr gene (g a, c t and t c) and inflammatory bowel disease (ibd) in german shepherd dogs (gsds). therefore the aim of this study was to determine the functional significance of the tlr snps in the breed of gsds. the tlr gene containing the risk haplotype for ibd (acc) and wild-type haplotype (gtt) were stably transfected into hek cells. nf-kb activity was measured by transiently transfecting the cells with nf-kb firefly and hsv-thymidine kinase promoter (prl-tk) renilla plasmids. the cells were stimulated with various tlr ligands ( . mg/ ml flagellin, . mg/ml flagellin, mg/ml lps, mg/ml pam csk and media control). firefly and renilla luciferase activities were measured using the dual-glo luciferase assay system (promega, uk). the supernatants were harvested and used in an il- elisa (r&d systems). peripheral whole blood from dogs carrying the wild type and mutant tlr genes was cultured and stimulated with tlr ligands as above. canine tnf-alpha was measured in the supernatant by commercially available elisa (r&d systems). t-test was used to determine differences of relative luciferase activity, il- concentration and tnf-alpha concentration between wild-type and mutated tlr cells. there was a significant increase in nf-kb activity when the cells with mutated tlr were stimulated with . mg/ml of flagellin compared to the cells expressing wild-type tlr (p . ), which correlated with il- expression in the supernatant (p . ). similarly, in the whole blood assay the tlr risk haplotype for ibd in gsds (acc) was significantly hyperresponsive to flagellin at a concentration of . mg/ml compared to the tlr wild-type haplotype (gtt) (p . ). we show for the first time that polymorphisms associated with canine ibd in gsds are functionally hyper-responsive to flagellin compared to the wild-type receptor. blocking the hyper-responsive receptor found in susceptible dogs with ibd may alleviate the inappropriate inflammation seen in this disease. proton pump inhibitors (ppi) are widely used in human and also veterinary medicine. side-effects of ppi treatment reported in people are atrophic gastritis, gastric and esophageal cancer, and rebound hyperacidity following cessation of treatment, which has been speculated to be due to a sustained increased in circulating gastrin concentration. moreover, long-term ppi treatment has been associated with an increased risk for osteoporosis in people. little is known about the effect of ppi treatment on serum gastrin concentration or calcium metabolism in dogs. eight healthy adult research dogs ( males and females) were enrolled into the study. the dogs received an average dose of . mg/ kg of omeprazole orally twice daily for days. blood samples were collected prior to initiating the treatment and every days during the days of treatment and during the days after discontinuation of treatment for determination of serum gastrin, ionized calcium, pth, and oh vitamin d . gastric fluid was collected via gastroscopy after an overnight fast for measurement of gastric ph prior to, during, and after the omeprazole treatment period. normally distributed data were compared with a repeated measures anova and post hoc dunnett's test. data that were not normally distributed were compared with a friedman's test and a post-hoc dunn's test. gastric fluid ph was significantly higher (p o . ) at the end of the treatment period (median: . ; range: . - . ) when compared to pretreatment values (median: . ; range: . - . ). serum gastrin concentrations increased significantly from a median baseline of . ng/l (range: . - . ) to a maximum median of . ng/l (range: . - . ) at day of treatment (p o . ). serum gastrin remained significantly increased above baseline values from day to day of the treatment, but was not different from pre-treatment values days after the end of the treatment. omeprazole treatment had no effect on ionized calcium or pth for the duration of the study. marginal, but significant changes of oh vitamin d were observed at day (end of the treatment period -increased by . %) and day ( days after the end of the treatment -decreased by . %). this study shows that treatment with omeprazole for weeks results in a profound and sustained increase in serum gastrin concentration in dogs. this effect is rapidly reversible after cessation of the treatment. no effect on calcium metabolism was observed. however, this study documents only the effect of short-term treatment and it is possible that the effects of long-term administration are different. omeprazole treatment has been associated with small intestinal bacterial overgrowth and a higher risk for infectious enteropathies in humans. using a semi-quantitative sequencing approach, we have previously shown that omeprazole treatment may lead to alterations in both duodenal and gastric bacterial populations in healthy dogs (acvim ). however, a sequencing approach can only estimate relative proportions of genomic bacterial targets. therefore, significant changes in the total number of bacteria could not be evaluated. the aim of this study was to quantify gastric and duodenal bacterial populations in dogs undergoing omeprazole treatment. eight month-old healthy research dogs ( males and females) were enrolled. the dogs received an average dose of . mg/kg of omeprazole orally twice a day for days. endoscopic gastric and duodenal biopsies were harvested and days before starting omeprazole treatment, on the last day of treatment (day ), and days after the end of treatment (day ). all biopsies were fixed in % formalin for hours, processed, and embedded in paraffin blocks. fluorescent in situ hybridization was used to quantify mucosa-associated bacteria using fluorescently-labeled probes targeting the s ribosomal rna. statistical analysis aimed to compare changes in helicobacter spp. in gastric biopsies and total bacteria in both gastric and duodenal biopsies using the glimmix and npar way procedures in sas s . . bacteria were counted in , and microscopic fields ( Â) obtained from and gastric and duodenal biopsies, respectively. in the stomach, omeprazole treatment led to a decrease in helicobacter spp. (log of average counts ae standard error: . ae . at day ) when compared to the counts ( . ae . , p . ) and ( . ae . , p . ) days before treatment. after completion of omeprazole treatment, helicobacter spp. increased and returned to baseline counts ( . ae . at day , p . vs day ). also, in the stomach, non-helicobacter spp. bacteria were observed more often during omeprazole treatment (median: , range: - ) than on days (median: , range: - ) and (median: , range: - ) before and days after (median: , range: - ) omeprazole treatment; however, statistical comparison across time points did not reach significance. in the duodenum, while the median number of bacteria for all time points was zero, non-parametric comparison of median scores (number of points above median) revealed significantly higher numbers of bacteria during omeprazole treatment (p . ). our results suggest that omeprazole treatment for weeks leads to a lower abundance of helicobacter spp. organisms in the stomach of healthy dogs. also, this transient decrease in helicobacter spp. was accompanied by a higher abundance of other bacteria in both the stomach and the proximal duodenum. the smartpill ph.p s capsule (the smartpill corporation) is a wireless motility capsule that measures ph, pressure, and temperature as it passes through the gastrointestinal (gi) tract. analysis of this data allows the calculation of gastric emptying time (get), small and large bowel transit time (slbtt), and total gi transit time (tgtt). this study evaluated the variability associated with repeated measurement of gi transit times and the effect of oral administration of ranitidine (zantac s ) on gi transit times in dogs using this system. it was hypothesized that ranitidine would reduce gi transit times. six privately owned healthy adult dogs weighing between . kg and . kg were used. on occasions each dog was fed a standard meal followed by oral administration of a capsule. data were recorded until the capsule had passed in the dog's feces. on a th occasion each dog was given mg of ranitidine po q hrs starting hrs prior to testing. the dogs were then fed the test meal and the capsule was administered as above. ranitidine was given until the capsule had passed in the dog's feces. proprietary smartpill software was used to calculate get, slbtt, tgtt, and the median gastric ph (mgph). mean intra-individual and inter-individual coefficients of variation (cv%) were calculated for get, slbtt, and ttt for the first time points. transit times and gastric ph recorded at all time points were compared using a repeated measures anova. where significant differences were identified, post-hoc testing was performed using a bonferroni's multiple comparisons test. significance was set at p o . . a sharp rise in ph indicating exit of the capsule from the stomach was identified in each experiment. mean (ae sd) get, slbtt, and tgtt without ranitidine were ae , ae , and ae min, respectively. mean get, slbtt, and tgtt during treatment with ranitidine were ae , ae , and ae min, respectively. mean intra-individual cv% before ranitidine for get, slbtt, and tgtt were . , . , and . %, respectively. mean inter-individual cv% before treatment with ranitidine for get, slbtt, and ttt were . , . , and . %, respectively. no significant differences in get, slbtt, or tgtt were found at any of the time points. the mean mgph during treatment with ranitidine (ph . ) was significantly higher than at all other time points (overall mean ph for the time points: . ; p o . ). the smartpill system is an easy to use, ambulatory, non-invasive, non-radioactive method for assessing gi transit times in medium to large breed dogs. measurements of gi transit times, especially slbtt, were subject to considerable intra-individual and interindividual variation. no significant effect of oral ranitidine on gi motility was identified in this group of dogs. however, as expected, oral ranitidine caused a significant increase in gastric ph. the intestinal microbiota has been implicated in the pathogenesis of various gastrointestinal disorders in both humans and dogs. recent metagenomic data suggest that specific bacterial groups, including bacteria within the clostridium clusters iv and xiva (i.e., faecalibacterium spp., ruminococcaceae, and lachnospiraceae) and bifidobacterium spp. are decreased, while proteobacteria are increased in dogs with clinical signs of gastrointestinal disease. the objective of this study was to establish quantitative polymerase chain reaction (qpcr) assays for these specific bacterial groups and evaluate their abundance in healthy dogs and dogs with clinical signs of gastrointestinal disease. fecal samples were collected from healthy dogs ( females and males) and dogs with clinical signs of gastrointestinal disease ( females and males). novel quantitative pcr assays were established for faecalibacterium spp., ruminococcaceae, and lachnospiraceae by aligning respective group specific sequences against canine specific sequences obtained from s rrna gene clone libraries and sequences available from the ribosomal database project. primers for bifidobacterium spp. and proteobacteria were selected from previously published studies. the specificity of the qpcr assays was confirmed by sequencing of obtained qpcr amplicons. the bacterial dna abundance in fecal samples was compared between healthy dogs and dogs with clinical signs of gastrointestinal disease using a mann-whitney u test. significance was set at p o . . a significantly lower abundance of faecalibacterium spp. (p o . ) and ruminococcaceae (p . ) was observed in dogs with clinical signs of gastrointestinal disease when compared to healthy dogs. proteobacteria were more abundant in dogs with clinical signs of gastrointestinal disease, but this difference did not reach statistical significance (p . ). there was no significant difference in the abundance of lachnospiraceae (p . ) and bifidobacterium spp. (p . ) between both groups. in conclusion, we established novel qpcr assays for faecalibacterium spp., ruminococcaceae, and lachnospiraceae. we observed significant decreases in the abundance of faecalibacterium spp. and ruminococcaceae in dogs with clinical signs of gastrointestinal disease. these bacterial groups are considered major short-chain fatty acid producers and studies are warranted to determine if a decrease in these bacterial groups is associated with decreases in short chain fatty acid production. further studies are also needed to determine if these bacterial shifts are associated with specific gastrointestinal disorders. the pathogenesis of chronic enteropathies (ce) in dogs likely involves complex interaction between the mucosal immune system and the intestinal microbiota. while the application of bacterial s rdna sequence-based analysis has shown an association between altered microbial composition and duodenal inflammation in dogs, relatively little is known about alterations in non-invasive mucosal and luminal bacteria seen with diseases involving the ileum and colon. the present study sought to evaluate the relationship of enteric bacteria to type and severity of mucosal inflammation affecting the ileum and colon of dogs with ce. eleven client-owned dogs with ce involving both the small and large intestines were prospectively enrolled. ce was diagnosed on the basis of a history of chronic gastrointestinal signs, exclusion of identifiable underlying disorders, and histopathologic evidence of intestinal inflammation. mucosal bacteria were detected in formalinfixed ileal and colonic tissue sections with fluorescence in situ hybridization (fish) using s rdna-targeted probes directed against all bacteria, enterobacteriaceae, e. coli, eubacterium rectale-clostridium coccoides group, bacteroides/prevotella, and helicobacter spp. sections were examined by epifluorescence microscopy and the number of bacteria and their spatial distribution (luminal, superficial mucus, epithelial adherent, within mucosa) was determined in ten x fields of each section. microbial composition in ce dogs was compared to the ileal/colonic microbiota of healthy control (hc) dogs using a mixed effect anova model. p values o . were considered significant. the final diagnoses for dogs with ce included ibd (n ) and lymphosarcoma (n ). when compared to hc dogs, dogs with ce showed regional (ileum versus colon) imbalances in microbiota composition characterized by selective enrichment of mucosa-associated populations. evaluation of colonic biopsies in dogs with ce showed that the total number of bacteria (p o . ), clostridium (p o . ), enterobacteriaceae (p o . ) and e. coli (p o . ) were increased in the adherent mucus regions of dogs with ibd as compared to hc dogs. total bacteria (p o . ) and e. coli (p o . ) were also more numerous in dogs with lsa versus hc and ibd dogs (p o . for e. coli). ileal biopsies from ce dogs similarly showed variable dysbiosis with increased total bacteria (p o . ) but decreased helicobacter spp (p o . ) and bacteroides (p o . ) observed within inflamed intestines as compared to hc tissues. the spatial distribution of these bacteria was also appreciably different from hc dogs, with higher numbers of bacteria generally found within the adherent mucus compartment as compared to other ileal regions. our data demonstrate that dogs with ce affecting the ileum and colon have altered microbiota composition that may be a cause or consequence of mucosal inflammation. recognition of these microbiota imbalances may provide new opportunities for therapeutic intervention. trichomonads have been rarely reported in the feces of dogs and their pathogenicity remains uncertain. although pentatrichomonas hominis (ph) is considered to be a commensal that may overgrow in dogs with other causes of diarrhea, little is known regarding the history, clinical presentation or prevalence of concurrent gi infections in dogs with trichomonosis. the aim of this study was to determine whether dogs with diarrhea and trichomonosis could be distinguished from dogs having diarrhea without trichomonosis on the basis of clinical signs or presence of concurrent enteric infections. fecal samples from dogs were submitted to ncsu from - for trichomonas spp. pcr testing. dna was extracted using a zr fecal dna mini-prep kit and absence of pcr inhibitors verified by amplification of bacterial s rdna. pcr for ph and tritrichomonas foetus (tf) was performed as well as real-time pcr assays for possible concurrent enteric infectious agents. obtainable medical records were reviewed. all submitted fecal samples were submitted from dogs with diarrhea that was variably described as soft, mucoid, hemorrhagic, or watery. mean age of the dogs was . years (median . ; range: . - months) and represented a total of breeds. ph, tf, or concurrent ph and tf were diagnosed in , , and dogs respectively (group a). the remaining dogs were negative for ph and tf by pcr no dogs were identified as infected with canine distemper virus or parvovirus. five samples from each group had insufficient quantity or quality of dna for concurrent infectious disease testing. in this large study of canine trichomonosis, no differences in age, clinical signs, or prevalence and identity of concurrent enteric infection between diarrheic dogs with or without ph were identified. thus, these findings do not appear to support a primary pathogenic role for ph as a causative agent of diarrhea in dogs. gastrointestinal motility disorders are a common clinical problem in domestic animals. many of the g.i. motility disorders have been treated previously with -ht agonists although limited availability of drugs in this classification have stimulated interest in the use of new (and old) drug therapies. the dopaminergic antagonists are a group of drugs with well-known anti-emetic effects at central dopamine d receptors, and putative gastrointestinal prokinetic effects at peripheral d receptors. domperidone has been shown, for example, to reverse gastric relaxation induced by dopamine infusion in the dog. similar studies have not been reported in the cat or rabbit, two species at risk for distal gastrointestinal motility disorders. our aim was to study the effects, mechanisms, and sites of action of domperidone in feline colonic and rabbit gastrointestinal smooth muscle contraction. portions of stomach (fundus and antrum), intestine (duodenum and ileum), cecum (rabbits only), and colon (ascending and descending) were obtained from healthy cats and rabbits from - months of age. longitudinal and circular smooth muscle strips from each site were suspended in physiologic (hepes) buffer solution, attached to isometric force transducers, and set to optimal muscle length (l o ) using acetylcholine (ach; À m). muscle strips were treated with domperidone (d; À to À m) in the presence or absence of ach ( À to À m), and maximal force output (p max ) was normalized for cross-sectional area (n  newtons/m ). domperidone (d) had a minor direct effect of inducing feline and rabbit gastric, cecal, and colonic smooth muscle contraction. direct effects were similar whether in the longitudinal or circular muscle orientation. the direct effect of domperidone was dose-dependent and maximal (feline colon p max . - . n; rabbit colon p max . - . n) at a dose of À m. domperidone had a much greater indirect effect in augmenting cholinergic (ach; À m) contractions in feline and rabbit gastric, cecal, and colonic smooth muscle. domperidone-augmented cholinergic contractions were - % (feline colon p max . ae . n ach only; feline colon p max . ae . n ach d) of baseline cholinergic contractions. domperidone contractions were of a similar magnitude to those induced by cisapride. domperidone effects were similar in mucosaintact and mucosa-dissected preparations. domperidone contractions were unaffected by prazosin (a receptor antagonist), yohimbine (a receptor antagonist), or terbutaline (b receptor agonist), but were somewhat attenuated by dopamine (d receptor agonist) and a non-specific cholinergic antagonist (atropine). in vitro studies show for the first time that domperidone has minor direct and major indirect effects in augmenting cholinergic contractions of feline and rabbit gastrointestinal (stomach, cecum and colon) smooth muscle. as recognition of acute and chronic pain in dogs has increased, so too has the use of non-steroidal anti-inflammatory drugs (nsaids) often in conjunction with tramadol. in people and rats, co-administration increases the risk of perforation and gastric injury over nsaids alone. using an ex vivo model of acid injury in canine gastric mucosa, we examined the effects of indomethacin and tramadol on gastric permeability and concentrations of gastroprotective prostaglandin e (pge ). mucosa from the gastric antrum was harvested from shelter dogs immediately after euthanasia, and mounted on ussing chambers. the tissues were equilibrated for -minutes prior to addition of acidic ringer's solution (ph, . ). after -minutes of injury, the acid was replaced with neutral ringer's and the tissues were treated with indomethacin, tramadol or both. tissues were maintained for minutes total, during which time permeability was assessed electrically. prostanoid concentrations were quantified using a commercially available elisa. western blots were performed for cox- and À . recovery of gastric barrier function after acid injury was inhibited by co-administration of tramadol and indomethacin ( figure ) but not by tramadol or indomethacin alone (data not shown). prostaglandin e increased with acid injury. the increase in pge was inhibited by co-administration of indomethacin and tramadol (in pg/ml: acid injury . ae . , indo tramadol . ae . ). there was no significant effect of treatment on cox- or À expression. co-administration of tramadol with a non-selective nsaid inhibits the return of gastric mucosal barrier function after acid injury in canine tissue, suggesting that caution is required in prescribing concurrent use of these drugs in dogs at risk for gastric ulcers. these drugs may exert this effect by decreasing levels of gastroprotective prostanoids. further study is needed to understand the mechanism of this drug interaction. an increased intestinal permeability (ip) has been suggested to be both cause and consequence of gastrointestinal (gi) disease, such as inflammatory bowel and celiac disease, in people. a novel tight junction regulator, larazotide acetate (alba therapeutics, baltimore, md) has been shown to significantly decrease ip in rats and in humans with celiac disease. the purpose of this study was to determine if larazotide acetate reduces ip in soft coated wheaten terriers (scwt) and norwegian lundehunds (nl) with chronic gi disease and ameliorates clinical signs. four nl ( females, males; median age: . yrs, range: . - . yrs) and scwt ( females, males; median age: . yrs, range: . - . yrs) were enrolled based on presence of clinical signs of gi disease and hypoalbuminemia, increased fecal alpha proteinase inhibitor (a -pi) concentrations, and/or hypocobalaminemia. scwt with protein-losing nephropathy were excluded. dogs were fed q hrs and received . mg ( nl and scwt) or . mg ( scwt) of larazotide acetate po before each meal for days. prior to start of treatment (day ) and at the end (day ), ip was evaluated by calculating the lactulose/rhamnose (l/r)-ratio in serum samples obtained at , , , and min after oral dosing. also, consecutive fecal samples each were collected prior to day and day for n-methylhistamine (nmh) measurement. pre-and post-treatment data were compared using a wilcoxon signed rank test. the . mg vs. . mg dose groups were compared using a mann-whitney u test. statistical significance was set at p o . . l/r-ratios (medians) for the min sampling time point were significantly lower on day ( . ) than on day ( . ; p . ). dogs treated with . mg q hrs had significantly lower min l/r ratios on day than dogs treated with . mg ( . vs. . ; p . ). no difference was found between breeds. fecal nmh concentrations were not different between time points, treatment groups, or breeds. fecal a -pi concentrations were available for of the dogs and were significantly higher on day compared to day (p . ). no differences were found between pre-and post-treatment serum albumin or cobalamin concentrations. weight gain was seen in all nl. resolution of diarrhea, vomiting, hyporexia, as well as an increased activity was seen in scwt. another scwt had resolution of diarrhea and a decrease in pruritus. no changes in clinical signs were reported in the remaining scwt. this study indicates that larazotide acetate might be able to reduce ip in dogs. this effect may be dose-dependent. however, not all dogs showed an improvement in clinical signs, suggesting that factors other than increased ip might have been responsible for the clinical signs in these dogs. breed-related effects cannot be ruled out, and further studies are warranted to determine the efficacy of larazotide acetate in dogs of other breeds with gi disease. to analyze different biochemical markers, calculate clinical activity scores, and assess survival in dogs with ple and compare them with those in dogs with food-responsive diarrhea (frd) without protein loss. dogs with ple and dogs with frd, referred to the university of bern, ch, were enrolled. selection criteria included a history of chronic diarrhea ( weeks), exclusion of identifiable underlying causes, and histopathologic evidence of intestinal inflammation, but not neoplasia. underlying disorders were excluded based on cbc, chemistry profile, urinalysis, fecal analysis, trypsinlike immunoreactivity, cobalamin, folate, and transabdominal ultrasound. also, canine pancreatic lipase immunoreactivity (spec cpl s ), c-reactive protein (crp), calprotectin and alpha -proteinase inhibitor (a -pi) were measured in serum from dogs and compared with dogs with frd without ple. all dogs were scored using the canine ibd (cibdai) and the canine chronic enteropathy (cce) clinical activity index (ccecai). total protein, albumin ( - . g/l), and total calcium ( . - . mmol/l) were decreased in all dogs. cobalamin was decreased in all but dogs ( o - ng/l). spec cpl was mildly increased in / dogs with ple and normal in / ple and all frd dogs. crp was normal in / dogs with ple ( / frd), mildly increased in / ( / frd), and moderately increased in / ple dogs ( / frd). calprotectin was slightly higher in dogs with ple, but all ple and frd dogs yielded values in the normal range. serum a -pi was significantly lower in dogs with ple than in those with frd (p o . ), with / ple dogs below the reference range ( / frd). cibdai ranged from to and ccecai from to . at the end of the study, / dogs were still alive with survival times between and days. / dogs died with a median survival of days (range - days). dogs with mildly increased crp died earlier than dogs with a normal or moderately increased crp (p . ), whereas albumin, calcium, spec cpl, calprotectin, cibdai, and ccecai had no significant impact on outcome and survival. in conclusion, dogs with ple have a significantly lower a -pi in the serum than dogs with frd. furthermore, most dogs with ple have an increased crp and a decreased cobalamin. a mild increase in crp appears to be a poor prognostic factor. while hypoalbuminemia is a common finding associated with chronic enteropathies, its impact on survival in this population is poorly defined. the aim of this study was to compare dogs with chronic enteropathies on the basis of their serum albumin concentration at the time of presentation. we hypothesized that dogs with a protein losing enteropathy (ple) have a significantly shorter survival time compared to dogs with chronic enteropathies which are not hypoalbuminemic (controls). information obtained from the medical records included signalment, duration and characteristics of clinical signs, physical examination findings, clinicopathologic data and survival time. one hundred seventeen cases fit the inclusion criteria; in the ple group and controls. there was no statistical significance between groups for age (p . ), weight (p . ), weight loss (p . ) and body condition score (p . ). compared to control dogs, ple dogs had decreased serum concentrations of cobalamin (p . ), total calcium (p o . ), globulin (p o . ), cholesterol (p o . ) and ionized calcium (p o . ). survival analysis revealed a significantly decreased survival time for ple dogs (p . ); median survival was days for ple dogs and , days for controls. while the ple group did not survive as long, survival was not directly associated with severity of hypoalbuminemia; patients with albumin concentration o . g/dl survived longer than those with mild hypoalbuminemia ( . - . g/dl). this study supports the observation that chronic enteropathy patients have decreased survival time when presented with hypoalbuminemia; however this study suggests the severity of hypoalbuminemia is not a reliable indicator of survival. cobalamin (vitamin b ) deficiency in the chinese shar pei (shar pei) is suspected to be hereditary. inherited causes of cobalamin deficiency have been reported in humans and may affect absorption, transport, or cellular processing of cobalamin. based on human and veterinary studies, an increased serum methylmalonic acid (mma) concentration has been suggested to reflect cobalamin deficiency at the cellular level. in this context, it has been shown in humans that mma concentrations are higher in patients with genetic disorders affecting intracellular processing than in patients with genetic defects affecting gastrointestinal processing and extracellular transport of cobalamin. therefore, the aim of this study was to evaluate serum mma concentrations in shar peis and dogs of six other breeds with cobalamin deficiency. from in conclusion, serum cobalamin deficient shar peis had a times higher median serum mma concentration compared to cobalamin deficient dogs of six other dog breeds. further studies are needed to investigate the intracellular processing of cobalamin in shar peis with cobalamin deficiency. chinese shar peis (shar peis) have a high prevalence of cobalamin deficiency. two other conditions reported frequently in this breed are shar pei fever and cutaneous mucinosis. shar pei fever is an autoimmune disorder causing periodic flare-ups and is associated with increased serum concentrations of c-reactive protein (crp), a nonspecific marker of inflammation. cutaneous mucinosis is characterized by excessive deposition of mucin in the dermis. also, hyaluronic acid (ha), the main component of mucin, was shown to be significantly higher in serum from shar peis with cutaneous mucinosis than in healthy controls. to date, a possible association between shar pei fever and/or cutaneous mucinosis on one side and cobalamin deficiency on the other has not been investigated in shar peis. thus, the aim of this study was to compare serum concentrations of ha (an indicator of cutaneous mucinosis) and inflammatory markers (crp, calprotectin, and s a ), assumed to be increased in episodes of shar pei fever, in shar peis with and without cobalamin deficiency. serum samples from shar peis, collected from to , were analyzed. serum ha and crp (reference interval (ri): . - . mg/l) were quantified by using commercial elisa kits (echelon biosciences, salt lake city, ut, usa and tridelta, maynooth, ireland; respectively). serum calgranulin concentrations were measured using an in-house elisa (calprotectin; ri: . - . mg/l) and ria (s a ; ri: . - . mg/l), respectively. mann-whitney u tests were used to compare serum ha, crp, calprotectin, and s a concentrations between shar peis with and without cobalamin deficiency. significance was set at p o . . fourteen shar peis were severely cobalamin deficient, defined by an undetectable serum cobalamin concentration ( o ng/l). in the remaining dogs, serum cobalamin concentrations were within the reference interval ( - ng/l). serum concentrations of ha, crp, calprotectin, and s a were not significantly different between cobalamin deficient shar peis (medians: . ng/ml, . . fifty percent of cobalamin deficient shar peis had serum calprotectin concentrations above the upper limit of the reference interval, and % had serum s a concentrations above the suggested upper reference limit. in this study, serum concentrations of ha, crp, and the calgranulins did not differ between cobalamin deficient shar peis and shar peis with a normal serum cobalamin concentration. this finding leads us to speculate that increased ha and/or inflammatory markers are not associated with cobalamin deficiency in shar peis. further studies are needed to investigate serum cobalamin concentrations in patients with shar pei fever or cutaneous mucinosis. cobalamin deficiency (cd) has been associated with gastrointestinal and pancreatic disease in dogs. hereditary cd has been demonstrated in giant schnauzers and single case reports have suggested congenital cd in the border collie (bc) breed. clinicopathologic findings of cd vary and can be unspecific as cobalamin acts as a co-factor for a multitude of enzymatic reactions. the two most important reactions concern the conversion of methylmalonyl-coa to succinyl-coa and the re-methylation of homocysteine (hcy). these two metabolites increase when cobalamin is lacking and act as markers for cobalamin availability on a cellular level. preliminary data from dogs suggested that measurement of methylmalonic acid (mma) may be a better diagnostic test for cd than serum cobalamin concentration. therefore the goals of the study were ( ) to establish reference values for serum cobalamin, urine mma and plasma hcy in healthy pet dogs, ( ) to screen a larger bc population from switzerland for cd, and ( ) to perform genomic analyses on bc with cd. for determination of reference values healthy pet dogs were used. serum cobalamin was measured using an automated chemiluminescence assay (immulite ), urine mma was determined using gas chromatography and expressed as a ratio to urine creatinine and plasma hcy was measured using high pressure liquid chromatography and fluorimetric detection. to calculate reference ranges the th and th percentile were used. data were analyzed using non-parametric tests. reference ranges for cobalamin, hcy, and mma were: cobalamin . - . ng/l; urine mma - . mmol/mol creatinine; and plasma hcy . - . mmol/l. the screened bc population comprised purebred dogs and bc (median . months; range - ) suffering from congenital cd could be identified. clinical signs differed and consisted of tiredness ( ), stunted growth ( ), anemia ( ), dysphagia ( ) and persistent fever ( ). median (ranges) results for healthy bc and bc with cd were: for cobalamin ( - ) and . ( - ) ng/l; for urine mma ( - ) and ( - ) mmol/mol creatinine; for hcy . ( . - . ) and . ( - . ) mmol/l. strikingly, healthy bc with cobalamin concentrations well within the reference range had significantly higher urine mma concentrations compared to control dogs. under the assumption that the four affected bc are inbred to a single founder animal, first results of genotyping on the k illumina canine_hd snp chip suggest that mutations in the cubn and amn gene can be excluded to cause the observed cd in these dogs. we conclude that cd is a rare familial disease in bc with variable clinical signs. to define the genomic region responsible for cd further genetic analysis is in progress. it remains to be determined why some bc have high urine mma concentrations despite a serum cobalamin concentration within the reference range. calprotectin is a protein complex that plays an important role in the innate immune response. preliminary data suggest that canine calprotectin (ccp) is a useful marker for the detection of inflammation in dogs. recently, a radioimmunoassay for the measurement of ccp has been developed and analytically validated, but this test requires the use of a radioactive tracer. therefore, the aim of this study was to develop and analytically validate an enzyme-linked immunosorbent assay (elisa) for the quantification of ccp in serum and fecal specimens from dogs. canine calprotectin (ccp) was purified, antiserum against purified ccp was raised in rabbits, monospecific antibodies were purified by affinity chromatography, and a sandwich-elisa was developed. purified antibodies were used for capturing and, after coupling with horseradish peroxidase (hrp), for reporting. a hrp substrate was used for color development. the assay was analytically validated by determination of analytical sensitivity and specificity, dilutional parallelism, spiking recovery, and intra-and inter-assay variability. control intervals for serum and fecal ccp were established from and healthy pet dogs, respectively, using the central th percentile. sensitivity of the assay for serum samples assayed in a : dilution and for fecal extracts assayed in a : , dilution was . mg/l and . mg/g, respectively. over a wide range of the assay, there was no cross-reactivity with cs a , the closest structural analogue of ccp available. observed to expected ratios (o/e) for serial dilutions ranged from . - . % (mean ae standard deviation [sd]: . ae . %) for four different serum samples, and from . - . % (mean ae sd: . ae . %) for five different fecal extracts. o/e for spiking recovery ranged from . - . % (mean ae sd: . ae . %) for four different serum samples and different spiking concentrations, and from . - . % (mean ae sd: . ae . %) for different fecal extracts and different spiking concentrations. intra-assay coefficients of variation (cv) for different serum samples were . , . , . , and . %, and . , . , . , and . % for different fecal extracts. inter-assay cv for different serum samples were . , . , . , and . %, and . , . , . , and . % for different fecal extracts. the control intervals for serum and fecal ccp were established as . - . mg/l and . - . mg/g, respectively. we conclude that this new elisa for the measurement of ccp is analytically sensitive, linear, accurate, precise, and reproducible, and does not cross-react with canine s a . further studies evaluating the clinical usefulness of measuring serum and/or fecal ccp are currently under way. the syndrome of hemorrhagic gastroenteritis (hge) is characterized by a peracute onset of hemorrhagic diarrhea, vomiting, depression, and anorexia, and can be associated with a high mortality if untreated. the etiology of hge is unknown, but it is speculated that an abnormal response to bacterial endotoxins, bacteria, or dietary components may play a role. hge is characterized by an increased vascular/mucosal permeability, thought to represent a type i-hypersensitivity reaction, whereas inflammation and necrosis appear to be rare. however, markers of gastrointestinal (gi) inflammation and changes in the intestinal microbiota have not been studied extensively in dogs with hge. therefore, the aim of this study was to evaluate fecal canine calprotectin (cp) and s a (a ), a -proteinase inhibitor (a -pi, a marker of gi protein loss), and bacterial groups that have previously been shown to be decreased (i.e., faecalibacterium spp., ruminococcaceae, bifidobacterium spp.) or increased (i.e., proteobacteria) in fecal samples from dogs with hge. fecal samples from consecutive days were collected from dogs with hge. fecal cp, a , and a -pi concentrations were measured by in-house immunoassays. bacterial dna was extracted from each fecal sample and was analyzed for faecalibacterium spp., proteobacteria, rumino-coccaceae, and bifidobacterium spp. using quantitative pcr assays. concentrations of fecal cp, a , and a -pi, and the abundance of bacterial dna were compared using a friedman test with dunn's post-hoc tests. significance was set at p o . . at the time of diagnosis (day ), fecal cp, a , and a -pi were above the suggested reference intervals in , , and of the dogs, respectively. until day , this number decreased to , , and , respectively. decreases in concentrations were significant between days and for a (p . ), and between days and for a -pi (p . ), but not for cp despite a trend (p . ). no differences in the abundance of faecalibacterium spp. (p . ), bifidobacterium spp. (p . ), or proteobacteria (p . ) were observed. however, the abundance of rumino-coccaceae was significantly lower on day when compared to day (p . ). in this study, fecal markers of inflammation and gi protein loss were increased in dogs with hge. although the number of patients was small, following initiation of treatment, two of the markers decreased significantly. these results suggest a loss of protein into the gi tract at the onset of hge. the lack of significant increases of faecalibacterium spp., bifidobacterium spp., and ruminococcaceae, and decreases in proteobacteria may suggest gi dysbiosis. further longitudinal studies are needed and are currently under way to evaluate gi dysbiosis in canine hge patients. the most recent antiemetic approved for use in dogs is maropitant citrate (cerenia s , pfizer animal health). maropitant is a selective nk receptor antagonist that acts by blocking the binding of substance-p within the emetic center and chemoreceptor trigger zone. label dosage recommendations for maropitant citrate are mg/ kg sc or mg/kg orally once daily for up to consecutive days (acute emesis) and mg/kg orally once daily for up to consecutive days (motion sickness). the study objective was to determine when steady-state is reached and the pharmacokinetics of maropitant administered at label oral dosages once daily for consecutive days. two groups of eight healthy beagles were administered maropitant citrate at or mg/kg orally once daily for days. concentrations of maropitant and its metabolite were measured in plasma using a lc-ms/ms assay. pharmacokinetic parameters were estimated using non-compartmental pharmacokinetic techniques and a modeling approach was used to estimate steady-state. the accumulation ratio for maropitant was . (auc - ) and . (cmax) for the mg/kg dose; and . (auc - ) and . (cmax) for the mg/kg dose after days. the model estimate for the number of doses required to reach % of steady-state was . for mg/kg and . for mg/kg. three dogs experienced a single episode of vomiting. dosing maropitant citrate beyond the label duration was well tolerated by healthy dogs. steady-state was reached after approximately doses for daily mg/kg and doses for daily mg/kg oral dosing. previously presented at the veterinary cancer society, november . cobalamin (vitamin b ) is involved in a variety of metabolic processes. altered serum cobalamin concentrations have been observed in dogs with gastrointestinal disorders, such as exocrine pancreatic insufficiency (epi) or severe and longstanding ileal disease. this study was conducted to identify breeds with a higher proportion of a decreased serum cobalamin concentration that were submitted to the gastrointestinal laboratory. the study was also aimed at investigating serum trypsin-like immunoreactivity (tli) concentrations that were diagnostic for epi in the dogs with a decreased serum cobalamin concentration. except for csp, breeds identified here, have not previously been identified to have a higher rate of a decreased serum cobalamin concentration. also, a possible association between an undetectable serum cobalamin and a decreased serum tli in ai needs to be further investigated. calprotectin (cp) is a widely used marker for the diagnosis and monitoring of gastrointestinal (gi) inflammation in humans. studies in humans usually report fecal cp concentrations based on a single stool sample although considerable day-to-day variability of fecal cp was found in patients with gi disease and in healthy controls. intra-individual variation of canine cp (ccp) was also substantial in a small number of healthy dogs but has not been determined in dogs with chronic gi disease. thus, the aim of this study was to compare the day-to-day variation of fecal ccp in dogs with chronic gi disease before and during treatment to that in healthy dogs. we hypothesized that fecal ccp would be less variable in patients with chronic gi disease than in healthy controls, and thus collection of a single fecal sample would be sufficient. fecal samples from consecutive days were prospectively collected from dogs (group a; median age: . years) referred for diagnostic work-up of chronic signs of gi disease, from dogs (group b; median age: . years) with stable gi disease while being treated, and from healthy adult dogs (group c; mean age: . years). fecal samples were extracted and ccp was measured by an in-house immunoassay. mean ccp, standard deviation, coefficient of variation (cv), and difference between maximum and minimum ccp for the -day sample collection period were calculated for each dog and were compared among groups using a kruskal-wallis test. fecal ccp ranged from . - . mg/g (median: . mg/g) in dogs with gi disease (group a), from . - . mg/g (median: . mg/g) in dogs of group b, and from . - . mg/g (median: . mg/g) in healthy controls (group c). cvs were - . % in group a (median: . %), . - . % in group b (median: . %), and - . % in group c (median: . %), respectively. patients in group a appeared to have less variable fecal ccp than dogs in group b and c, but this difference was not significant (p . ). the difference between maximum and minimum ccp for the -day sample collection ranged from - . mg/g in group a (median: . mg/g), from . - . mg/g in group b (median: . mg/g), and from - . mg/g in group c (median: . mg/g), and were not significantly different between any of the groups (p . ). in this study, considerable day-to-day variation of fecal ccp was found in dogs with chronic gi disease (regardless of treatment) and was comparable to that in healthy dogs. results of this study suggest that for evaluating fecal ccp in dogs with clinical signs of gi disease, three consecutive fecal samples rather than a single fecal sample should be analyzed. because we did not intend to evaluate the clinical usefulness of fecal ccp as a marker of gi disease in dogs, disease severity, quality, and location differed among dogs in groups a and b. the diagnostic utility of fecal ccp in dogs with gi disease is currently being investigated. it has been suggested that diagnosis of clostridium perfringens related enteropathy should be based on the detection of the c. perfringens enterotoxin gene (cpe-gene) by pcr and/or c. perfringens enterotoxin (cpe) by elisa in feces. however, the prevalence of the cpe-gene and cpe in dogs and especially cats with gastrointestinal disease has not yet been reported. also, there is limited information about the stability of cpe in fecal samples at various storage conditions. the aim of this study was to evaluate the prevalence of the cpe-gene and cpe and the stability of cpe in fecal samples from dogs and cats. to evaluate the prevalence of the cpe-gene, a total of fecal samples from dogs and cats with clinical signs of gastrointestinal disease ( dogs and cats) and fecal samples from those without such signs ( dogs and cats) were examined using pcr. to evaluate the prevalence of cpe, a total of fecal samples from dogs and cats with clinical signs of gastrointestinal disease ( dogs and cats) and dogs without such signs were evaluated using a commercially available elisa kit (techlab, blacksburg, va). the results were analyzed using a fisher's exact test. significance was set at p o . . to evaluate the stability of cpe, fecal samples from dogs and from cats with clinical signs of gastrointestinal disease that were positive for cpe were examined. also, cpe negative samples from dogs were evaluated as negative controls. each sample was subdivided into aliquots and evaluated on day ; on days , , and after being stored at room temperature (rt) or c; and on day after being stored at À c. the prevalence of the cpe-gene was not significantly different between dogs with signs of gastrointestinal disease ( / ; . %) and dogs without ( / ; . %; p . ). also, the prevalence of the cpe-gene in cats with signs of gastrointestinal disease ( / ; . %) was not significantly different compared to cats without ( / ; . %; p . ). pcr and elisa results were available for samples. of the pcr positive samples, only ( . %) were elisa positive. of the pcr negative samples, only ( . %) was elisa positive. the prevalence of cpe was not significantly different between dogs with clinical signs of gastrointestinal disease ( / ; . %) and those without ( / ; . %; p . ). the prevalence of cpe in cats with signs of gastrointestinal disease was / ( . %), but no samples from cats without such signs were available. when evaluating the stability of cpe, results for all aliquots were consistent with the initial result, except for one sample (on day , stored at rt, which was initially cpe positive). these results indicate that only a small proportion of samples that are pcr positive for the cpe-gene are also positive for cpe. studies are warranted to further compare the prevalence of cpe among animals with gastrointestinal disease and those without. furthermore, the results indicate that cpe is relatively stable in fecal samples at various storage temperatures. clostridium perfringens has been implicated as a cause of diarrhea in dogs. the main study objective was to compare two culture methods for the identification of c. perfringens. a secondary objective was to evaluate c. perfringens toxin genes a, b, b , e, ı and cpe from canine isolates using a multiplex pcr and determine their prevalence in a group of normal and diarrheic dogs. fecal samples were collected from clinically normal (nd, n ) and diarrheic dogs (dd, n ) at a primary care veterinary facility. isolation of c. perfringens was performed using direct inoculation of feces onto % sheep blood agar (sba) as well as enrichment of stool in bhi broth followed by inoculation onto sba. isolates were tested by multiplex pcr for the presence of a, b, b , e, ı and cpe genes. c. perfringens was isolated from % ( / ) of nd fecal samples using direct culture and . % ( / ) with bhi enrichment (p . ). in the dd, corresponding isolation rates were . % and . % (p . ). all isolates possessed a toxin gene. b, b , e, ı and cpe toxin genes were identified in . %, . %, . %, . % and . % of nd isolates, respectively. in the dd group, b and b were identified in %, e and ı were not identified and the cpe gene in . % of isolates. bhi enrichment did not significantly increase the yield of c. perfringens compared to sba but increased time and cost involved. c. perfringens (p . ) and c. perfringens toxin genes were present in equal proportions in nd and dd groups (p ! . ). culture of c. perfringens and pcr for toxin genes are of limited diagnostic utility due to the high prevalence of c.perfringens in normal dogs and the lack of apparent difference in toxin gene distribution between normal and diarrheic dogs. endoscopic biopsies are a relatively convenient, non-invasive test for feline infiltrative intestinal disorders. commonly, only the duodenum is examined due to cost, risks and time required to prepare the colon using lavage solutions, cathartics and/or enemas. the purpose of this study was to evaluate the consistency between endoscopic biopsies of the duodenum and ileum in cats. endoscopic biopsies from cats which had duodenal and ileal tissue specimens were evaluated retrospectively. all slides were randomized and reviewed by a single pathologist (jm) for quality, number of biopsies, and diagnosis according to wsava standards. no information regarding history, clinical signs, endoscopic findings, or previous histological diagnosis was made available to the pathologist. statistical comparison of the diagnosis of sc-lsa and ibd by intestinal location was conducted using fisher's exact test (p o . significant). of cats ( . %) were diagnosed with sc-lsa in the duodenum and/or ileum. of these cats, ( . %) were diagnosed with only duodenal sc-lsa, ( . %) were diagnosed with only ileal sc-lsa, and ( . %) had sc-lsa in both duodenum and ileum. in cats with only ileal sc-lsa, had severe ibd in duodenal biopsies, possibly consistent with early sc-lsa. of these had duodenal biopsies without evidence of sc-lsa. our results suggest there is a population of cats in which diagnosis of sc-lsa may only be found by evaluating ileal biopsies. clinicians should consider performing both upper and lower gi endoscopic biopsies in cats with suspected infiltrative small bowel disease. periodontitis is one of the most common diseases in cats and is mainly due to the presence of plaque and calculus. in this study, we investigated putative correlations between dental tartar and gingivitis and also between gingivitis and subgingival bacteria in cats. twelve cats (median age: years; range: - years; dsh and persians; females and males) were enrolled. dental tartar was obtained during scaling for a dental prophylactic procedure. all cats were negative for felv and fiv infection as assessed by a commercial elisa test (snap s fiv/felv combo test). severity of gingivitis (scores: - ; normal, mild, moderate, and severe) and dental tartar (scores: - ) were scored in each cat. endodontic paper points were applied for collecting a bacterial sample from the subgingival area and transferred to thioglycollate transporting media for bacterial culture. the relationship between gingivitis and tartar thickness scores was analyzed by spearman correlation. a student's t-test was used to compare the mean differences (gingivitis and tartar thickness scores) between upper and lower teeth. the association between severity of gingivitis and bacterial type was tested by chi square test. the spearman correlation coefficient for the average gingivitis score and the average tartar thickness score was . (p o . ). interestingly, the average tartar thickness scores from the upper jaw were significantly higher than those from the lower jaw (p o . ). the highest scores were found for the molar teeth in all cats. bacterial culture revealed . % anaerobic bacteria species (i.e., bacteroides spp., peptostreptococcus anaerobius, and eubacterium aerofaciens) and . % aerobic bacteria species (i.e., pasteurella multocida, streptococcus spp., enterococcus spp., staphylococcus spp., bacillus cereus, escherichia coli, and pseudomonas aeruginosa). anaerobic bacteria were found mostly in cats with higher gingivitis scores ( - ; chi square: p o . ), while pasteurella multocida was found mostly in cats with lower gingivitis scores ( - ; chi square: p o . ). antimicrobial sensitivity testing indicated that all of the anaerobic bacteria were sensitive to clindamycin, chloramphenicol, metronidazole, cefoxitin, or tetracycline, % were sensitive to erythromycin, and % were sensitive to penicillin. the most abundant aerobic bacterial species, pasteurella multocida, was sensitive to cefoxitin in all cases in which it had been cultured. these results suggest that anaerobic bacteria may be associated in the pathogenesis of severe gingivitis. these data warrant further studies of the prophylactic use of antibiotics in cats undergoing dental prophylactic procedures. inflammatory bowel disease is the most common cause of vomiting and diarrhea in dogs. although it can occur in any canine breed, certain breeds are more susceptible. we have previously shown that polymorphisms in the tlr and tlr gene are significantly associated with inflammatory bowel disease (ibd) in the german shepherd dog (gsd), a breed at risk of developing this disease. it would be useful to determine if these polymorphisms are significant in other canine breeds as this may allow the development of novel diagnostics and therapeutics to be applied to all canine breeds with ibd. therefore the aim of this study was to investigate whether polymorphisms in canine tlr and tlr genes are associated with ibd in other non-gsd canine breeds. four non-synonymous snps in the tlr gene; t c, g a, a t and g a and three non-synonymous snps in the tlr gene; g a, c t and t c previously identified in a mutational analysis in gsds with ibd were evaluated in a case-control study using a snapshot multiplex reaction. sequencing information from unrelated dogs with ibd consisting of different non-gsd breeds from the uk were compared to a breed-matched control group consisting of unrelated dogs from patients treated for noninflammatory disease at the royal veterinary college, london, uk. as in the gsd ibd population the two tlr snps; c t and t c were found to be significantly protective for ibd in other breeds included in this study (p . and p . respectively). this study confirms the protective effects of the two tlr snps (c t and t c) in other canine breeds with ibd. this highlights the importance of tlr in the pathogenesis of canine ibd and may represent common pathological pathways of ibd in different canine breeds due to the high degree of haplotype sharing seen among breeds. this may allow for the future expansion of novel diagnostics and therapeutics to be applied to all canine breeds with ibd. further functional studies looking at the role of tlr in the pathogenesis of canine ibd are needed to confirm these findings. toll-like receptor (tlr ) is an extracellular pattern recognition receptor belonging to the innate immune system. we have recently shown that three non-synonymous single nucleotide polymorphisms (snps) in the tlr gene (g a, c t and t c) are significantly associated with inflammatory bowel disease (ibd) in german shepherd dogs. in addition, we confirmed that two of these tlr snps (c t and t c) were significantly associated with ibd in a population consisting of different dog breeds. in order to determine if other novel snps exist in the tlr gene in addition to the ones identified in the gsd population, mutational analysis was carried out in seven boxer dogs with ibd. polymerase chain reaction was carried out to amplify the tlr coding region in the seven dogs with ibd. sequencing was carried out using sequence based typing with the abi prism sequencing kit (applied biosystems, uk) and analyzed using an abi automated sequencer (pe applied biosystems). sequencing information from seven boxer dogs with ibd from the uk were compared to the reference sequence published on the ensemble webserver (www. ensembl.org/canis_familiaris). in addition to the three snps identified previously in the tlr gene, a novel non-synonymous snp; t c was identified in the boxer dog population with ibd. this snp has never been reported before and was present as the homozygote genotype in three dogs with ibd and in one dog as the heterozygote genotype. using the simple modular architecture research tool (smart) web server (http:// smart.embl.de/) we were able to map the t c snp to the leucine rich repeat domain of the tlr protein. the leucine rich repeat domain is involved with ligand binding and therefore a change in the amino acid in this region may affect function, especially as the t c snp results in a change in the amino acid from non-polar to polar. our study further confirms the role of tlr in the pathogenesis of canine ibd. our results suggest that in addition to shared risk polymorphisms amongst breeds, individual breeds may harbor unique snps arising after breed formation which may further affect their susceptibility to this disease. however, a case-control study would be needed in the boxer dog to confirm the significance of the tlr t c snp and further functional data would be needed to elucidate the exact role of this polymorphism in canine ibd. an automated power driver device (oncontrol, vidacare) has recently become available for bone marrow aspiration (bma) and bone marrow biopsy (bmb) in humans. the purpose of our study was to compare this automated technique to the traditional manual technique for bone marrow sampling in cats. twelve healthy research cats were anesthetized using a standardized protocol on different occasions, days apart, to have bmas and bmbs performed by the same operator. on day , half of the cats were randomized to have a bma performed at both the proximal humerus and the iliac crest, and a bmb performed at the iliac wing, using the oncontrol device ( -gauge needle for bma; -gauge needle for bmb). the other half of the cats had the same procedures performed using a manual technique ( -gauge illinois needle for bma; -gauge jamshidi needle for bmb). on day , each cat had bma performed at the opposite humerus and iliac crest, and a bmb performed at the proximal humerus using the opposite technique from day . for each procedure, the operator was given a maximum of attempts to successfully collect a sample. the rate of success, as well as the number of attempts were recorded. the ''ease of use'' of the device was rated by the operator on a -point scale after each procedure. using previously determined criteria, the macroscopic and microscopic qualities of the bma and bmb samples were assessed by a board-certified pathologist, blinded to the technique used. the level of pain experienced by each cat was evaluated , , , , and hours following each set of procedures, using a previously validated pain scoring system. two sample t-tests were used to compare the automated technique to the manual technique and to compare the humerus to the iliac crest site for bmas and the humerus to the iliac wing site for bmbs. for all procedures, at all sites, the ''ease of use'' was better for the automated technique than for the manual technique (p o . ). the duration of the procedure and the number of attempts to collect a sample were significantly lower with the automated technique for bma at the proximal humerus (p o . ). there was no significant difference in the level of pain at any time point following each set of procedures with either technique. performing bma at the proximal humerus was associated with a higher rate of success (p o . ), a lower number of attempts (p o . ), a shorter duration of the procedure (p o . ), a higher-rated ''ease of use'' of the technique (p o . ), and a better quality sample (p o . ) when compared to sampling from the iliac crest, in conclusion, we found the automated bone marrow sampling technique suitable for use in adult cats. this technique was easier to use than the manual technique for both bma and bmb, and reduced the duration of the procedure and the number of attempts for successful bma at the proximal humerus. performing bma at the proximal humerus was faster, easier and allowed collection of better quality samples than at the iliac crest, independently of the technique used. the fractious nature of some feline patients sometimes makes sedation or general anesthesia necessary for routine procedures such as blood collection for hematologic analyses. it has been anecdotally reported that sedation or general anesthesia could induce variations in hematologic parameters in cats, making it important for the clinician to be able to anticipate potential changes on hematologic parameters that could result from chemical restraint. this study evaluated the effects of a standardizecd anesthetic protocol using ketamine ( mg/kg, iv), midazolam ( . mg/kg, iv) and buprenorphine ( mg/kg, im) on the hematologic parameters of healthy adult research cats. each cat had blood samples collected before and after induction of anesthesia on different occasions, days apart. in total, pairs of complete blood counts were obtained. analyses were performed at a certified veterinary laboratory. paired sample t-tests were used to determine whether there were any statistical differences between hematologic parameters before and after induction of general anesthesia, for each cat, on different occasions. compared to preanesthetic values there was a significant decrease in red blood cell count, hemoglobin concentration, hematocrit, lymphocyte count and plasma total protein concentration after induction of anesthesia. there was no significant difference in the segmented or band neutrophil, eosinophil, basophil, monocyte and platelet counts between the samples taken before and after induction of anesthesia. on average, there was a . % decrease in the red blood cell count ( .  /l to .  /l) (p o . ), a % decrease in hemoglobin concentration ( . g/l to . g/ l) (p o . ), a . % decrease in the hematocrit ( . l/l to . l/l) (p o . ), a . % decrease in the lymphocyte count ( .  /l to .  /l) (p . ), and a . % decrease in the plasma total protein concentration ( . g/l to . g/l) (p o . ) when samples taken before and after induction of anesthesia were compared. if only the hematocrit was considered as a marker of anemia, % of the samples from these healthy cats, taken while they were under general anesthesia, would have been misinterpreted as belonging to anemic patients (hematocrit o . l/l), using the reference interval established in our laboratory. none of the cats would have been considered anemic before induction of general anesthesia. in practice, the decrease in lymphocyte count following anesthesia is unlikely to be of clinical relevance, as all the samples except had a lymphocyte count that was within the reference interval for cats established by our laboratory. this study suggests that complete blood counts performed on blood taken under general anesthesia with this combination of anesthetic drugs in cats should be interpreted cautiously in order not to make a false diagnosis of anemia. the mechanism responsible for the decrease in circulating red blood cell mass following anesthesia induction in cats is unknown and requires further investigation. rivaroxaban is an oral inhibitor of activated coagulation factor x (xa). it is expected to have similar coagulation effects as low molecular weight heparin, without the need for injection, making it an attractive alternative for long-term anticoagulant therapy in cats. citrated blood obtained from five healthy adult cats was exposed in vitro to varying concentrations of rivaroxaban, followed by coagulation testing. the rivaroxaban was extracted from commercially available tablets (xarelto s ) and dissolved in dmso prior to addition to the blood. tests performed included kaolin-activated thrombelastography (teg), prothrombin time (pt), dilute pt (dpt), activated partial thromboplastin time (aptt), and anti-factor xa (axa) activity. dose-dependent prolongations were seen in all coagulation parameters. similar to human data, therapeutic axa levels (between . - . axa units) were achieved at in vitro concentrations between and mg/l. at mg/l, dpt measurements were clinically prolonged in all cats ( . ae sec vs. . ae . sec, p . ), while aptt values were only mildly prolonged from baseline ( . ae sec vs. . ae sec, p . ). significant prolongations were seen in dpt at ( . ae ec, p . ). teg r time did not prolong from baseline values until concentrations of mg/l were reached ( . ae min compared to . ae . min, p . ). rivaroxaban has similar coagulation effects in the cat as in other species and may play a role in feline thromboprophylaxis. kaolinactivated teg does not appear to be sensitive to low concentrations of rivaroxaban in the cat. anticoagulated blood is required for platelet function studies. sodium citrate, a calcium chelater, is the most commonly used anticoagulant to measure coagulation parameters including platelet aggregation but it may have a negative effect on platelet responsiveness. dogs are generally considered moderate responders to collagen on platelet aggregation and are notorious for being poor or inconsistent responders to adp-induced platelet aggregation using citrated whole blood. hirudin, a selective thrombin inhibitor, can also be used as an anticoagulant for coagulation assays and is the anticoagulant of choice for certain assays including the multiplate s platelet function analyzer. ten adult healthy dogs were used to compare whole blood platelet aggregation between citrated and hirudinated blood samples. venous blood was collected atraumatically from the external jugular vein directly into tubes containing . % trisodium citrate or hirudin. whole blood platelet aggregation was performed (whole-blood lumi-aggregometer, chrono-log corporation, havertown, pa, usa) with collagen ( mg/ml) and adp ( mm) as agonists. maximal platelet aggregation (ohms) was recorded. there was a significant increase in collagen-induced platelet aggregation from the hirudinated samples compared to the citrated samples ( . ae . vs. . ae . o, p o . ). there was also a significant increase in adp-induced platelet aggregation from the hirudinated samples compared to the citrated samples ( . ae . vs. . ae . o, p . ). the results of this study show a significant difference in platelet responsiveness between citrated and hirudinated whole blood using the chrono-log impedance aggregometer. while both collagen and adp-induced platelet aggregation was attenuated from citrated blood samples, this was most notable for adpinduced aggregation where almost all samples had no objective measurable platelet aggregation. it is suggested from this data that future whole blood platelet aggregation studies performed on the chrono-log impedance aggregometer should use hirudinated blood samples although new reference limits would need to be established. low-molecular-weight heparin (lmwh) is now used to prevent thrombotic complications in dogs. a functional assay such as the calibrated automated thrombogram (cat) may provide a new approach for monitoring lmwh therapy. we hypothesized that cat would detect decreased endogenous thrombin potential (etp) in healthy dogs receiving lmwh (fragmin s ). twenty-four healthy adult beagles were included in this study and divided equally in four groups. one dose of u/kg, u/kg or u/kg of lmwh were given subcutaneously to healthy dogs and compared to a control group. platelet poor plasma (ppp) was collected over a hour period. using a repeated-measure linear model, effect of lmwh on etp was time and dose dependent with a significant interaction (p o . ). compared to control dogs, significant differences were obtained for group u/kg at t (p . ), for group u/kg at t (p . ) and between t -t minutes (p o . ) respectively, and for group u/ at t (p . ), between t -t minutes (p o . ) respectively and at t (p . the cat assay can be employed to measure the effects of lmwh at different doses in healthy dogs, resulting in significant time and dose-dependent decreases in etp and warrants further investigation as a tool for monitoring lmwh therapy in dogs. the purpose of this study was to determine the effects of prednisone and prednisone plus ultralow-dose aspirin on coagulation parameters in healthy dogs, with an emphasis on thromboelastography (teg). this was a prospective, randomized, blinded study utilizing fourteen dogs determined to be healthy based on normal physical examination, complete blood count, biochemistry, urinalysis, and fecal floatation. dogs were evenly divided into either prednisone plus aspirin (pa) or prednisone plus placebo (pp) groups. baseline values for teg parameters (r, k, angle, ma, ly , ly , g, ci) were measured twice two days apart, and thrombin-antithrombin complexes (tat), and traditional coagulation parameters (prothrombin time, activated partial thromboplastin time, d-dimer, antithrombin (at), fibrinogen) were measured once. each dog received mg/kg/ day of prednisone, and either . mg/kg/day of aspirin (pa group) or placebo (pp group) for days. a complete blood count, biochemistry profile, teg, tat, and traditional coagulation parameters were then repeated on each dog. day to day variation was calculated for the teg parameters using the two baseline measurements. the change from baseline between and within each group were compared using t-tests, or wilcoxon sample test where appropriate, for teg, tat, traditional coagulation parameters, and hematocrit. day to day variation in teg was acceptable ( %) for ma, g, and angle, unacceptable ( %) for r, k, ly and ly , and not meaningful for ci. within both groups, ma, g, ci and fibrinogen significantly increased from baseline (p o . ). within both groups, ly and at significantly decreased from baseline (p o . ). for the pp group, ly significantly decreased from baseline (p . ), and approached significance for the pa group (p . ). all other within group changes from baseline were not statistically significant (p-values . ). for all parameters, there was no difference between groups for change from baseline (p values . ). day to day variation in some teg parameters is high and may preclude their clinical utility. prednisone causes hypercoagulability in healthy dogs based on increased g, ma, and ci. the addition of ultra-low dose aspirin to prednisone has no effect on the parameters measured in this study. 'aspirin resistance' has been identified in people and dogs that develop thrombi despite low dose aspirin therapy. variability in platelet cyclooxygenase (cox) isoform expression is one proposed mechanism for aspirin resistance in people. two isoforms (cox- and cox- ) have been identified in canine platelets. high (antiinflammatory) dose aspirin inhibits platelet function and alters expression of both cox isoforms in most dogs. this study evaluated the effects of low dose aspirin on platelet function and cox expression in normal dogs. twenty-five healthy client-owned dogs were evaluated before and at two time points (days and ) during aspirin therapy ( mg/kg po sid). platelet response to aspirin (siemens pfa- s ; collagen/ epinephrine cartridges), was stratified into one of three groups [aspirin responders ( dogs), non-responders ( dogs), or inconsistent responders ( dogs)]. flow cytometry identified platelet cox- and cox- expression. an elisa was used to measure urine -dehydro-thromboxane b ( -dtxb ). there were no significant differences between groups for cox- , cox- or -dtxb at any time point. when all dogs were considered as a single group, there was a significant increase (p o . Ã) in cox- and cox- mean fluorescent intensity (mfi) from baseline to day , . % ae . (mean ae sd) and . % ae . , respectively. there was a significant decrease in mean urine -dtxb :creatinine on day and by . % (p . à ) and % (p o . à ). as with our previous high dose studies, cox- expression was increased with aspirin exposure. however, there was a significant increase in cox- expression with low dose aspirin in contrast to the decrease seen at higher doses. our study suggests that levels of platelet cox- and cox- expression do not influence aspirin response in dogs. although thromboxane levels decreased in most ( of ) dogs on low dose aspirin, platelet function was consistently affected in only % of dogs, suggesting that differences in response to thromboxane may play a role in the variable affects of low dose aspirin on canine platelet function. delayed postoperative bleeding is common in retired racing greyhounds (rrgs), despite normal results of routine hemostasis assays. the excessive postoperative bleeding in the rrgs is not due to primary or secondary hemostatic defects, and may be due to enhanced fibrinolysis or to a clot maintenance dysfunction. providing a method to prevent or minimize the severity of postoperative bleeding in rrgs will not only have major economic impact for owners, but also will markedly decrease the associated complications of minor or major surgeries in the breed. epsilon aminocaproic acid (eaca) is a potent inhibitor of fibrinolysis that also supports clot maintenance due to unknown mechanisms. the objective of this double-blinded, prospective, randomized study was to evaluate the effects of eaca versus placebo on the prevalence of bleeding in rrgs, and to investigate its mechanism of action by using thromboelastography (teg). we compared the effects of eaca and placebo in rrgs that underwent elective ovariohysterectomy or orchiectomy at the veterinary medical center, the ohio state university during years. the main endpoint was bleeding (prevalence and severity); minor endpoints included most teg parameters. thirty percent ( / ) of the rrgs in the placebo group had delayed postoperative bleeding starting to hours after surgery, compared to % ( / ) in the eaca group (p . ). on the teg parameters, the r time (clot formation time) was significantly different between treatment groups (p . ). the postoperative administration of eaca significantly decreased the prevalence of postoperative bleeding in rrgs. thromboembolism associated with protein losing nephropathy (pln) has been long recognized as a serious and unpredictable complication in dogs, however its prevalence remains unknown. in humans, surrogate indicators are frequently used to assess thromboembolic risk. this study aimed to investigate the prevalence of hypercoagulability in pln dogs based on thromboelastography (teg), and to determine whether hypercoagulability in these patients could be predicted by clinical assessments that identify systemic hypertension (systolic blood pressure mmhg), hypoalbuminemia (serum albumin o . mg/dl), antithrombin activity (o %), and degree of proteinuria (urine protein:creatinine ratio [upc] ! ). between march -september , twenty-seven dogs were identified with pln at the animal medical center. the prevalence of hypercoagulability based on a teg g-value . was . %. there was no statistically significant relationship, either categorically or continuously, in univariate as well as multivariate analyses of all variables. univariate logistic regression (odds ratio; lower and upper confidence limit; p value) for hypertension was À . ; . , . ; . ; for albumin - . ; . , . ; . ; and for antithrombin activity - . ; . , . ; . . thus, in this patient population, in the absence of teg, prediction of hypercoagulability using abnormalities in commonly measured clinicopathologic variables was not helpful. however, given the documented high prevalence of hypercoagulability in patients with pln, early institution of prophylactic anti-platelet or anticoagulant therapies should be considered. thromboelastography (teg) is a test of global hemostasis. due to the effects of extrinsic factors on whole blood coagulation, sample collection method (scm) may influence results. the purpose was to determine if scm influenced teg using kaolin-activated citrated whole blood (wb). healthy dogs with normal platelet counts were prospectively enrolled. three wb samples were obtained from each dog at least hours apart from alternating jugular veins in a randomized order of three methods: ) vacutainer s into citrated tube (vac), ) citrated syringe with transfer into plain tube (cit), or ) plain syringe with transfer into citrated tube (plain). draw time was recorded in seconds. kaolin-activated teg was performed, with measurement of reaction time (r), clot formation time (k), maximum amplitude (ma), and alpha angle. eleven dogs were enrolled. there were no significant differences in teg indices between vac samples and either cit or plain samples. cit samples had a significantly higher k value (p . ) and a lower alpha angle (p . ) compared to plain samples. draw times ranged from - seconds. a longer draw time was significantly correlated (p . ; r À . ) with a shorter r time. higher platelet count was significantly correlated (p . ; r . ) with a higher ma. scm did not have a significant effect on teg parameters when comparing vac samples to either cit or plain samples. minimizing sample collection time and trauma during venipuncture may be important in minimizing hypercoagulable changes in teg indices. liquid plasma (lp) is defined as either plasma collected and refrigerated immediately after collection or fresh frozen plasma (ffp) that is thawed and stored refrigerated until use. stability studies in people have shown that adequate clotting factor activity is preserved for at least days. lp is transfused in human level i trauma centers to critically ill people requiring rapid infusion of clotting factors as the time required to defrost ffp is considered prohibitive. the use of lp has not been described in veterinary critical care. the purposes of this study were to ) determine the length of time required in a water bath for a unit of canine ffp to thaw and ) describe the use of lp in a busy university emergency room (er). for part : six units ( ml) of canine ffp were individually thawed in a c water bath. the duration of time (in minutes) to thaw was recorded. for part : the transfusion log was reviewed for dogs receiving lp in the last months. the indications and outcome were recorded. the mean time ae sd thaw time was . ae . minutes. ten units of lp were transfused to critically ill or injured dogs during the study time. indications for lp transfusion included hypovolemic shock due to intra-abdominal hemorrhage in dogs ( traumatic, non-traumatic) and rapid correction of hemorrhage following parenteral tissue plasminogen activator administration in dog. lp volume transfused ranged from . to . ml/kg. no transfusion reactions were identified. effect on coagulation was not consistently evaluated. time required to thaw a unit of ffp is greater than minutes which could be detrimental in a bleeding, coagulopathic dog. lp was transfused without incident to critically ill and injured dogs and represents a potential new addition to the armamentarium of treatments in a veterinary er setting. further investigation of canine lp is warranted including evaluation of in vitro factor stability and in vivo efficacy in correcting coagulopathy. immune mediated thrombocytopenia (imt) is associated with increased morbidity and mortality. large prospective research studies in dog platelet antibodies and clinical utilization of platelet immunoglobulin assays are limited. potential explanations include limited availability and low specificity due to nonspecific binding. the focus of this study is to evaluate optimized direct and indirect platelet surface associated immunoglobulin (psaig) and staining with anti-cd antibodies (cd ab) for the utilization in classifying thrombocytopenic dogs. one hundred clinically ill and apparently healthy dogs were prospectively evaluated. data collected included a history of hemorrhage, physical examination evidence of bleeding, complete blood count, and measurement of psaig and cd ab. the psaig assay utilized polyvalent antibodies with correction for non-specific binding by subtraction of background fluorescence with control antiserum. thrombocytopenia was defined as o , /ml and all dogs were clinically classified into of groups (g): g imt, n , g thrombocytopenia from non-immune mediated diseases, n , g ill with normal platelet counts, n , g healthy dogs, n . median platelet counts, by groups, were g , , ; g , , ; g , , ; and g , , /ml. for the direct and indirect psaigs in dogs with itp (g ), more dogs (n and n ) with clinical evidence of bleeding had antibodies compared to those who were not bleeding (n and n ). considering only direct psaig the sensitivity and specificity was % and %, respectively for the diagnosis of imt. for indirect psaig the sensitivity and specificity was % and %, respectively, for the diagnosis of imt. when considering both direct and indirect psaig together, the sensitivity was % with a specificity of %. in g interference from high control antiserum background staining was noted in . % of dogs and resulted in a negative direct psaig classification. minimal background interference was noted in g , g , or g . the percentage of platelets stained with cd ab was significantly less in g (median , p . ) vs. g (median , p . ) vs. g (median . , p . ) and g (median . ). these findings indicate the optimized platelet surface associated immunoglobulin assay has a high specificity, however poor sensitivity, for the diagnosis of imt. the decreased cd staining in g (imt group) may reflect decreased surface gpiiia expression, blocking by anti-gpiiia antibodies or other proteins, clearance by macrophages, or increased non-platelet debris and has potential applications in the diagnosis and treatment of imt. greyhounds have lower serum concentrations of a-globulin than other breeds, explained by negligible levels of haptoglobin (hp) measured using different methods (colorimetric, immunoturbidimetric and protein electrophoresis). the purpose of the present study was to characterize the hp gene in greyhounds. we isolated dna and rna from blood samples of akc-registered and retired racing greyhounds (akcg, rrg), and a german shepherd dog (gsd). we sequenced the hp exons and splice sites, and conducted array comparative genomic hybridization to identify associated dna structural variation (custom m agilent oligonucleotide array). additionally, we tested for the presence of one or multiple haplotypes spanning hp in greyhounds using a high density snp array ( k illumina hd). sequencing results of hp in both dna and cdna revealed three synonymous snps in the racing greyhound. we did not identify structural variation overlapping or near the hp gene. notably, we detected that the rrg and akcg do not appear to share a specific haplotype spanning hp. despite having low or undetectable serum concentrations of hp, we did find that rrg hp mrna is expressed and lacks amino acid variation. this suggests that the clinical absence of the hp is attributable to post-transcriptional hp effects or to an unknown physiological interaction. finally, given the existence of distinct rrg and akcg haplotypes spanning hp, it is important to characterize serum levels of hp in akcg in follow on studies. we reported that hemoglobin in retired racing greyhounds (rrg) has higher oxygen carrying properties and affinity than other breeds. surprisingly, very little is known about canine hemoglobin genetics. the purpose of this study was to characterize genetics of canine beta globins. using computational blast analysis of the dog genome, we identified five beta globin genes in a single locus: two human hbelike followed by three hbb-like genes. we isolated dna and rna from blood of rrgs, akc registered greyhounds (akcg), and german shepherd dog (gsd). all beta globin exons and splice sites were sequenced, and the beta globin locus was examined by array comparative genomic hybridization (custom m agilent array). additionally, we determined the number of common haplotypes that span this locus in rrgs and akcgs using high density snp array ( k illumina hd). expression and sequence analysis of cdna showed all five beta globin genes are actively expressed in adults. canhbb and were created by relatively recent segmental duplication and have identical protein sequence. canhbb / are abundantly expressed in adults; canhbb is expressed at greatly reduced levels. sequencing results revealed one rare non-synonymous single nucleotide polymorphism (snp) in hbe of rrgs, but no variation that could explain their abnormal hemoglobin. we did not detect structural variation overlapping or near the beta globin locus. notably, rrg and akcg do not share haplotypes spanning the beta globin locus. this is the first characterization of canine hemoglobin genetics, and the first report of canine embryonic hemoglobins and their expression in adults. sampling of the bone marrow in the dog from the costochondral (cc) junction can be performed with minimal to no sedation and readily available equipment but is not in widespread use in the united states. the aim of this study was to compare the number of attempts needed to successfully obtain a sample, the time needed for the procedure, and the sample quality between aspirates obtained from the cc junction and more traditional sites (humerus or femur) in healthy dogs when performed by novice and seasoned practitioners. samples were obtained from healthy anesthetized laboratory reared adult dogs after undergoing terminal endoscopic surgery. paired samples from separate dogs were obtained by each practitioner using either a gauge needle and cc syringe at the cc junction or an gauge rosenthal needle and cc syringe from either the proximal humerus or femur (clinician preference). three small animal veterinary interns, one experienced technician and one boarded internist were monitored for number of attempts to success and length of time needed for success of each procedure. slides were prepared by a single investigator and read by a blinded clinical pathologist. data were compared using the paired t-test for normally distributed data and wilcoxen signed rank test for non-gaussian distributions. five pairs of samples from three dogs were evaluated. two dogs had two pairs drawn from opposite limbs and ribs. mean number of attempts to success for traditional sampling sites ( . /À . ) and time to success ( . minutes /À . ) did not differ significantly from attempts ( . /- . , p . ) or time ( . /- . , . ) needed when aspirating from the cc junction. subjectively, samples were of similar quality with regards to cellularity and number of particles present when compared within practitioners. myeloid: erythroid ratio and percentage of lymphocytes were also not significantly different between sites (m:e ratio p . , lymphocyte % p . ) and were within normal limits. while there were no significant differences between the two sites in terms of number of attempts or time to success, it should be noted that the ''seasoned'' practitioners had never performed an aspirate at the cc site and had an increased number of attempts compared to the traditional sites. if the number of attempts needed for success decreases with experience, it is likely the time required would decrease as well. both subjectively and objectively, there were no significant differences in quality or cell populations between the two sampling sites in healthy dogs. based on this data, bone marrow aspiration from the cc junction appears to be equivalent to more traditional sampling sites in healthy dogs. larger studies in clinically ill dogs should be performed before routinely using the site in the clinical setting. recent research on iron homeostasis has elucidated the tightly controlled intestinal uptake of iron. hepcidin, the major hormone limiting iron absorption and release from macrophages, is downregulated by matriptase- , a transmembrane serine protease (tmprss ) produced by the liver. while iron deficiency is commonly caused by chronic blood loss anemia and rarely dietary deficiency or intestinal disorders in dogs and other species, we report here the clinical to molecular investigations of a dog with iron-refractory iron deficiency anemia (irida) caused by a matriptase- deficiency homologous to a recently described autosomal recessive disorder in humans. the proband, a spayed female cocker spaniel without any clinical signs except for recent occasional idiopathic seizures, exhibited a lifelong history of microcytosis and hypochromasia but not anemia. there was no evidence of any blood loss and the dog was receiving an appropriate meat-based diet. mean values of complete blood cell counts, performed from . - years of age, were: hematocrit % (normal reference range - ); rbc count .  /ml ( . - . x ); mcv fl ( - ); mchc g/dl ( - ). serum iron parameters revealed severe iron deficiency with serum iron mg/dl ( - ); total iron binding capacity mg/dl ( - ); serum iron saturation o % ( - %), and ferritin ng/dl ( - ). prolonged courses of oral ferrous sulfate supplementation and several short courses of intramuscular (dextran) injections and intravenous iron infusions did not result in improvement of any red cell or serum iron parameters. however, this dog was never anemic and the partial seizures could not be directly related to the iron deficiency status. no family members were available for further studies. genomic dna was extracted from the proband's edta blood and the exons of the tmprss gene were amplified with flanking primers and then sequenced. in comparison to the normal canine tmprss sequence and that of a sequenced control dog we found a homozygous missense muation, r h, toward the c-terminal end of the protein in the proband's gene. in conclusion, the severe microcytosis and hypochromasia, low serum iron parameters and lack of a response to oral and parenteral iron therapy led to the diagnosis of irida. the missense mutation in the matriptase- at position from an arginine, which is conserved across all species currently deposited in the genbank, to a histidine is likely the disease-causing mutation. this is the first report of an irida in the dog with features very similar to those observed in humans. dogs with naturally-occurring irida may be helpful in developing and assessing novel therapies. accidental ingestion of copper-coated zinc pennies minted after is the most common causes of zinc toxicity anemia in the dog. zinc toxicity anemia may also be seen with ingestion of zinc from other sources as ingestion of metallic foreign material other than pennies, medicines containing zinc, and zinc supplements. the purpose of this study was to determine if there is a weight below which dogs are more susceptible to zinc toxicity anemia secondary to metallic foreign body ingestion. records of dogs presented to the internal medicine service at the veterinary medical center of long island for metallic foreign body ingestion were reviewed for signalment, weight, presenting pcv, and type of metallic foreign body ingested. eighteen dogs met the inclusion criteria and were compared. of the dogs, there were cases of coin ingestion ( %), with ( %) involving ingestion of or more pennies. the other cases involved ingestion of a metallic object ( ), decorative garland ( ), and bb pellets ( ).of the dogs exposed to zinc, ( %) were less than pounds ( . kgs). of those cases ( %) had ingested one or more pennies. eleven out of the ( %) zinc exposure dogs were anemic at presentation. the average weight of the dogs was . pounds ( kg). this study showed that dogs less than pounds appear to be more susceptible to developing anemia secondary to zinc toxicosis, with the majority of cases due to ingestion of pennies minted after . zinc toxicity anemia secondary to penny ingestion is more commonly seen in small dogs. we suspect larger dogs are able to pass pennies through the pyloric sphincter and thus not develop clinical signs. although thrombocytopenia is common in hospitalized dogs, canine cryopreserved platelet concentrate (pc) is used infrequently. data suggest in vitro efficacy of pc and when administered to research dogs, but efficacy is unknown in clinical patients. study objectives were to determine clinical characteristics of dogs receiving pc as well as safety and efficacy of pc in thrombocytopenic dogs. medical records were evaluated retrospectively to identify dogs that received pc. information evaluated included patient characteristics, platelet count, acute transfusion reactions, and survival. twenty six dogs met study criteria. dogs receiving pc ranged in age from - years (mean . years) and / ( . %) were spayed or intact females. hemorrhage was reported in / dogs ( . %) prior to pc transfusion. platelet counts prior to transfusion ranged from to  /ul (mean . /À .  /ul). change in platelet count was measured in dogs and the mean change was . /À .  /ul. dose of pc administered ranged from . to ml/kg with a mean of . /À . ml/kg. no acute adverse reactions were reported. there was no correlation between transfusion dosage and platelet count change post transfusion. survival to discharge occurred in / ( . %) of dogs. the only variable correlated with survival was age with survivors being younger than non-survivors ( . years-old ae . vs. . years-old ae . .; p . ). efficacy of cryopreserved pc transfusions for improving clinical outcome in dogs with thrombocytopenia is yet to be determined; however, pc is well tolerated in clinical patients. fresh frozen plasma (ffp) is used to treat coagulopathies in dogs. current transfusion guidelines recommend that ffp be administered within hours of thawing to avoid decreasing clotting factor function and bacterial contamination. the purpose of this study was to assess clotting factor activity and bacterial contamination of ffp that had been thawed and refrigerated for days. blood was collected from client-owned healthy dogs with no known history of coagulopathy or of administration of drugs affecting coagulation. plasma was separated from whole blood and frozen (À c) within minutes of collection. thawed plasma was maintained at c ( /À c). aerobic and anaerobic bacterial culture, prothrombin time (pt), activated partial thromboplastin time (ptt), and factor ii, vii, ix, and x analyses were tested at time of whole blood collection, ffp thaw, hours post-thaw, hours post-thaw, and hours post-thaw. there were no statistically significant differences in pt and ptt at any of the measured time points. statistically significant differences occurred between initial measurements of factors ii, vii, ix, and x and subsequent time points, but there was no difference in activity levels of the factors once ffp was thawed. one bacterial colony was grown from each of two samples from post-thaw plasma. thawed plasma protocols do not significantly decrease the function of factors ii, vii, ix, and x or prolong pt and ptt. bacterial contamination of the plasma supply seems unlikely, but strict aseptic technique should be used when obtaining plasma for patient use. erythrocyte pyruvate kinase (pk) deficiency is the first and most common erythroenzymopathy described in dogs, cats, and humans. the pk enzyme plays a crucial role in the erythrocyte energy metabolism and its absence causes severe hemolytic anemia, often misdiagnosed as autoimmune hemolytic anemia. the disease is inherited as an autosomal recessive trait and affected dogs also develop osteosclerosis. in dogs, the enzymatic diagnosis is complicated by the anomalous expression of malfunctioning m -pk expression, but breed-specific r-pk mutation tests have been reported for basenjis, west highland white terriers (whwt), and beagles. we report here on a survey of canine pk deficiency studied at the penngen laboratory. a biased group of samples were received for screening from dog breeds with known mutations as well as from dogs with chronic, prednisone-and antibiotic-resistant hemolytic anemia and their relatives. edta blood samples and/or cheek swabs as well as medical record information were received and genomic dna and/ or enzyme activity testing were performed. among the whwts % and % were found to be homozygous deficient dogs or carriers, respectively, with a mutant allele frequency of . . the average age at the time of diagnosis was . years ranging from months to years of age; some samples came from europe and south america. of the beagles studied, % were affected and % were carriers (mutant allele frequency . ). the average age at the time of diagnosis was years ranging from months to years. surprisingly, very few samples from basenjis were received for screening, and none showed the mutant allele. while pk-deficient basenjis lived o years, whwt and beagles often show milder signs and can reach years of age. several dogs from other breeds were also examined because of chronic regenerative anemia and none had any of the known mutations seen in the other breeds. however, based upon pk enzyme activity studies, chihuahua, dachshund, miniature poodle, spitz, eskimo toy, and labrador retriever dogs were found to be affected; they also had osteosclerosis and at least one labrador retriever developed severe hemochromatosis (hepatic iron , ppm; normal o , ppm, analyzed on a dry weight basis). moreover, sequencing of the r-pk cdna from a pk-deficient labrador retriever revealed a new nonsense mutation in exon . in conclusion, pk deficiency appears to be a common cause for hemolytic anemia in certain breeds, and mutation testing makes screening simple. pk deficiency should also be considered in dogs of other breeds which may require the more cumbersome enzyme testing. studies to identify new mutations will confirm and simplify the diagnosis. supported in part by nih grant rr . immune-mediated hemolytic anemia (imha) is a common hematological condition observed in dogs. the diagnosis is based on clinical history, presenting signs and hematological evidence of imha such as regenerative anemia, leucocytosis and presence of spherocytes. the definitive diagnostic procedure is the coomb's test (direct antiglobulin test, dat) which is known to be highly specific but lacks diagnostic sensitivity. direct flow cytometric assay (fca) for igg, igm or c coated red blood cells (rbcs) detection might be more sensitive and thus could be introduced as an alternative diagnostic tool. to investigate the usefulness of fca for imha diagnosis, evaluation of igg, igm or c coated rbcs was performed from dogs presented at the veterinary hospital at usp that fulfilled clinical and hematological criteria for imha. thirty eight healthy dogs were included as controls. dat was performed with polyvalent and monovalent anti-dog sera with twofold serial dilutions of each one, incubated with % rbcs suspension at c and c. for fca, % rbcs from anemic and healthy dogs were incubated with fitc anti-dog igg, anti-dog igm and anti-dog c and submitted to flow cytometry evaluation. specific software and mann whitney u test were used for data analysis. five dogs showed positive results for dat with polyvalent coombs reagent at c (titer to ) and c (titer to ) but only three of them had agglutination titer for anti-igg at c ( to ) and c ( to ). no positive results were observed for anti-igm and anti-c dat. by fc, percentage of igg, igm and c coated rbcs in normal and anemic dogs were, respectively, , % and , % (p o , ); , % and , % (p o , ); , % and , % (p o , ). igg coated rbcs percentage were higher in dogs showing dat positive results (min. , %; max. , %; median , %). direct flow cytometric erythrocyte immunofluorescence assay is more sensitive than dat for detection of antibodies coated rbc in anemic dogs and may provide quantitative data useful for laboratorial diagnosis of imzha. bone marrow aspirates from cats are typically obtained from the ilium, humerus or femur, but may be difficult to obtain and/or of poor quality. in this study the feasibility, safety, and nature of sternal aspiration in cats was investigated. under general anesthesia, bone marrow aspirates were obtained in a randomized order by a single investigator from the sternum and ilium of healthy cats weighing . - . kg, with body condition scores of - (on a scale of - ). for sternal aspirates, cats were positioned in sternal recumbency and a -inch, - ga hypodermic needle attached to a cc syringe was inserted into the cranial manubrium and directed caudally along the long axis of the sternum. aspirates were also obtained from the right iliac crest using an ga illinois needle attached to a cc syringe. difficulty of site localization, needle insertion and advancement, and specimen aspiration, were scored from (easiest) to (hardest). bone marrow smears were prepared by one investigator and reviewed by a pathologist blinded to aspiration site and cat. sample quality was scored from (no marrow particles) to (excellent) based on the number of wellsmeared marrow particles on the slide. particle cellularity was scored from ( % fat) to (o % fat). post-procedure, cats were treated with tramadol ( - mg/kg, po, q h) for days, and assessed for post-biopsy pain (colorado state university feline acute pain scale, range [no pain] - [maximum]) and site swelling (range [none] - [marked]). data were analyzed by ancova accounting for effects of weight and body condition score. pneumothorax was not identified. it was significantly easier to perform sternal than iliac aspiration, but the quality of the sample was significantly better for iliac than for sternal aspirates. because of limitations due to sample quality, bone marrow morphology in sternal samples could not be compared to iliac samples in all cats. for samples that could be compared, cellularity was identical for sternal and iliac samples from cat but underestimated in the sternal sample from another cat. myeloid:erythroid ratios and lymphocyte numbers were the same for sternal and iliac samples in and cats, respectively. megakaryocyte numbers were the same in one sample, less in sternal samples compared to iliac samples from cats, and overestimated in the sternal sample from cat. bone marrow cell morphology was normal in all acceptable samples. it was concluded that sternal aspiration of bone marrow using a - ga hypodermic needle is ) easier to perform than iliac aspiration; ) safe; but ) provides samples of lower quality than iliac aspiration in cats. the diameter of - ga jamshidi-type needles makes bone marrow core biopsy difficult in cats. in this study, biopsies of the left humeral head were taken under anesthesia using a -inch, ga needle (ez-io s intraosseous infusion system, vidacare) from healthy cats weighing . - . kg with body condition scores of - (on a scale of - ). humeral biopsies were compared to biopsies taken from the left iliac crest using a -inch, ga jamshidi needle. biopsies were performed in randomized order by one investigator. biopsy was repeated to a maximum of attempts until a specimen ! mm long was obtained. difficulty of site localization, needle insertion and needle advancement were scored from (easiest) to (hardest). specimens were wrapped in tissue paper and placed in davidson's fixative for min and then transferred to formalin. biopsy sections were reviewed by a pathologist blinded to biopsy site and cat. biopsy length on the slide was measured, and biopsy quality was scored from (no hematopoietic tissue) to (! intertrabecular spaces free of artifact). post-procedure, cats were treated with tramadol ( - mg/kg, po, q h) for days, and assessed for postbiopsy pain (colorado state university feline acute pain scale, range [no pain] - [maximum]) and swelling of biopsy sites (range [none] - [marked]). data were analyzed by ancova accounting for effects of weight and body condition score. there were no significant differences between ga and ga biopsies except for post-biopsy swelling, and there were no significant effects of body weight and body condition. six ( %) of ga and ( %) of ga biopsies were considered acceptable specimens for assessment of bone marrow architecture and morphology; all intact spaces in these biopsies had normal hematopoiesis and cell morphology. comparison of acceptable ga to ga biopsy specimens from cats showed no significant differences for cell density and lymphocytes/plasma cells, while cellularity, assessed as high in of the ga biopsies, was assessed as medium in corresponding ga biopsies; and megakaryocytes, assessed as - /low-power field in one ga biopsy, were assessed as /low-power field in the ga biopsy. myeloid:erythroid ratios were greater in ga biopsies compared to ga biopsies in cats, and less in the ga biopsy in one cat. discordant results between biopsies were not related to differences in quality. in conclusion, ga bone marrow biopsy of the humerus was as likely to yield a specimen of acceptable quality as was ga biopsy of the ilium, and resulted in less post-biopsy swelling. reports on canine acute liver failure (alf) include individual or small case series of animals with a specific diagnosis. the aim of this study was to describe the clinical course, outcome and etiology of alf in dogs presenting to a referral hospital. medical records ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) were reviewed for a diagnosis of alf (elevated serum bilirubin or icterus with concurrent coagulopathy or hepatic encephalopathy (he)). fifty cases were identified representing breeds: labradors retrievers, golden retrievers, german shepherds, and cocker spaniels. median age was years ( m to humerus, ga . ae . . ae . . ae . . ae . à ( . - . ) ( - ) ( - ) ( - ) ilium, ga . ae . . ae . . ae . . ae . . ae . . ae . à ( . - . ) ( - ) ( - ) ( - ) ( - ) ( - ) yrs). presenting signs included anorexia ( / ), vomiting ( / ), polydipsia ( / ) and neurologic signs ( / granulomatous hepatitis (gh) is a histopathological diagnosis characterized by focal aggregations of activated macrophages mixed with other inflammatory cells that is usually part of a systemic disease process (wsava). published case reports describe many potential infectious causes, but only one retrospective study involving nine dogs with gh has detailed clinically relevant findings. the aims of this study were to describe the clinical and clinicopathologic findings in dogs with a histopathological diagnosis of gh, and to identify infectious agents using differential staining techniques, pcr, and fluorescence in-situ hybridization (fish) in archival paraffin-embedded tissues from dogs with gh. medical records of dogs with a histopathological diagnosis of gh (n ) were reviewed and signalment, historical toxin exposure or evidence of other systemic diseases, clinical signs, physical exam findings, clinicopathologic test results, imaging findings, concurrent diagnoses, treatments administered, and case outcome, when available, were extracted and summarized. twelve archival formalin-fixed, paraffin-embedded hepatic tissue samples were available for special staining and molecular diagnostic testing. two of these samples had sufficient tissue for only pcr. the mean age of dogs with gh was years (median . years; range to years) and included males and females representing different breeds. common presenting complaints included inappetance or anorexia (n ), weight loss (n ), lethargy (n ), fever (n ), and vomiting (n ). high mixed liver enzyme activity ( / ) was the most common clinicopathologic abnormality. leukemia was diagnosed in one dog and copper-associated hepatopathy in dogs. no infectious agents were identified using differential staining techniques. bartonella species dna was not pcr amplified from the extracted archival tissue. furthermore, no bacteria were identified by means of fish using a universal eubacterial probe. these data suggest a possible role for copper accumulation in the genesis of gh in dogs and support further evaluation of dogs with gh for evidence of copper-associated hepatopathy. future studies should include detailed environmental histories, the collection of adequate sample volumes for quantification of hepatic copper content and the examination of frozen tissues using novel molecular diagnostic platforms. hepatocyte copper and iron accumulation contribute to cell loss, inflammation, and fibrosis. the purpose of this study was to compare copper and iron accumulation in feline liver samples with different disease processes. liver biopsies (n ) submitted between july , and june , were evaluated using wsava guidelines and categorized as non-hepatic/normal, congenital, inflammatory/infectious, neoplastic, and other. copper (by rubeanic acid) and iron (by prussian blue) accumulation were graded by increasing amounts ( - ) and location (centrilobular cl, midzonal mz, periportal pp, random r). associations between metal scores and diagnosis category were assessed using the kruskal-wallis test. histologic diagnoses were non-hepatic/normal (n ), congenital (n ), neoplastic (n ), infectious/inflammatory (n ), and other (n ). ninety-two samples were negative for copper; remaining samples were graded (n ), (n ), and (n ). histologic diagnoses (pattern) for positive samples were congenital ( cl), infectious/inflammatory ( : cl, mz, pp, r), neoplastic ( pp), and other ( cl). iron staining was negative in samples; remaining samples were graded (n ), (n ), and (n ). distribution was primarily cl (n ) or r (n ), though mz (n ) and pp (n ) distribution occurred. there were no significant differences by kruskal-wallis analysis for amount or location of hepatocellular iron or copper for the different disease categories. in this study, copper accumulation was rare, had variable distribution and occurred primarily in samples with inflammatory/ infectious disease. in contrast, iron accumulation was common and did not correlate with disease category. further prospective evaluation of copper and iron accumulation in feline liver disease and association with outcome may be warranted. chronic hepatitis (ch) in dogs is a progressive condition without clearly defined treatment. glucocorticoids are commonly used to stop progressive inflammation and fibrosis but are associated with significant side effects including a steroid hepatopathy that complicates enzyme monitoring. cyclosporine is proposed as an alternative therapy, but there are no published reports of its use for canine ch. patient records at the csu veterinary teaching hospital were searched for histologically confirmed cases of ch treated with cyclosporine. data were compiled on cyclosporine dosing, concurrent medications, clinical course and biochemical parameters. patients over a -month period were identified. serum alanine aminotransferase (alt) decreased by an average of % in dogs. the alt normalized completely in of dogs treated for days. in of dogs on mg/kg/day, the alt also normalized. five of the patients that exhibited clinical signs prior to treatment showed measurable improvement (weight gain, fewer gastrointestinal signs). eight patients had hyperbilirubinemia or ascites prior to treatment; these resolved in . post-treatment histopathology, available in one patient, revealed decreased severity of ch. five patients exhibited adverse effects including gastrointestinal signs ( ), gingival hyperplasia ( ), and papillomatosis ( ). cyclosporine was discontinued in dogs with gastrointestinal signs. cyclosporine was an effective therapy for many cases of ch and should be considered for patients who are refractory to or cannot tolerate glucocorticoids. prospective clinical trials with histological documentation are needed to better define cyclosporine's effectiveness in ch. insertion of the veress needle and establishment of pneumoperitoneum is associated with to % of all laparoscopic complications in humans. the purpose of this study was to determine the accuracy of interpretation of tissue impedance measurements for veress needle location. two laparoscopists, blinded to impedance measurements, placed reusable veress needles in cadaverous dogs euthanized for reasons unrelated to the study. placement order was randomized. a third individual evaluated impedance measurements using a handheld device (sensormed, knoxville tn) to determine correct versus incorrect needle placement. veress needle locations were marked using contrasting colors of india ink; tissues were dissected to determine ink locations. impedance measurement interpretation identified / correct and / incorrect placements, respectively. sensitivity, specificity, accuracy, and precision for correct veress needle placement are listed below. agreement was moderate (kappa . , p . ) for placements by operator and very high (kappa . , p o . ) for placements by operator . results for tissue impedance measurement interpretation are superior to published data for currently available tests. impedance measurements accurately detected all incorrect needle placements. comparison of needle placement with and without tissue impedance feedback will be necessary to determine whether it increases operator detection of inappropriate veress needle placement and decreases installment phase complication rates. delayed detection of intestinal perforation during veress needle insertion is associated with high mortality. the purpose of this study was to evaluate the accuracy of tissue impedance measurement interpretation for veress needle location. two laparoscopists, blinded to impedance measurements, placed reusable veress needles in cadaverous cats. placement order was randomized. a third individual evaluated impedance measurements (sensormed, knoxville, tn) to determine placement location. needle locations were marked using india ink; tissues were dissected to determine ink locations. impedance measurement interpretation identified / correct and / incorrect placements. all undetected incorrect placements were located within the retroperitoneal fat pad. sensitivity, specificity, accuracy, and precision for correct veress needle placement are listed below. correlation was absent (kappa À . , p . ) for placements by operator and substantial (kappa . , p o . ) for operator . there was no association between correct or incorrect placement and operator on chi-squared analysis. failure of impedance measurements to identify placement in the retroperitoneal fat pad resulted in poor accuracy and discordant kappa statistics. small cat size limited the number of appropriate placement sites, perhaps resulting in excessively dorsal placement. comparison of needle placement with and without tissue impedance feedback will be necessary to determine whether impedance measurements increase detection of inappropriate veress needle placements or decrease installment phase complication rates. best clinicopathologic tests detecting portosystemic shunting (pss) in dogs remains controversial. this retrospective study examined performance of single random "fasting" and paired serum bile acids (sba; pre-and -hr post-feeding) in a large population of non-icteric dogs with confirmed pss (abdominal ultrasound, colorectal scintigraphy, radiographic or spiral-ct portography, laparotomy, or necropsy). sba were measured by enzymatic colorimetric method with normal o mmol/l. dogs meeting inclusion criteria (n ) included portosystemic vascular anomalies (psva; extrahepatic [e-psva], intrahepatic [i-psva]), and acquired pss (apss). signalment and laboratory parameters were recorded. non-parametric statistical analyses were used, two-tailed p o . applied with bonferroni corrections. median age and weight of breeds were . ( . - ) yrs and . ( . - ) kg, with equal gender distribution. random "fasting" sba detected % psva and % apss, whereas post-feeding sba detected % psva and % apss. low protein-c (o % activity) occurred in % psva and % apss. low mcv and creatinine occurred in % and % of psva dogs, respectively; other tests were less helpful. in apss, post-feeding sba was superior. compared to apss, psva had significantly (p . ) lower mcv, cholesterol, bun, creatinine, glucose, and protein-c. compared to e-psva, i-psva had significantly (p . ) lower post-feeding sba, mcv, albumin, urine specific gravity, and protein-c but higher cholesterol and glucose. post-feeding sba reflect physiologically provoked bile acid challenge and should be the preferred sba test in non-icteric dogs for pss detection. protein-c assists in identifying psva but its utility in apss may be complicated by concurrent coagulopathies and inflammation. this study compared outcomes of treatment with adjunctive nonsteroidal anti-inflammatory drugs (nsaids) or anti-inflammatory glucocorticoids in dogs with severe pulmonary blastomycosis. medical records were reviewed for dogs diagnosed with blastomycosis at the university of illinois veterinary teaching hospital between and . dogs with a presenting pao of mmhg, and clinical or radiographic signs of respiratory blastomycosis were included. all dogs were treated with either itraconazole, fluconazole, amphotericin b, or a combination of these. group (g ) dogs were treated with nsaids and group (g ) dogs were treated with glucocorticoids as anti-inflammatory adjunctive therapy. the following comparisons were made: days of oxygen supplementation, days in hospital, survival to discharge, and long term patient survival. mann-whitney u tests and chi-squared tests were performed on continuous and categorical data, respectively. p o . was considered significant. sixty-eight dogs fit the inclusion criteria. g consisted of dogs and g consisted of dogs. the two groups were found to be similar in weight, age, and sex distribution. there was no significant difference between the two groups with regard to duration of oxygen supplementation, duration of hospitalization, survival to discharge, and patient survival. there does not appear to be a difference between the clinical course or patient outcomes between groups of dogs with severe pulmonary blastomycosis treated with nsaids or anti-inflammatory glucocorticoids. further studies need to be performed to fully evaluate the impact these adjunct treatments have on prevention of ards and additional respiratory complications. diagnosis of feline histoplasma capsulatum infection traditionally relies upon identification of organisms in circulating monocytes or affected organs. in recent years, an antigen assay (aa) was developed for the diagnosis of disseminated histoplasmosis in human patients, but there is little information describing this test in cats. the goal of this study was to determine the sensitivity and specificity of h. capsulatum aa in cats with clinical disorders suggestive of histoplasmosis. urine and serum h. capsulatum aa results for feline patients from veterinary hospitals were evaluated. medical records were reviewed for confirmatory evidence of histoplasmosis (based on cytological or histopathological findings) or an appropriately supported alternate diagnosis. aa results were available for cats; initial testing was performed on urine samples, serum samples, and unspecified sample. of these cats, / had a definitive diagnosis of histoplasmosis based on organism identification, and had a definitive alternate diagnosis (e.g., neoplasia, other infection) based on necropsy findings (n ) or other clinical data (n ). an additional cats had a clinical alternate diagnosis with no cytological or histopathological evidence of histoplasmosis in the affected body system(s). the remaining cats had unverified histoplasmosis (n ) or an open diagnosis (n ). of the cats with confirmed histoplasmosis, were positive on initial urine aa. one cat (with rectal involvement) was negative, indicating a test sensitivity of %. one cat was positive on urine aa but negative on serum aa. all of the cats with definitive or clinical alternate diagnoses had negative results on the aa, suggesting an excellent specificity ( %). however, this result should be interpreted with caution, as the possibility of primary or concurrent histoplasmosis was only definitively excluded in the patients who underwent necropsy examination. these findings suggest that the aa for h. capsulatum is a reliable diagnostic tool in this species. a positive result appears to reliably support the presence of infection, but a small percentage of infected cats may be negative on aa. in addition, tests performed on urine may be more sensitive that those performed on serum. the purpose of this study was to evaluate the sensitivity and specificity of an aspergillus galactomannan antigen enzyme immunoassay (ga-eia) for the diagnosis of canine systemic aspergillosis. serum and urine samples were collected from sick dogs at hospitals (ucd and tamu). group dogs were diagnosed with systemic aspergillosis using culture (sterile site) or microscopy and culture (non-sterile site). group dogs had clinical findings suggestive of aspergillosis but an alternate diagnosis was established. group dogs were not suspected to have aspergillosis. samples were tested using the ga-eia and results expressed as a galactomannan index (gmi). gmis . were considered positive. comparisons were performed using the mann-whitney test. there were dogs in group , in group , and in group . serum was collected from all dogs, and urine from , , and dogs, respectively. serum gmis did not differ from urine gmis across groups. serum gmis of group dogs were higher than those of group and group dogs (p o . ). results from dogs in group did not differ from those in group (p . ). two dogs in group tested negative, but had localized pulmonary infections. one dog in group , which had paecilomycosis, tested positive. two dogs in group tested positive. one was being treated with plasmalyte. the other had a cutaneous opportunistic mycosis. these data support the utility of this assay to aid in the diagnosis of systemic aspergillosis in dogs. anaplasma phagocytophilum, an ixodes tick transmitted rickettsial bacterium has a wide mammalian host range that is not commonly reported in cats. clinical signs in humans, dogs and cats are often vague and include lethargy, anorexia and malaise. the purpose of this retrospective study was to describe the clinical signs, laboratory data and response to treatment in cats that tested positive for a. phagocytophilum on a commercially available pcr of peripheral blood (fastpanel tm ). this study describes and reports the appearance of intracellular morulae in feline neutrophils contributing to the diagnosis of a. phagocytophilum. the a. phagocytophilum real-time pcr (rt pcr) assay consists of four multiplexed primer systems designed to detect a total of three distinct genes. amplicons were confirmed as a. phagocytophilum by dna sequencing. clinicopathologic data was obtained by review of medical records and interview of primary veterinarians. complete blood counts were available from / cats and / blood smears were reviewed. the cats included in this study were all positive for a. phagocytophilum by real-time pcr. the cats ranged from months to years of age with an average age of . years. fifteen of cats had a history of tick exposure and lived in the northeastern region of the us, an ixodes endemic area. all cats presented with lethargy, / were anorexic and / had a fever (temperature o f). other clinical findings included hepatomegaly, splenomegaly, ataxia and ocular changes of conjunctivitis and elevation of the nictitating membrane. hematologic findings included leukopenia ( / ), neutropenia ( / ) and lymphopenia ( / ). thrombocytopenia was not noted in any case. morulae were seen within neutrophils in / cases. all cases in this report responded to treatment with doxycycline. this is the first report of the identification of morulae within neutrophils via peripheral blood smear review in cats confirmed by rt pcr to be infected with anaplasma phagocytophilum in north america. infection with anaplasma phagocytophilum should be considered in a clinically ill cat with tick exposure, living in an ixodes endemic area that presents to a veterinarian for lethargy, anorexia and fever. the spectrum of disease manifestations and the accompanying clinicopathological abnormalities indicative of bartonellosis in dogs have not been thoroughly characterized. the objective of this unmatched case-control study was to compare signalment, clinical and pathologic findings in clinically-ill dogs suspected of a tick-borne disease that were negative for bartonella sp. dna (controls) as were the dogs diagnosed with bartonellosis by pcr amplification, dna sequencing and the bapgm (bartonella alpha proteobacteria growth medium) enrichment culture approach. both groups were tested under the same laboratory conditions and in the same time frame. medical records were reviewed for information regarding signalment, medical history, physical examination findings, clinicopathological abnormalities, microbiological data and treatment. the study population consisted of bartonella-infected dogs and non-infected dogs. healthy dogs with no historical illnesses, such as blood donors, were excluded. the following species were amplified: b. henselae (n , . %), b. vinsonii subsp. berkhoffii (n , . %), b. koehlerae (n , . %), b. volans-like (n , . %), b. bovis (n , . %). nineteen ( . %) bartonella-infected dogs were febrile and lethargic and ten ( . %) had neurological signs. laboratory abnormalities for both groups are summarized below (number of affected dogs provided in parenthesis): multivariate logistic regression using confounding factors was performed to establish potential associations between specific variables and bartonella sp. infection. there were no differences in signalment, age, sex, body weight and duration of clinical signs between the two groups. compared to the control population, infection with the genus bartonella was associated with a diagnosis of endocarditis (p . , or . , %ci . - . ) and hypoglobulinemia (p . , or . , % ci . - . ). controls were more likely to have joint effusion (p . , or . , % ci . - . ) and azotemia (p . , or . , %ci . - . ) than were the bartonella sp. infected dogs. bartonella was detected in dogs with signs such as fever, anemia, thrombocytopenia, hyperglobulinemia and proteinuria that are typically associated with tick-borne diseases. when endocarditis or hypoglobulinemia are detected, testing for bartonella should be prioritized. likewise, the detection of bartonella should prompt further testing for endocarditis, if not already investigated. surveillance studies in other species depend on detection of antibodies to the highly conserved influenza a nucleoprotein (np); however, no such antibody detection assay is approved for canine use in the u.s. the purpose of this study was to determine the diagnostic accuracy of a commercial blocking elisa used for avian species in detecting influenza a np antibody in dogs. since the blocking elisa is not a species-specific or viral subtype-specific format, we hypothesized that it would detect np antibodies in dogs infected by influenza a virus. serum samples from uninfected dogs (n ) and dogs naturally infected with canine influenza h n (n ) were tested using the idexx flockchek blocking elisa for influenza a np antibody according to manufacturer instructions. the sample/negative control (s/n) absorbance ratios for infected dogs ranged from . to . compared to . to . for uninfected dogs. a receiver operating characteristic (roc) curve analysis determined optimum diagnostic sensitivity ( . %) and specificity ( . %) at a s/n cutoff ratio of . . using this cutoff ratio, the overall diagnostic accuracy was . %. coefficients of variation for intra-assay ( . %) and inter-assay ( . %) testing demonstrated good repeatability with canine sera. the excellent diagnostic accuracy of the commercial blocking elisa makes it a suitable tool for large-scale surveillance of influenza a virus exposure in dogs. upper respiratory disease (urd) can affect a majority of cats in shelters and is one of the leading reasons for euthanasia of otherwise adoptable cats. the purpose of this study was to determine prevalence and risk factors for upper respiratory pathogens in four different models for managing unowned cats: short-term animal shelters (shel), long-term sanctuaries (sanc), home-based foster care (fost), and trap-neuter-return (tnr) programs. conjunctival and oropharyngeal swabs were collected from cats, half of which had clinical signs of urd, and tested for feline herpesvirus (fhv), feline calicivirus (fcv), chlamydiophila felis, bordetella bronchiseptica (bb) and mycoplasma felis by real-time pcr. management model, vaccination, sex, age, body condition, and clinical signs were evaluated as risk factors for infection. a majority of cats in all management models carried one or more organisms capable of causing urd. in many cases, prevalence was similar in cats with or without clinical signs. unlike diseases that can be controlled by segregation of symptomatic animals, the lack of strong correlation between the presence of pathogens with the presence of clinical signs suggests that feline urd control should be managed by vaccination before or at the time of intake ,biosecurity protocols that presume all cats may be shedding pathogens, and minimizing stressful conditions that contribute to disease susceptibility. depending on geographical location, sex, age and environment, - % of cats worldwide are infected with the feline immunodeficiency virus (fiv). knowledge of the fiv status of cats is important to limit the spread of disease and to institute appropriate health management. however, like all lentiviruses, fiv is highly variable in nucleotide sequence, and viral load in cats is variable during different disease stages. detection of antibodies is the most widely employed diagnostic approach, but does not distinguish fiv-infected from fiv-vaccinated cats. in this study, samples from fiv-seronegative cats, fivseropositive cats, and fiv-historically seronegative but vaccinated cats, were analyzed by a commercial quantitative pcr (qpcr) assay and virus isolation. replicate blood samples were coded, and then submitted for ) qpcr (idexx); and ) mononuclear cell isolation with -day culture and viral p antigen detection by elisa. for the p antigen elisa, cutoff absorbance values were established from analysis of fiv-negative samples. fiv infection status was pre-determined based on antibody-elisa results and vaccination history. results indicated that qpcr had a sensitivity of % for samples from fiv-seropositive cats, and a specificity of % and . % for samples from fiv-seronegative and fiv-vaccinated cats, respectively. at a cutoff value of standard deviations above the mean absorbance for p antigen elisa, results from fiv-negative samples yielded a sensitivity of . % for samples from fiv-seropositive cats, and a specificity of . % and . % for samples from fiv-seronegative and fiv-vaccinated cats, respectively. conclusions from this study are ) the commercial fiv qpcr assay has high specificity but limited sensitivity for diagnosis of fiv infection; ) -day virus culture has limited sensitivity and specificity. hence, detection of antibodies remains the most reliable test for diagnosis of fiv infection, but qpcr may be suitable to rule out infection. oral disease is an important clinical problem in feline medicine and includes common painful conditions such as oropharyngeal inflammation (formerly known as gingivostomatitis) and tooth resorptive lesions. a number of infectious agents have been associated with private veterinary clinics in the u.s. were recruited to test feline patients presenting with oral disease. presenting cases included cats with plaque, calculus, gingivitis, stomatitis, periodontal disease, tooth resorptive lesions and other oral diseases as defined by the practitioner. all cats were tested using a commercially available point-of-care elisa test (idexx snap combo). confirmatory tests were not performed as part of the study. seroprevalence was calculated as the percentage of positive tests in the study population for each virus. a total of , cats were tested. seroprevalence for felv was . % and for fiv was . %. of these, cats ( . %) were infected with both viruses. seroprevalence was higher in cats with inflammatory oral disease than in cats characterized with other types of oral disease. of , cats with gingivitis, seroprevalence for felv was . % and for fiv was . %, with . % of cats co-infected. of , cats with stomatitis, seroprevalence for felv was . % and for fiv was . %, with . % of cats co-infected. the seroprevalence for felv and fiv reported in this population of cats with oral disease was higher than in a recent large study where samples from u.s. cats not specifically selected for oral disease were tested (felv . %, fiv . %). results of this study indicate that further investigation of the role of retroviruses in cats with oropharyngeal inflammation is warranted. reliable tests and preventive vaccines and medications for feline retroviral and heartworm (hw) infections are available, but compliance with protocols to reduce transmission is unknown. no largescale longitudinal studies evaluating prevalence over time have been reported. the purpose of this study was to determine the prevalence and risk factors for infection compared with a similar study completed for the first time years previously. veterinary clinics and animal shelters in the us and canada submitted results of testing using a point-of-care elisa for felv antigen, fiv antibody, and hw antigen (idexx snap triple) and risk factor information for cats tested during march-september . bivariable and multivariable analyses were used to evaluate risk factors for infections. a total of , cats were tested. only % of owned cats were prescribed hw preventive. risk of retroviral infections was increased by outdoor access, adulthood, and male gender. the most important risk factor associated with all infections was clinical disease; in particular, respiratory and oral diseases and abscesses or bite wounds. multivariate analysis revealed differences among geodivisions and across infection types. feline retroviral and heartworm infections are easily prevented, but difficult to treat. despite availability of effective management protocols, compliance remains inadequate to reduce the prevalence of these infections. improved use of preventive care and testing to identify and segregate contagious cats, particularly those at high-risk, is required to reduce the morbidity of these preventable infections. infectious disease outbreaks are common in animal shelters and are frequently managed by depopulation when risk-assessment tools are not available. during a canine distemper virus (cdv) and parvovirus (cpv) outbreak in sheltered dogs, we used a cdv/cpv point-of-care antibody titer elisa, a cdv quantitative rt-pcr test, and a cpv fecal antigen test as risk assessment tools to guide release of exposed dogs from quarantine and euthanasia of diseased dogs. serum samples (for antibody titers) and swabs of the conjunctiva and upper respiratory tract (for cdv pcr) were collected from asymptomatic dogs starting on day of the outbreak. dogs with positive cdv pcr tests were retested every weeks until euthanized for progressive disease or released following recovery from infection. dogs with clinical signs of parvoviral infection were tested using a cpv fecal antigen test. for dogs ! months old, protective antibody titers correlated with resistance to clinical disease, but % of dogs shed cdv. lack of protective cdv antibody titers correlated with susceptibility to clinical infection, but most dogs recovered. risk assessment and outcome in dogs ! months of age feline herpesvirus (fhv- ) is a common ocular and respiratory pathogen of cats that can have clinical illness exacerbated by stress. cyclosporine (csa) is commonly used for the treatment of a number of inflammatory diseases in cats and can induce immune suppression. a small number of cats administered csa to block renal transplant rejection have developed clinical signs of upper respiratory tract disease that may have been from activated fhv- . in this study, young adult cats experimentally inoculated with fhv- several months previously were divided into three groups and administered methylprednisolone acetate ( cats, mg/kg, im, day and day ), csa ( cats, . mg/kg, po, daily for days), or a placebo ( cats, corn syrup; . ml/kg, po, daily for days). each cat was assigned a daily individual clinical score by a trained, masked observer using a standardized score sheet during the initial pre-treatment time period (day - to day ) and throughout the day treatment period. each individual clinical score (conjunctivitis, blepharospasm, ocular discharge, sneezing, nasal discharge, nasal congestion, and body temperature scores), the total clinical score (sum of all parameters), the total ocular score (sum of conjunctivitis, blepharospasm, ocular discharge), and total respiratory score (sum of ocular discharge, sneezing, nasal discharge, nasal congestion) were analyzed using sas proc glimmix with 'treatment', 'time', and the two-way interaction 'treatment by time' all as fixed effects. statistical significance was defined as p o . . on day of the study, all of the csa treated cats had detectable concentrations of csa in serum (mean . ng/ml; standard deviation . ng/ml; median . ng/ml). when group mean values for clinical signs were compared over time as described, significant differences in individual clinical score measurements, in total score, total ocular score, or total respiratory score were not detected over time among any of the treatment groups. while clinical signs of activated fhv- occurred in some cats administered methylprednisolone or csa, disease was mild and self-limited in most cats and there were no significant csa sideeffects. these results suggest that the csa protocol described here is unlikely to reactivate latent fhv- infection and cause significant clinical illness. the purpose of this study was to determine the prevalence and risk factors for enteropathogens in four different models for managing unowned cats: short-term shelter, long-term sanctuary, home-based foster care, and trap-neuter-return (tnr) programs. fecal samples were collected from cats, half with diarrhea (d) and half with normal feces (n), and tested for a panel of feline and zoonotic enteropathogens by polymerase chain reaction, antigen, and fecal flotation. risk factors for infection evaluated include management practices, fecal consistency, and signalment. a majority of cats had at least one enteropathogen of feline or zoonotic importance, regardless of management model or preventive healthcare protocol. for most enteropathogens, the presence or absence of diarrhea did not correlate with infection, the exceptions being t. foetus in sanctuary cats and fcov in foster cats. prevalence of specific enteropathogens varied between management models, reflecting differences in preventive healthcare and housing conditions. management protocols for unowned cats were inadequate for elimination of infections present at the time of intake and for prevention of transmission of enteropathogens among shelter cats. improved compliance with effective vaccination, deworming, sanitation, and housing protocols is needed to reduce zoonotic and feline health risks. several allergic diseases of cats, including atopy and gingivostomatitis, can be resistant to glucocorticoids but responsive to cyclosporine. toxoplasma gondii infection occurs in approximately % of cats and the effect cyclosporine therapy has on the t. gondii oocyst shedding period is unknown. the objective of this study was to determine whether administration of cyclosporine before or after t. gondii infection influences the oocyst shedding period. the young adult cats were t. gondii seronegative when administered , t. gondii tissue cysts orally on day . group cats (n ) were never administered cyclosporine; group cats (n ) were administered cyclosporine ( . mg/kg, po) daily on days - ; and group cats (n ) were administered cyclosporine ( . mg/kg, po) daily from days - . available feces from individual cages were collected daily and fecal flotation by sugar centrifugation was performed for days after t. gondii inoculation. group shed oocysts for a significantly shorter period than groups or and had a significantly lower oocyst shedding scores than groups and on days - after t. gondii inoculation. group cats had completed the oocyst shedding period prior to being administered cyclosporine and repeat oocyst shedding was not detected during administration of the drug. administration of cyclosporine prior to t. gondii infection lessened oocyst shedding which is likely from the anti-t. gondii effects of the drug. administration of cyclosporine using this protocol is unlikely to induce repeat t. gondii oocyst shedding in client-owned cats. à group with diarrhea significantly different than group with normal feces p o . known about its metabolic pathways or mechanism of pathogenicity and whole genome sequencing of feline hemoplasmas has not yet been reported. the aim of this study was to completely sequence the genome of m. haemofelis to further characterise this important pathogen. mycoplasma haemofelis genomic dna was purified and subjected to whole shotgun roche sequencing. gaps were closed using targeted pcr and amplicon sequencing. ribosomal genes and potential open reading frames (orfs) were predicted in silico. putative orfs were annotated and orthologous groups identified. analysis showed a circular genome of . mbp with a gc content of . %. thirty-one transfer rnas (trnas) were identified, accounting for all amino acids, including a tryptophan trna for the opal codon (uga). of the , putative proteins identified, ( . %) matched to proteins from other bacterial species. in common with the pneumoniae group of mycoplasmas, the closest phylogenetic relatives of the hemoplasmas, genes involved in carbohydrate metabolism were limited to enzymes of the glycolytic pathway, with glucose appearing to be the sole energy source for m. haemofelis. the majority of the pentose phosphate pathway genes present in other cultivatable mycoplasmas appear to be incomplete or absent in m. haemofelis, suggesting an alternative mechanism for sourcing purine and pyramidine bases such as scavenging from the host. a gene encoding a glyceraldehyde- -phosphate dehydrogenase homolog of the immunogenic msg protein of mycoplasma suis was present. of the uncharacterized hypothetical proteins, , were arranged in series of orthologous repeats, or comprised fragments there-of, encoding putative proteins of approximately amino acids. the predicted motifs of the majority of these putative proteins were consistent with these proteins being presented on the cell surface; an n' terminal signal peptide or transmembrane region followed by a non-cytoplasmic tail. these data have provided valuable information as to why this pathogen remains highly fastidious; it lacks some of the metabolic pathways found in cultivatable mycoplasmas. we have also identified a homolog of a known m. suis immunogenic protein, and identified a potential mechanism for host immune system evasion by way of highly repetitive, putatively surface-expressed hypothetical proteins with variable sequences. canine leptospirosis has been recognized as a re-emerging disease in the u.s. over the past years, and several serosurveys of the prevalence of leptospiral antibodies in dogs have been published during that time. the role of cats in the epidemiology of leptospirosis has received little attention. serosurveys of cats for exposure to or infection with leptospires have been published from other geographic areas, but none for cats in the u.s. in the past four decades. the new england states have been found to have a high incidence of canine leptospirosis. the purpose of this pilot study was to determine the prevalence of leptospiral antibodies in a population of feral cats in central massachusetts. blood was collected from sexually intact feral cats presented to a spay and neuter program. microagglutination titers to leptospira serovars autumnalis, hardjo, bratislava, icterohaemorrhagiae, canicola, pomona, and grippotyphosa were determined. three of cats ( . %) had a positive titer to one or more serovars, with autumnalis being the most common. these results are consistent with previously published prevalence rates in feral cats. further studies are required to determine the role of leptosporosis in clinical disease in the domestic cat. since years the rivalta's test is routinely used in several european countries as a tool to diagnose feline infectious peritonitis (fip) in cats with effusion. it is inexpensive and easy to perform in private practice. there is, however, only little information about mode of action or its diagnostic value. the objectives of this study were to evaluate sensitivity, specificity, positive (ppv) and negative predic-tive values of the rivalta's test to diagnosis of fip and to examine if there is a correlation with any effusion or blood parameters. medical records of cats with effusion in which the rivalta's test was performed between and were reviewed concerning diagnosis, blood and effusion parameters, and survival time. effusion and blood parameters were compared between rivalta-positive and -negative effusions using the mann whitney u test. prevalence of fip in cats with effusion was . %. the rivalta's test showed a sensitivity of . %, a specificity of . %, a ppv of . %, and a npv of . % for the diagnosis of fip. the ppv improved, when cats with lymphoma or bacterial infection were excluded (ppv . %) and also, when only cats younger than years (ppv . %) or year (ppv . %) of age were included. the most important significantly different parameters between rivalta-positive and -negative effusions were specific gravity as well as cholesterol, triglyceride, and glucose concentration in the effusion. the rivalta's test in general is a useful tool to diagnose fip, but its sensitivity and specificity are not as high as previously assumed. if the rivalta's test, however, is performed in young cats or if certain diseases have been ruled-out, its diagnostic value is high. effusion total protein is not highly correlated with test outcome. therefore, it is still unclear, which components in the effusion of cats with fip lead to a positive rivalta's test. canine parvovirus (cpv) and canine distemper virus (cdv) infections are relatively common in animal shelters and are important population management issues since the immune status of incoming dogs is usually unknown. our study aimed to determine the antibody protection status of dogs at the time of admission into an animal shelter (pre-vaccination) and over the following weeks after vaccination. serum samples were obtained from incoming shelter dogs aged months and older with no known history of vaccination. immediately following serum collection, the dogs were vaccinated against cpv and cdv using a modified live vaccine (mlv). cpv and cdv antibody protection status was determined using synbiotics titerchek. dogs with unprotective serum antibody levels against cpv and/or cdv were retested at - days post-vaccination and again at - days post-vaccination, if antibody levels were still unprotective against cpv and /or cdv. at the conclusion of the study, stored duplicate sera were submitted for batch 'gold standard' testing to determine canine distemper virus serum neutralization and canine parvovirus hemagglutination inhibition antibody titers. based on the synbiotics titerchek results, / dogs ( . %) were protected against cpv and / ( . %) were protected against cdv at intake. older incoming dogs were more likely to be protected against cpv (p o . ) and cdv (p . ). dogs that were spayed/neutered were more likely to be protected against cpv on intake than intact animals, although this result was not statistically significant (p . ). the number of dogs with protective titers against cpv/cdv was increased at - days post-mlv (cpv - / , . %; cdv - / , . %) and further increased at - days post-mlv (cpv - / , . %; cdv - / , . %). we conclude that incoming shelter dogs often do not have protective antibody titers against cpv and cdv, but older shelter dogs are more likely to be protected against cpv. based on this population, we further conclude that a large percentage of dogs develop protective antibody titers to cpv and cdv within to weeks when vaccinated with a mlv. mycoplasma spp. are common inhabitants of the feline oral cavity and so likely contaminate many cat bite abscesses. mycoplasma spp. are cell-wall deficient and so do not respond to beta-lactam class antibiotics, the class most commonly use for the treatment of cat bite abscesses. the objectives of this study was to determine whether mycoplasma spp. are common contaminants of cat bite abscesses and are associated with beta-lactam resistant clinical disease. privately owned cats with clinical evidence of an acute abscess suspected to be from a cat bite were included in the study. participants were given a free aerobic and anaerobic culture as well as mycoplasma spp. culture and polymerase chain reaction using mycoplasma genus specific primers. mycoplasma spp. amplicons were sequenced to determine the species. all cats were initially treated with appropriate wound management, were administered an antibiotic in the beta lactam class (amoxicillin-clavulanate or cefovicin), and were rechecked in person or by phone days after beginning treatment. of the cats entered into the study to date, mycoplasma spp. were amplified from cats ( . %). of the positive samples with adequate dna for sequencing, one was consistent with m. felis and the other was consistent with m. equigenitalium. of the cats, responded by day to the initial treatment, including of the mycoplasma spp. positive cats. the cat that failed initial treatment was positive for m. equigenitalium on both day and day and ultimately responded to administration of a fluoroquinolone. the results suggest that while mycoplasma spp. commonly contaminate cat bite abscesses, routine wound management and antibiotic therapy is adequate for control. however, as mycoplasma spp. infections do not respond to beta lactam class antibiotic therapy, these organisms should be on the differential list for cats with abscesses that fail treatment with this antibiotic class. molecular diagnostic assays are frequently used in clinical practice to aid in the diagnosis of suspected infectious respiratory diseases in dogs. however, most currently available assays cannot distinguish strains of the organisms used in vaccines from naturally occurring strains. our prior studies demonstrated that previously immune adult dogs are unlikely to shed nucleic acids of vaccine strains of adenovirus , parainfluenza, or bordetella bronchiseptica. however, whether this is true for puppies is unknown. puppies (n ) at a breeding facility were moved into area without other dogs at weeks of age. swabs of the nasal and pharyngeal mucosa were collected prior to vaccination and on days , , , , , , , , , , , , , and after vaccination with an intranasal adenovirus , parainfluenza, and b. bronchiseptica vaccine (intratrac , schering plough). the swabs were shipped on cold packs by overnight express for dna/rna extraction and assay in the fastpanel tm pcr canine respiratory disease profile at antech diagnostics. all puppies were negative for the infectious agents prior to vaccination. after vaccination, positive assay results for parainfluenza and b. bronchiseptica were first detected on day and on day for adenovirus . by day , dna or rna of the agents were amplified from all puppies from both sample sites and most samples were positive for all agents through day . by day , only one dog was still positive for b. bronchiseptica. the results indicate that intranasal administration of adenovirus , parainfluenza, or bordetella bronchiseptica vaccines commonly leads to positive molecular diagnostic assay results for a short time period after primary vaccination. these findings should be considered when assessing the results of these assays in client-owned puppies with respiratory disease. antimicrobial resistance in escherichia coli is an increasing concern in both human and veterinary hospitals' patients. the choice drug for treatment in dogs is enrofloxacin, a second generation fluoroquinolone (fq) whose activity reflects, in part, ciprofloxacin. among the difficulties in effective e. coli treatment is rapid detection of fq resistance. the purpose of this study was to determine the specificity and sensitivity of a fret based assay for the rapid detection of urinary tract infections caused by fq associated multi-drug resistant e.coli. clinical e. coli isolated from canine urine and clinical veterinary urine samples being examined for e. coli were subjected to susceptibility testing for drugs representing drug classes. pure isolates were designated ndr (no drug resistance), sdr (single drug resistance) and mdr (multi-drug resistant) (n mdr, sdr and ndr). minimum inhibitory concentration (mic) for enrofloxacin ranged from . mg/ml to mg/ml, with high mic generally associated with mdr. extracted dna from culture and from urine were subjected to fret-pcr targeting single nucleotide polymorphisms in gyra. the resulting product was sequenced to detect other polymorphisms. further, to determine the level of detection, microbial free canine urine was inoculated with to cfu/ml of isolates characterized by variable susceptibility to enrofloxacin (mic enro . , . , . , , , , mg/ml). of pure isolates, were confirmed positive for enrofloxacin resistance (mic enro mg/ml), of which were positively identified by the fret-pcr assay giving a sensitivity of . %. only isolate that was resistant was not detected (specificity of . %). however, of the isolates expressing high level resistance (mic x breakpoint [ mcg/ml]), and mdr (n ), sensitivity . %. of the urine samples contained e. coli of which determined to be fq-resistant by the assay. colony dilutions of e. coli confirmed the assay able to detect enrofloxacin resistance at as low as cfu/ml. the relationship between cfus and the peak of the -(d/dt) fluorescence of the melting curve was r . . these results conclude that the assay is capable of detecting not only the presence of escherichia coli in clinical samples, but also detecting severity of fluroquinolone resistance and infection. the fluoroquinolones (fqs) are a key class of synthetic antimicrobial agents with an established history in both humans and companion animals of efficacy for treatment of urinary tract infections (utis) caused by e. coli, and fluoroquinolones are common therapy. among the commonly used fqs in dogs and cats are the nd generation drugs, enrofloxacin, marbofloxacin, orbifloxacin (all veterinary approved) and the human drug ciprofloxacin; no rd and th generation fq is routinely used. the purpose of this study was to assess the in vitro activity of different generation fqs toward e.coli uropathogens whose phenotype ranges from no resistance to multidrug resistance. a total number of canine uropathogenic canine or feline e.coli isolates had been subjected to susceptibility testing to drugs classes ( drugs) and phenotyped as to resistance: none (ndr, n ), single (sdr, n ), or multiple, mdr (resistance to - drug classes; n ). mdr included isolates susceptible (enr s -mdr, n ) or resistant (enr r -mdr) to enrofloxacin. the minimum inhibition concentrations (mics) for quinolones ( - st generation, - nd generation, - rd generation and - th generation) were determined for these isolates using broth microdilution methods according to clsi guidelines (e. coli atcc s served as a negative control). mic statistics were generated for each drug among phenotypes. the results showed that companion animal e. coli expressing ndr or sdr are largely susceptible to nd to th generation fqs. however, isolates expressing resistance to st or nd generation quinolone also express high level resistance based on the mic to rd and th generation fqs. the overall potency (mic) for the drugs for isolates not expressing enr resistance (that is, ndr, sdr and enr s -mdr) is gat canine leproid granuloma (clg) was first reported in brazil in . over the past years, cases of clg were diagnosed in sa˜o paulo, brazil, and clinical and epidemiological findings were similar to those reported in australia. all dogs presented with one or more, uni or bilateral, ulcerated or not, papular, nodular or tumoral lesions, mainly observed in the dorsal surface of the ear, site usually more affected. in general, the lesions are painless and confined to the subcutis and skin, and it does not involve regional lymph nodes, nerves or internal organs, and systemic clinical signs frequently are absent. short-coated breeds show a marked predisposition for this disease. the definitive diagnosis of clg was obtained by histological examination of skin biopsies that were stained with acid fast (ziehl-neelsen) and diffquik s . thirty one ( . %) of the dogs were purebred; in this study the breed pattern comprised ( . %) boxers, ( . %) german shepherd and labrador retriever, ( . %) dobermann, ( . %) brazilian terrier, ( . %) golden retriever, ( . %) bulldog, ( . %) american pitbull, ( . %) mastiff, ( . %) fila brasileiro and ( . %) cocker spaniel, ( . %) were of unknown breed. nineteen ( . %) of the thirty seven dogs were males. twenty ( . %) dogs were - years old. in most cases, dogs presented with unilateral or bilateral ear lesions, but rarely thoracic, foot and caudal lesions. the animals were successfully treated by use of rifampicin orally (''the brazilian protocol'') or enrofloxacin orally and topical rifamicin. anaplasma phagocytophilum is being recognized more frequently in dogs in endemic areas. currently, most suspected cases are evaluated for a. phagocytophilum antibodies by immunofluorescence assay (ifa) or elisa. since a. phagocytophilum is an acute disease, detection by antibody measurement may be negative on initial evaluation. it is possible that a. phagocytophilum dna can be amplified from blood or synovial fluid prior to seroconversion. wild caught ixodes scapularis adult ticks from rhode island were allowed to feed on young adult ( - years), mixed sex beagles for up to days. blood (weekly for weeks), serum (weekly for weeks), and synovial fluid (radiocarpal joint; alternating arthrocentesis weekly for weeks) were collected prior to tick attachment and then weekly after tick attachment. joint fluid cytology was performed and total dna was extracted from blood and synovial fluid and assayed in a proprietary real time pcr assay (fastpanel tm ) that amplifies the dna of anaplasma phagocytophilum, a. platys, ehrlichia canis, e. chaffeensis, and e. ewingii. serum was assayed for a. phagocytophilum antibodies by ifa. time to first positive results for serology and pcr were compared by paired student's t test. none of the beagles developed clinical evidence of disease, and no major changes in synovial fluid cytology were detected over time. of the beagles, were positive for a. phagocytophilum dna in blood or synovial fluid or ifa antibodies in at least one sample after tick attachment. antibody titers appeared in of dogs from weeks to (median to st positive weeks ae ). titer magnitude ranged from : to : , . anaplasma phagocytophilum dna was amplified from the blood of of dogs with positive test results ranging from to weeks (median to st positive weeks ae . ). anaplasma phagocytophilum dna was amplified from synovial fluid from of dogs between weeks to (median to st positive weeks ae ). of the dogs, were pcr positive for only one week and dog was pcr positive for two consecutive weeks. of the dogs, were positive for a. phagocytophilum in both blood and joints by dna analysis. anaplasma phagocytophilum dna was amplified from blood more quickly than seroconversion was detected by ifa antibody titer (t À . , p o . ) or dna was amplified from synovial fluid (t . , p o . ). anaplasma phagocytophilum dna can be amplified from the blood prior to development of detectable antibody titers by ifa. amplification of a. phagocytophilum dna from synovial fluid does not occur in all dogs, appears to be transient in most dogs, and a negative test result does not preclude a diagnosis of a. phagocytophilum infection. canine granulocytic anaplasmosis and granulocytic ehrlichiosis are tick-transmitted infections caused by anaplasma phagocytophilum (aph) and ehrlichia ewingii (eew), respectively. both organisms induce an acute clinical disease, frequently accompanied by fever, polyarthropathy and thrombocytopenia. however, aph and eew have different tick vectors, i.e. ixodes scapularis and ambylomma americanum, respectively, with different, but overlapping geographic distributions. in addition, infection outcome may be affected by other regional ticktransmitted pathogens, such as borrelia burgdorferi (mn) or ehrlichia chaffeensis (ar). therefore, we compared serology and pcr results derived from dogs examined at two private practices located in highly endemic areas for either aph or eew. serum collected between april-december, from minnesota dogs (n ) was tested by snap s dx s and whole blood was tested by aph pcr. serum collected from arkansas dogs (n ) for year beginning in august was tested using microtiter plate elisas for antibodies to eew, e. canis, and e. chaffeensis (ech) while whole blood was tested by ehrlichia pcr. comparisons were evaluated using chi square (Ã) and binomial (w) tests with an alpha of %. the above results indicated that dogs are frequently exposed to both aph and bb in mn, whereas ar dogs are often exposed to eew, but less frequently to ech. antibodies to e. canis peptides were found infrequently in both mn and ar with only seroreactive dogs detected in both locations. active eew infection, as determined by pcr, was four times more frequent in ar pet dog seroreactors as compared to active aph infections among aph seroreactors. although both organisms induce acute disease, the number of aph and eew pcr positive dogs that were also seropositive was relatively high suggesting that both organisms induce persistent infections or that dogs are frequently re-infected, despite the presence of a measurable humoral immune response. additional studies are needed to determine regional infection profiles in other areas that are endemic for these pathogens. anaplasma phagocytophilum and ehrlichia canis are two of the most common vector borne disease agents that infect dogs and cats. while pcr assays that amplify the dna of these agents from blood are currently available, there is minimal information concerning the performance of these assays in different commercial laboratories that utilize different techniques. the purpose of this study was to compare the e. canis and a. phagocytophilum results of two different laboratories on the same samples collected from client-owned animals. veterinarians in states (az, md, ct) were recruited to participate in the study based on high prevalence rates for e. canis or a. phagocytophilum infection. blood in edta was collected from dogs or cats with fever, thrombocytopenia, or clinical evidence of polyarthritis and an equal volume of the same blood sample was simultaneously shipped on cold packs by overnight express to colorado state and to antech diagnostics. standard operating procedures at each laboratory were followed for total dna extraction and amplification of gapdh as the dna control. at colorado state university, a previously published pcr assay that amplifies the dna of ehrlichia spp., anaplasma spp., neorickettsia spp., and wolbachia was performed on each sample with positive amplicons sequenced to determine the species. at antech diagnostics, a proprietary real time pcr assay (fastpanel tm ) that amplifies the dna of anaplasma phagocytophilum, a. platys, ehrlichia canis, e. chaffeensis, and e. ewingii was performed. in the study to date, samples from animals ( dogs and cats) have been assayed at both laboratories. dna of a. phagocytophilum ( cats and dogs) and e. canis ( dog) were amplified at both laboratories with a percentage agreement between laboratories of %. the results to date suggest that the assay results of the two laboratories for a. phagocytophilum and e. canis are comparable. ehrlichiosis and bartonellosis are zoonotic diseases caused by extremely small, obligate intracellular bacteria that require a mammalian reservoir and a blood sucking arthropod vector. human ehrlichiosis is present in peru, with a seroprevalence as high as % in the highlands. bartonella species in humans were also identified in peru since (b. bacilliformis). recently, a new species (b. rochalimae) was isolated from an american woman who became febrile after travelling to peru. dogs can become infected with the same ehrlichia species, and the majority of bartonella species that affect human beings. the role of dogs as reservoirs for human infections has not been clearly established, but exposure and/or infection in dogs has been used to monitor human exposure to tick-borne disease (tbd), since they share the same environment. the objective of this study was to determine the serological and molecular prevalence of anaplasmosis, ehrlichiosis and bartonellosis in rural dogs in the highlands of peru. a total of healthy adult dogs were enrolled in this study from four communities in the central highlands of peru: ondores, pachacayo, san juan de pachayo, and canchayllo. edta-blood samples were collected from dogs, whereas serum samples were available from dogs. serum samples were tested for ehrlichia canis, anaplasma, borrelia burgdorferi and dirofilaria immitis infections using a qualitative dot-elisa (snap s dx). the edta-blood samples were screened by conventional pcr for the groel gene of the genus anaplasma and ehrlichia, and for the intergenic transcribed spacer of the genus bartonella. speciation was conducted by nucleotide sequencing. bartonella genus dna was detected from seven of the dogs ( . %) and ehrlichia canis dna was detected and sequenced from one dog ( . %). four of the bartonella positive samples were identified by dna sequencing as b. rochalimae (genbank accession numbers hq and hq ). the other three bartonella positive samples were identified as b. vinsonii subspecies berkhoffii, the causative agent of endocarditis in dogs and humans. no dog was infected with anaplasma species by dna amplification, but one dog was seroreactive for this genus ( . %). no specific antibodies against ehrlichia canis and borrelia burgdorferi and no antigens of dirofilaria immitis were detected. this study expands the current knowledge about tbd in peru and describes for the first time the infection of b. rochalimae in dogs in peru. the results suggest that dogs may play an important role in the epidemiology of this infection in humans, since they can be asymptomatic but bacteremic. bartonella spp. dna is commonly amplified from the blood of cats exposed to ctenocephalides felis. in previous work, it was shown that cats administered imidacloprid and experimentally exposed to b. henselae infected cats and c. felis did not become pcr positive for b. henselae whereas untreated cats all developed infection. the purpose of this study was to determine if administration of imidacloprid to clientowned cats likely to be exposed to bartonella spp. and c. felis in the field lessens prevalence of bartonella spp. infection. veterinary students in tennessee and florida that owned cats that spent at least days per month outside and that were willing to apply imidacloprid to their cats monthly for six months were recruited for the study. blood for bartonella spp. pcr assay was collected from the cats seven months after starting imidacloprid administration and assayed at colorado state university. to serve as a control group that was unlikely to have been administered flea control products in the previous months, blood was collected from feral cats during tnr programs in each of the two cities and assayed for bartonella spp. dna. the bartonella spp. dna prevalence rates between the groups were compared by chi square analysis with significance defined as p o . . the overall prevalence rates for bartonella spp. dna in the blood of veterinary student cats ( . %) and the feral cats ( . %) were significantly different (p o . ). the distribution of results is shown in table . the results suggest that florida feral cats were more commonly exposed to c. felis than tennessee feral cats. while the cats in the groups were not exactly matched, the student cats were allowed outdoors for approximately days per month and lived in the same cities as the feral cats, so c. felis exposure rates were likely similar. as previously shown in experimentally-exposed cats, the use of imidacloprid monthly may influence transmission rates of bartonella spp. amongst naturally-exposed cats. in an endemic area for leishmaniosis and filariosis, coinfection can occur and immunomodulation produced by wolbachia might influence the clinical signs and progression of both diseases. the aims of the present study were ) to determine the prevalence of wolbachia in dogs infected with dirofilaria immitis (di) and other filarial nematodes, ) to evaluate the level of coinfection of leishmaniosis and filariosis by molecular assays and ) to evaluate any associations between leishmania infantum (li) infection, filariosis with or without wolbachia and clinical presentation and outcome. statistical differences between groups were tested for significance by the fisher exact test using spss v. . software (significance: p-value o . ). one-hundred and eighteen owned dogs from southeastern spain presenting for clinical evaluation were included in the study. criteria the results of this study highlight the increased sensitivity of pcr for diagnosis of filariosis, confirm the presence of wolbachia in dogs from the mediterranean basin, show the increased severity of hwd when li-filaria coinfection is present and suggest that wolbachia could play a protective role for leishmaniosis. wolbachia antigens can stimulate a th -type immune response, as has been previously described. however other factors (as treatment with doxycicline) might be responsible for the lower prevalence of wolbachia among filaremic dogs infected with li and further studies must be done to clarify this interaction. the purpose of the present study was investigate the occurrence of leishmaniasis in cats in the municipality of arac¸atuba, sa˜o paulo, brazil, an endemic area for canine visceral leishmaniasis. animals were evaluated by direct parasitological examination of lymphoid organs and serology for visceral leishmaniosis by immunosorbent assay (elisa) and indirect immunofluorescence (ifat). thirteen ( . %) out of cats studied were diagnosed with visceral leishmaniasis; eight ( %) by parasitological diagnosis through cytological examination of lymphoid organs, six ( %) were considered positive by elisa and one ( . %) by ifat. only two ( . %) out of the thirteen infected cats had clinical signs, characterized by the presence of crusty lesions on the dorsal cervical region and hepatosplenomegaly. regarding age five cats ( . %) had between six months and two years, being the others older than years ( . %). only one cat ( . %) was positive for the three employed methods. pcr confirmed leishmania sp infection in nine ( . %) cats, of which six were diagnosed previously by cytological examination, two by elisa and one by the three techniques employed. since its first description in feline leishmaniosis has been reported in several countries. the purpose of this study was to assess the prevalence of leishmania chagasi infection in cats showing dermatologic lesions from an endemic area for visceral leishmaniasis in brazil. animals were evaluated by direct parasitological examination of lymphoid organs, immunohistochemical technique for detection of amastigotes in lesioned skin and serology for visceral leishmaniosis by immunosorbent assay (elisa) and indirect immunofluorescence (ifat). twenty seven ( . %) out of the cats studied were diagnosed with visceral leishmaniosis. twelve ( . %) were positive by parasitological diagnosis; amastigote forms of leishmania sp were identified in lymphoid organs from / ( . %) infected cats, and immunohistochemical technique allowed the identification of nine ( . %) positive animals. the seroprevalence of leishmaniosis was . % ( / ) by elisa and . % ( / ) by ifat. fiv specific antibodies were found in / cats ( . %), of which / ( . %) had leishmaniosis. real time pcr confirmed leishmania chagasi infection in three cats. based on the evidence of the high occurrence of leishmaniosis in cats in this study, this disease should be included in the differential diagnosis of skin diseases of felines living in endemic areas. blastomyces dermatiditis is a dimorphic fungus that commonly affects large-breed hunting dogs. a recent advancement in diagnosis has come with the advent of a urine antigen screening test that has both high sensitivity and moderately high specificity. therapy for the disease involves use of antifungal agents, usually itraconazole, and length of treatment is based chiefly on resolution of clinical and radiologic signs. with the new urine antigen test, however, a noninvasive route of monitoring treatment progress is available and could be an adjunct device utilized to determine treatment efficacy and may even reveal a need for prolonged treatment. therefore, the purpose of this study was to determine if monitoring the blastomyces urine antigen test and comparing to pulmonary radiographic signs would elucidate the necessity for prolonged antifungal therapy, even after resolution of radiologic signs. to this end, a retrospective case review was performed that identified a series of client-owned animals with naturally occurring blastomycosis. the inclusion criteria were radiographic pulmonary parenchymal signs consistent with fungal disease and urine antigenconfirmed blastomycosis with repeated testing of both radiographs and urine antigen quantification as monitoring parameters until negative results achieved in each. ideally, intervals between testing dates would be between two and five months. radiographs were considered negative if all radiographic changes had resolved or if repeated radiographs separated by at least one month were considered static after documented improvement had occurred from original diagnostic radiographs (suspected scarring). urine antigen testing was considered negative if concentrations were less than . enzyme immunoassay units, a reference interval set by the testing laboratory. preliminary data analysis reveals resolution of radiographic signs of blastomycosis occurred earlier in many of the cases presented than did attaining a negative urine antigen concentration. ceasing treatment month after radiographic resolution of signs as has been recommended in the past might have resulted in premature discontinuation of therapy in many of the cases. monitoring of urine antigen concentrations may be of additional clinical use for determining when cessation of treatment should occur in cases of blastomycosis. persistent elevation of urine antigen concentrations after radiographic resolution of infection may account for apparent recrudescence of blastomycosis after suspected clinical resolution. giardia spp. and cryptosporidium spp. are both known to cause infections in dogs and humans in the united states. nevertheless, prevalence rates for dual infection in dogs had not been widely reported. in this study, fecal samples from dogs housed in a northern colorado animal shelter (n ), dogs owned by veterinary students in northern colorado (n ), and dogs from the pine ridge reservation in south dakota (n ) were collected. each sample was assayed with a commercially available fluorescent antibody assay that detects giardia spp. cysts and cryptosporidium spp. oocysts. those samples that were positive for giardia spp. or cryptosporidium spp. with adequate dna available for sequencing were genotyped by the glutamate dehydrogenase [gdh] and by the heat shock protein- [hsp- ] genes, respectively. overall, ( . %) of the dogs had current evidence of a protozoal infection ( table ). the dogs from pine ridge reservation had the highest prevalence rates for giardia infection and also for dual infections. from the student dogs, sequencing was successful for the three giardia isolates (assemblage d from dogs; assemblage c from one dog) and one cryptosporidium isolate (c. canis). from the reservation dogs, sequencing was successful for nine giardia isolates (assemblage d from dogs; assemblage c from dogs) and one cryptosporidium isolate (c. canis). cryptosporidium and giardia co-infections are commonly detected in dogs; in this study dual infections were more common than cryptosporidium infections alone. further studies will be required to determine the clinical importance of this finding. although the giardia and cryptosporidium isolates that were sequenced were the dog specific assemblages/genotypes, more samples should be analyzed in order to assess the potential for zoonotic transmission of either parasite. the current study was conducted to determine the prevalence of intestinal parasites in dogs visiting the veterinary teaching hospital, chiang mai university, northern thailand. fecal samples (n ) were collected and submitted by owners between august to february . demographic and geographic data were recorded. intestinal parasitic infection was diagnosed by both microscopic examination after zinc sulfate centrifugation flotation and commercially available ifa for giardia spp. and cryptosporidium spp. polymerase chain reaction and dna sequencing were performed on all giardia and cryptosporidium positive samples to provide genotyic information. overall prevalence of intestinal parasitic infection in dogs in chiang mai was . %. the most prevalent parasite was giardia spp. ( . %) followed by ancylostoma spp. ( . %), cryptosporidium spp. ( . %), cystoisospora spp. ( . %), toxocara canis ( . %), trichuris vulpis ( . %), coccidian-like ( . %), toxascaris leonina ( . %), and strongyloides spp. ( . %). the prevalence of having at least one parasite in dogs o year, - years, and years were . %, . %, and . %, respectively. of these infected dogs, . %, . %, . %, and . % were infected with one, two, three, and four organisms, respectively. available dna sequences from giardia spp. positive samples were shown to be dog specific. only one adequate dna sequence was available for cryptosporidium spp., which was shown to be c. canis. the findings suggested that intestinal parasitic infection was common in dogs in chiang mai, thailand. dogs could be potential source for zoonotic intestinal parasitic infection since dogs in this area are allowed for free roaming. regular deworming program is indicated to prevent not only transmission among dogs but also to human. a retrospective study was conducted on parasite positive fecal specimens consisting of canine, feline, equine and from other host species, comparing recovery of eggs, protozoan cysts and coccidian oocysts using standardized methods of parasite concentration: the formalin/ethyl acetate (f/ea) sedimentation concentration and the commercial fecalyzer (flotation) kit procedures. specimens were processed by each technique either according to manufacturer's instructions or according to standard laboratory procedures. formalin/ethyl acetate concentrations used at a ratio of ml normal saline to ml ethyl acetate for extraction of lipophilic material from pelleted stool samples, previously fixed in sodium acetate/acetic/acid/formalin (saf) solution. flotations with the fecalyzer kit were performed with concentrated zinc sulfate solution (s. g. . ) . the range of parasites recovered from these specimens included flagellate cysts ( total), coccidian oocysts ( total), ova and larvae of nematodes ( total), and ova of trematodes ( total) , and cestodes ( total). recovery rates by fecalyzer flotation were good for protozoan cysts, coccidian oocysts and nematode eggs and larvae, but very poor for cestode and trematode eggs. formalin/ethyl acetate concentration showed excellent recovery of all parasites and consistently outperformed fecalyzer in recovery rates. recoveries by f/ea concentrations were higher by . % for giardia, by . % for coccidia and by . % for nematode eggs and larvae. with the exception of coccidian oocysts, based on z-test analyses, recovery rates were significantly higher, at a confidence level of at least %, for all parasites, using formalin/ethyl acetate sedimentation concentration. although capc recommends the use of flotation with centrifugation methods for standard fecal ova and parasite examination for veterinary patients, sedimentation concentration methods are widely and effectively used in human diagnostic parasitology laboratories. these results provide good evidence for the use of f/ea concentration as a preferred method to flotation procedures for stool ova and parasite examinations in veterinary laboratories. cyclosporine and glucocorticoids are powerful immunosuppressive agents used to treat many inflammatory diseases. cyclosporine inhibits calcineurin-dependent pathways of t-cell activation and the resultant cytokine production, and glucocorticoids directly inhibit genes coding for cytokines. little work has been done comparing the effects of these agents on cytokine production in dogs. our study assessed these effects by measuring t-cell cytokine production using flow cytometry, and cytokine gene expression using quantitative reverse transcriptase polymerase chain reaction (qrt-pcr) in activated canine t-cells treated with cyclosporine and dexamethasone. for flow cytometric assays, peripheral blood mononuclear cells were separated using density gradients and cultured for hours in the presence of cyclosporine ( , , or ng/ml), dexamethasone ( À , À , À m), or cyclosporine plus dexamethasone. for qrt-pcr, whole blood was cultured for hours with the same drugs at the same concentrations, and rna was then extracted from leukocytes. expression of cytokines il- and ifn-g was analyzed in pma/ionomycinactivated t-cells by flow cytometry, and gene expression for il- and ifn-g in activated t-cell populations was assessed via qrt-pcr. flow cytometry and qrt-pcr both demonstrated inhibition of il- and ifn-g that was generally dose-dependent in response to both cyclosporine and dexamethasone. flow cytometry results from the average of samples collected from different dogs are shown in figure a . similar results were achieved using qrt-pcr ( figure b ). suppression of il- and ifn-g in activated t-cells has potential as an indicator of the efficacy of cyclosporine and glucocorticoids in suppressing canine t-cell function in vivo, and may therefore be of value for characterizing the immunosuppression induced by these drugs in clinical patients. idiopathic eosinophilic diseases are described in several breeds, but are over represented in rottweilers. the immunopathogenesis of idiopathic eosinophilic disorders is poorly characterised. studies in people highlight the importance of cytokines, particularly interleukin- (il- ), in mediating eosinophil maturation, differentiation, egress from the bone marrow, migration and polyclonal expansion. eotaxin- and eotaxin- also appear important for induction of chemotaxis and release of reactive oxygen species from eosinophils. the aim of the current study was to establish whether definable differences in specific cytokines associated with mediation of eosinophil production and survival are present between healthy rottweilers, non-rottweilers and rottweilers with non-parasitic eosinophilia. secondly, by evaluating cytokine profiles the study aimed to improve understanding of the pathophysiology of eosinophilia therefore assisting development of potential molecular treatment options. quantitative real-time reverse transcriptase polymerase chain reaction (qrt-pcr) assays were used to quantify messenger rna (mrna) encoding cytokines il- , il- , il- , il- p , il- p , il- p , il- , interferon gamma (ifn-g) and chemokines eotaxin- and eotaxin- from peripheral blood mononuclear cell (pmbc) samples obtained from healthy non-rottweiler dogs with normal eosinophil counts (n ) and rottweilers with normal (n ), mildly increased (n ) and high (n ) eosinophil counts. quantification of serum ifn-g was also performed using a commercially available canine-specific elisa. all samples were positive for housekeeping genes and all cytokines could be quantified with the exception of eotaxin- and - . results were normalised using three stably expressed housekeeper genes (rpl a, sdha and ywaz) and a relative copy number was calculated for each sample with the sample with the fewest copies given a value of . no significant differences were found between groups but there was a tendency for ifn-g mrna expression to be lower in the rottweilers with moderate to severe eosinophilia versus control dogs (p . ). this trend was not seen in the concentration of serum ifn-g quantified by elisa as there were no significant differences between normal and diseased animals. in conclusion, there were no significant differences in cytokine mrna profiles between normal dogs and rottweilers with varying degrees of eosinophilia. additional studies including larger numbers of affected dogs are warranted before any accurate conclusions can be made. the presence of large amount of antibody on erythrocyte membrane can accelerate red blood cell (rbc) removal process by the mononuclear phagocyte system. an antigenic stimulus such as the one promoted by vaccines, for example, can induce hypersensitivity reactions and may accelerate rbc destruction. the study objective was to evaluate the erythrocytic membrane potential in inducing lymphocyte proliferative response of recently immunized dogs. healthy adult dogs (n ) were immunized with multiple antigens (commercial vaccine with eight antigens: distemper virus, parvovirus, coronavirus, parainfluenza virus, adenovirus, infectious hepatitis virus and leptospire; and anti-rabies). blood samples from each animal were collected into edta tubes in two moments: pre (immediately before vaccination) and pos ( to days after vaccination). mononuclear cells were separated by gradient, marked with cfse-fitc and cultured. the stimuli for lymphocyte proliferation used were autologous erythrocytic membrane (aem) and concanavalin a (cona). aem was obtained by hypotonic lysis and tested in two concentrations (m : . ug/ ul; m : . ug/ ul). the proliferation assay was evaluated by flow cytometry and analyzed with specific software. the proliferation index (pi) was calculated dividing the fluorescence intensity of the basal sample by the stimulated one. statistical analysis was performed using paired t-test for parametric samples and wilcoxon test for non-parametric samples (a . ). the for the tested concentrations, autologous erythrocytic membrane does not constitute a stimulus for lymphocyte proliferation in vitro, either before or after vaccination procedure. additionally, there was no evidence of self-reagent lymphocytes to erythrocyte membrane after vaccination. e. coli is a common cause of canine urinary tract infection. current treatment emphasizes eradication of established infection rather than infection prevention but increased antibiotic resistance necessitates strategies to prevent infection. proanthocyanidins found in cranberry juice inhibit e. coli attachment to human uroepithelial cells, impairing bacterial adherence and colonization. we hypothesized that purified cranberry extract (ce) inhibits bacterial adhesion to canine uroepithelial cells. five healthy female dogs received an oral ce supplement (vetoquinol; mg ce/tablet) according to body weight for days. voided urine collected from each dog before (pre) and after ( -day) completion of the protocol was membrane filtered ( mm) and stored frozen (- c). bacterial adhesion was determined using an in vitro assay. briefly, urine samples were incubated with an uropathogenic e. coli strain that had been subcultured to promote fimbriae expression. urine samples containing e. coli were next incubated in -well plates containing methanol-fixed madin-darby canine kidney (mdck) cells for -hr ( c) to permit bacterial attachment. after incubation, plates were washed to remove nonadherent bacteria and fresh media added. plates were incubated ( c) for -hr to grow attached bacteria to detection level. bacterial concentration in each well was determined using a spectrophotometer ( nm). results were analyzed using the chi-square test. ce significantly reduced bacterial adhesion by % (n ; p . ) in -day urine samples compared with pre samples. the results show that ce supplementation can reduce adhesion of uropathogenic e. coli to canine uroepithelium and suggests one mechanism by which ce might improve urinary tract health. the purpose of this study was to determine prevalence of urovirulence factors (uvfs) and antimicrobial resistance in canine uropathogenic e. coli (upec) and to evaluate associations between uvfs and antimicrobial resistance. two hundred and twenty-one upec isolates from samples collected from different canine patients submitted to the university of tennessee microbiology laboratory in were evaluated. a multiplex pcr assay was used to detect cnf, hlyd, sfa/foc, and papgiii in dna lysate. in vitro susceptibility was evaluated and if the isolate was resistant to any antimicrobial in a class, it was considered resistant to that class. of the samples, the number of uvf expressed per isolate was: / ( %), / ( %), / ( %), - / ( %), and / ( %). expression of uvf was sfa ( %), hly ( %), cnf ( %), and pap ( %). presence of uvfs was associated with less resistance (p o . ). the combination of hly, cnf, and sfa was associated with less resistance (p o . ). when sfa was present alone, resistance was less (p o . ). average resistance to antimicrobial class by number of uvfexpressed was: uvf . ae . classes, uvf . ae . classes, uvf . ae . classes, uvf . ae . classes, and uvf . ae . classes. urovirulence factors were present in a moderate number of upec and correlated negatively with resistance. neither individual nor combinations of uvfs were associated with increased resistance. obesity is associated with several comorbidities in dogs including pancreatitis, osteoarthritis, oral disease, neoplasia, and lower urinary tract disease. investigator observations led to the hypothesis that morbidly obese dogs are more likely to have asymptomatic bacterial urinary tract infections (abuti) than overweight and moderately obese dogs. therefore, a pilot study was conducted to screen for abuti in obese dogs. urinalysis with urine culture and dual energy x-ray absorptiometry (dxa) were performed on fortythree dogs with body fat (bf) percentages ranging from to %. following dxa, subjects were categorized as obese (o)(bf - %, n ) and morbidly obese (mo)(bf %, n ). no dogs had owner-reported symptoms indicative of uti. the prevalence of abuti in o dogs was % (n ) and % (n ) in mo dogs. the dog in the o group with abuti was close to being mo with a bf equaling . %. of the nine dogs with positive cultures, were neutered males and were spayed females. the prevalence ratio of abuti in mo dogs was . , indicating dogs with % or greater bf are . times more likely to have the condition then dogs o % bf. the results of this pilot study coincide with other surveillance data describing an increased prevalence of lower urinary tract disease in obese dogs. in conclusion, dogs with body fat percentages greater than % are at risk for abuti, and veterinarians should consider screening all morbidly obese patients for urinary tract infections. calcium carbonate (cac) is recommended to decrease phosphate intake in chronic kidney disease. however, its effect is poorly documented in dogs. our objectives were to assess within-day, postprandial and cac effects on phosphatemia variations in healthy dogs. phosphatemia was measured every hours for hours in eight adult healthy beagle dogs in i) fasted condition and ii) a  crossover design. one group received cac mixed with maintenance diet ( . % phosphorus), while the second group received the diet alone. after a -week wash-out period, groups were switched. a general linear model was used to test the period, sequence, treatment, dog and time effects on phosphatemia and the area under the phosphatemia versus time curve (auc - ). a significant (p o . ) circadian variation existed in fasted dogs. the maximum difference (mean: À . mg/dl; % c.i.: À . mg/dl; À . mg/dl) was observed between a.m. and midnight. the auc - with cac ( ae mg.min/dl) was mildly but significantly lower (p . ) than without cac ( ae mg.min/dl). however, it was similar to the auc - in fasted conditions. feeding, with and without cac, has minor effect on phosphatemia. however, circadian variation of fasted phosphatemia might affect its interpretation. gfr measurement permits diagnosis of kidney injury prior to development of azotemia, and is the gold standard for kidney function assessment. accurate and rapid (o min) gfr measurement has been performed in rats by simultaneous transcutaneous assay of two intravascular fluorescently-labeled markers. a recently developed analyzer assays fluorescence via a fiberoptic cable introduced through a peripheral catheter, and thus should also allow rapid gfr determination in larger species. the purpose of this study was to determine correlation and agreement between fluorescent ratiometry (fr) and iohexol plasma clearance (ipc) in dogs over a range of gfrs. acute kidney injury (aki) was induced in female hound-type dogs ( mg/kg gentamicin iv q h), and fr and ipc gfr were simulta-neously determined on days , , and . a -sample, -hr protocol was used for ipc; fr was determined following bolus injection of a dextran conjugate mixture ( -sulfohexamine rhodamine-carboxymethyl kd dextran, -aminofluorescein-carboxymethyl kd dextran) with fluorescence measured over min. gfr was calculated using -compartment model concentration-vs.-time curves for both techniques. correlation was determined via spearman's rho; agreement was analyzed via bland-altman plots. ipc gfr and serum creatinine confirmed progressive aki in all dogs. correlation between fr and ipc was . (p o . ). bland-altman plots confirmed good agreement between techniques with slight underestimation of gfr by fr across most observed values. these results suggest fr is suitable for gfr determination in dogs with aki. importantly, the portable analyzer allowed for point-of-care gfr determination in o min using a peripheral vein. previously presented at the american society of nephrology renal week (related but not identical abstract). dogs with protein-losing nephropathy (pln) are at risk of thromboembolic disease, but the mechanism of hypercoagulability and the population of dogs at risk are unknown. the purpose of this study was to characterize thromboelastography (teg) in dogs with pln. twenty-eight client-owned dogs with pln (urine protein:creatinine ratio (upc) . ) and control dogs were enrolled. teg parameters, antithrombin activity, serum biochemical profiles, and upc were measured. teg analyses were run in duplicate with kaolin activation; reaction time (r), clot formation time (k), maximal amplitude (ma), and g (global clot strength) were analyzed. a wilcoxon sum rank test was used to evaluate differences between groups. twelve pln dogs ( . %) were azotemic. nineteen pln dogs ( . %) were hypoalbuminemic [serum albumin (salb) o . g/dl]; had salb o . g/dl. dogs with pln had higher k (p o . ), ma (p o . ) and g (p o . ) than controls. r was similar between the two groups. pln dogs with salb o . g/dl had higher g (p o . ) values than dogs with salb . g/dl; however, even pln dogs with normal salb ( . g/dl) had significantly higher g values than controls (p o . ). no significant relationship between upc and g, salb and g, antithrombin and g, or salb and antithrombin was noted using linear regression analysis. these results indicate that antithrombin, salb, and upc cannot be used alone to predict hypercoagulability as assessed by teg in dogs with pln. a comprehensive evaluation of the coagulation system in individual patients may be necessary to predict the point at which to initiate anti-thrombotic therapy. cystinuria is a hereditary renal tubular reabsorption defect of cystine, ornithine, lysine and arginine (collectively, cola). the low solubility of cystine in acidic urine predisposes to the formation of uroliths. type i cystinuria in newfoundland and labrador retriever dogs is an autosomal recessive trait caused by mutations in the slc a gene, whereas in other breeds, the cause of cystinuria has not yet been determined. we report here on the clinical, biochemical and molecular features of cystinuria in irish terriers. urine and edta blood were collected from irish terriers from europe and australia. a nitroprusside screening test was used to identify increased cystine in urine. urinary amino acid concentrations were determined by high-pressure liquid chromatography. cystinuric dogs were defined as having cystine calculi, a positive nitroprusside result, urinary cystine ( mmol/g creatinine) and/ or a cola concentration of mmol/g creatinine. all females tested nitroprusside negative and had normal urinary cystine (o mmol/g creatinine) and cola (o mmol/g creatinine) concentrations. the intact males that formed calculi as adults exhibited cystine concentrations ranging from - and cola from - mmol/g creatinine. an additional males had similarly high cola values with cystine levels from - mmol/g creatinine. among the affecteds tested, % were nitroprusside positive. the negative nitroprusside results and/or low urinary cystine levels of affecteds may be due to precipitation of cystine in acidic urine. sequencing the coding regions of the slc a and slc a genes from edta blood identified no mutations. the mode of inheritance remains undetermined. however, castration appears to lower the urinary cystine and cola concentrations and to prevent cystine calculi formation, while diet changes have lesser effects. in conclusion, non-type i cystinuria in irish terriers (and several other breeds like mastiffs and scottish deerhounds) is a unique form characterized by increased aminoaciduria only in males, with lower cystine and cola excretion and fewer and later urolith formation compared to type i cystinuria. castrating cystinuric irish terriers lowers their cystine and cola excretion and thus their risk for calculi formation. cats and dogs that are diagnosed with acute kidney injury (aki) and resultant uremia that is not responsive to standard medical therapy are likely to benefit from renal replacement therapies, such as intermittent hemodialysis (ihd). the purpose of this study was to evaluate the long-term outcome of patients with aki treated with ihd, and to establish whether renal function, as determined by serum or plasma creatinine concentrations, is associated with longterm survival. medical records of cats and dogs that were diagnosed with aki, treated with ihd, and survived longer than days following the last ihd treatment were retrospectively analyzed. standard methods of survival analysis using kaplan-meier product limit curves and the log-rank test were performed. for all-cause mortality, the median survival time was days ( % confidence interval: , ) for cats and days ( % confidence interval: , ) for dogs. when only renal-related causes of death were taken into account, the median survival time was not reached for cats or dogs. survival time for all-cause mortality was inversely associated with the lowest creatinine concentration within the to day period following the last ihd treatment (p o . for cats, p o . for dogs). this study demonstrates that veterinary patients that are diagnosed with aki, treated with ihd, and survive greater than days after the last ihd treatment have a good longterm prognosis and frequently die from causes that are unrelated to renal impairment. renal fine-needle aspiration (r-fna) is oftentimes attempted during evaluation of dogs and cats with renomegaly, mass lesions, or suspected infiltrative processes. diagnostic utility of fna is dependent upon the organ being sampled; additionally, in some organs, certain diagnostic imaging findings are associated with improved concordance of fna with final diagnosis. objectives of this study were to evaluate the diagnostic utility of r-fna and determine whether concordance with final diagnosis is associated with specific clinicopathologic or diagnostic imaging findings. we hypothesized that r-fna is most useful in patients with diagnostic imaging results suggestive of renal neoplasia (i.e. masses or suspected infiltrative processes). dogs and cats that had undergone r-fna from jan , to dec , were identified by database search. patient signalment, serum creatinine and blood urea nitrogen concentration, urine specific gravity, dipstick protein, r-fna result, and final diagnosis were recorded. patients were excluded if abdominal radiographs or sonographic images were not available for review, or if diagnostic test results were insufficient for determination of final diagnosis. a single coauthor blinded to final diagnoses interpreted all abdominal images using a pre-set list of descriptors and grading criteria. radiographic kidney shape, margin distortion, and ventrodorsal kidney-to-l ratio were evaluated. sonographic kidney margin distortion, cortical echogenicity, and corticomedullary junction distinction were described, and presence of nodules or masses, peri-renal effusion, or a peripheral sonolucent rim was noted. concordance of r-fna and final diagnosis was determined, and the chi-squared or fisher's exact test were used to determine association of concordance with the above variables; p o . was considered significant. dogs and cats ( animals) met all inclusion criteria. r-fna results were concordant with the final diagnosis in ( . %) patients, discordant in ( . %) patients, and inadequate for cytologic interpretation in ( . %) patients. neoplasia or fip were the final diagnoses in of ( . %) and of ( . %) patients with concordant results, respectively. renal lymphoma (p . ), renal carcinoma (p . ), and renal neoplasia in general (p . ) were not associated with a higher likelihood of r-fna and final diagnosis concordance. there was no association noted between likelihood of r-fna and final diagnosis concordance when patients were stratified by species, serum creatinine or blood urea nitrogen concentration, urine specific gravity, dipstick proteinuria, or any diagnostic imaging variables. this study failed to identify concurrent clinicopathologic or diagnostic imaging findings that enhanced the diagnostic utility of r-fna. future studies should use standardized criteria to prospectively identify patients in which r-fna will be performed, evaluate additional variables that may be associated with increased r-fna diagnostic utility, and directly compare the utility of r-fna with that of other diagnostic techniques. feline lower urinary tract disease (flutd) is a disease with increasing prevalence in private practices and veterinary teaching hospitals. although several underlying causes can cause the obstructive form in male cats, the idiopathic form (feline interstitial cystitis) often is diagnosed as underlying reason in cats o years. the goal of this retrospective study was to identify possible predisposing factors in order to optimize the therapy of these patients. as a study group, cats hospitalized with obstructive flutd at the veterinary university of vienna were examined during a year period ( ) ( ) ( ) . as a control group cats presented for other reasons were randomly chosen during the same time period. the data were examined concerning the signalment and history. furthermore, the long-term outcome was evaluated with a questionnaire. based on assumptions a student's t-test or a chi-square test was used. there were no significant differences in age and breed. the body weight was significant higher in the flutd group than in the control group (p o . ). we could observe a significant risk for the disease of a weight of kg (p o . ). there were significant less cat toilets in the flutd group compared to the control group (p o . ). furthermore we could observe that in the households of flutd cats there was significant less than one toilet per cat (p o . ) and more cats diseased on flutd lived strictly indoor than outdoor (p . ).there were no significant differences at the time of hospitalization in age, breed, number of cats per household or season of the year between the two groups. in summary, we could observe that cats over kg body weight kept indoor with less than one toilet per cat have a significant higher possibility to be affected by obstructive flutd. further studies with an extensive history of animal husbandry are needed to identify risks predispoing cats to this frequent and cost-intensive disease. although purine uroliths (ammonium urate, sodium urate, xanthine, uric acid, etc.) represent the third most common stone type in cats, purine uroliths have the highest rate of recurrence ( % in months). in dogs, mutation of the urate transporter (slc a ) and portovascular anomalies are common risk factors. however the underlying cause(s) for purine urolith formation in cats is unknown. the purpose of this study was to test the hypothesis that hyperuricosuria without alterations in liver function is common in cats with urate uroliths. urine concentrations of purine metabolites were measured by high-performance liquid chromatography in cats with ammonium uroliths (cases), clinically healthy, breed and gender matched cats (negative controls), and cats with naturally occurring xanthine uroliths (positive controls). prior to urine collection, all cats were fed a standard maintenance food (protein g/ kcal) for weeks. urinary xanthine, uric acid, and allantoin concentrations and concentration to creatinine ratios were calculated and compared between groups. also, serum pre-and post-prandial bile acid concentrations were measured. when compared to control cats, urinary uric acid concentration was significantly higher in case cats (p . ). xanthine was not detected in the urine of cases or negative controls. a significant difference in fasted and post-prandial serum bile acid concentrations was not detected in cases or controls (p . , . ).hyperuricosuria without increased concentrations of urinary xanthine or allantoin appears to be a risk factor for ammonium urate urolith formation in cats. an association between portovascular shunts and purine urolithiasis was not observed in this population of cats. studies indicate that proteinuria is predictive, on a population basis, of those cats at risk of developing azotemia. seldi-tof-ms is a sensitive, high-throughput, proteomic technique utilising chromatographic surfaces to facilitate separation and detection of proteins and peptides within biological fluids such as urine. individual low molecular weight (lmw) urinary proteins have been considered as potential biomarkers for renal damage but provide only a limited representation of the urinary proteome; seldi-tof-ms may provide a more global assessment. normotensive, non-azotemic geriatric cats ( years) were recruited prospectively from two first-opinion clinics for routine health screening. at entry cats received a full physical examination, plasma biochemistry, evaluation of total t concentration and urinalysis including urine protein to creatinine ratio. re-examination was offered at and months. cats were divided into two groups based on clinical status at the month re-examination (azotemic; creatinine concentration ! . mg/dl and non-azotemic). optimisation studies were performed to facilitate the automated preparation (biomek ) of cm (weak cation exchange) arrays for seldi-tof-ms analysis (ciphergen enterprise ) of urine samples from cats at entry to the study. results are reported as median [ th , th percentile]. mann whitney u-test and wilcoxon signed rank test were used to compare variables between groups and between timepoints, respectively. ciphergen express ( . ) software was used to analyse spectral data and a mann whitney u-test was used to identify clusters which differed significantly between groups (p o . ) at entry to the study. twenty non-azotemic cats were recruited, of which cats developed azotemia by months. no significant differences in age, body weight, biochemical or urinalysis variables were identified between groups at entry to the study. as might be expected creatinine increased significantly ( . mg/dl [ . , . ], . [ . , . ], p . ) between study entry and months in the cats that developed azotaemia and there was a commensurate increase in phosphate concentration ( . mg/dl [ . , . ], . [ . , . ], p . ). creatinine and phosphorus did not change significantly over time in the cats that did not develop azotaemia. seven clusters with m/z values of , , , , , were found to differ significantly between groups at entry to the study. the low protein concentration of feline urine makes the use of proteomic techniques challenging. however, this pilot study indicates that seldi-tof-ms can be utilised to examine the feline urinary proteome and that differences in low molecular weight protein patterns may be useful to differentiate those cats which are at risk of the development of azotemia. further work is necessary to identify these proteins/peptides. fibroblastic growth factor (fgf- ) is a phosphotonin with an important physiological role in the regulation of phosphorous and vitamin d metabolism, and may therefore play a part in the development of renal secondary hyperparathyroidism. previous studies in cats have shown parathyroid hormone (pth) to be elevated prior to the development of azotemia. the study objectives were to explore the hypothesis that fgf- is a mediator of the development of renal secondary hyperparathyroidism in the nonazotemic stages of feline ckd. healthy, non-azotemic (plasma creatinine concentrations (cr) o . mg/dl) geriatric cats were recruited into the study prospectively and followed for months. at the study end point cats were categorised into the following groups: group (n )-cr . mg/dl, group (n )-cr ! . mg/dl but did not meet the criteria for group and group (n )-cr . mg/dl in association with reduced urine concentrating ability (usg o . ) or demonstration of persistent azotemia (cr . mg/dl). plasma samples were subjected to routine biochemical analysis, intact pth, calcitriol and intact fgf- assay. variables were compared between the groups at the baseline time point. gfr was measured in an additional group of cats ( non-azotemic, iris stage ii, iris stage iii) using a corrected slope-intercept iohexol clearance method. relationships were explored using linear regression analysis and determining the coefficient of determination (r ). results are presented as median [range] . at the baseline time point fgf- concentrations were significantly higher in group ( . [ . - . ], p . ) and group ( . [ . - . ], p . ) compared to group ( . [ . - . ] ). weak positive relationships were identified between fgf- and pth (r . , p . , n ) and fgf- and cr (r . , p . , n ). however, the positive relationships between fgf- and phosphate (r . , p . , n ) and fgf- and calcitriol (r . , p . , n ) were not significant. the additional group of cats in which gfr measurement was performed there was an inverse relationship between fgf- and gfr (r . , p . ). in conclusion, fgf- was elevated in cats prior to the development of azotemia. the role of fgf- in the development of feline renal secondary hyperparathyroidism remains to be determined and should be explored through interventional studies. however, considering the relationship between fgf- and gfr, it cannot be excluded that the phosphotonin is simply a marker of reduced filtration. chronic kidney disease (ckd) is common in geriatric cats and hypoxia might contribute to the progression of this disease. the aim of this study was to evaluate urinary vascular endothelial growth factor (vegf) as a marker of renal hypoxia. cats were recruited through geriatric clinics held at two first opinion london practices. vegf was measured in stored samples using a canine elisa kit validated for use on feline urine and indexed to creatinine concentration to yield a vegf to creatinine ratio (vcr). two studies were undertaken -firstly a cross-sectional analysis of clinical variables associated with vcr in cats with ckd. diagnosis of ckd was based on concurrent findings of plasma creatinine ! mg/dl and usg . , with persistence of azotemia for ! weeks. only patients receiving no medical therapies were included. normotensive and (pre-treatment) hypertensive cats were included, but borderline cases (mean systolic blood pressure - mmhg on the date of sampling) were not. hyperthyroid cats were also excluded from this cross-sectional study. associations between vcr and clinical data were initially assessed using the spearman's coefficient and mann whitney test. linear regression was then used for multivariate analysis. the second study used samples from a trial in which hypertensive cats that had been treated with amlodipine for at least months were entered into a randomised cross-over study where they received placebo or benazepril ( . to mg/kg daily) for weeks in turn. vcr on placebo was compared with that on benazepril using the wilcoxon signed ranks test. cats with well controlled hyperthyroidism were included in this intervention study. results are reported as median [ th, th percentile]. vcr was higher ( . [ . , . ] vs. . [ . , . ] fg/g, p . ) in untreated hypertensives (n ) than normotensives (n ). vcr was correlated with pcv (r À . , p . , n ), upc (r . , p o . , n ), plasma phosphate (r . , p . , n ), and usg (r À . , p . , n ), but not plasma creatinine concentration. in the best multivariate model, pcv was associated with vcr independently of upc (r . , n ). vcr was significantly reduced by benazepril therapy ( . [ . , . ] fg/g) compared with placebo ( . [ . , . ] fg/g; p . , n ) with a reduction seen in % of cases. these results suggest urinary vegf excretion is associated with proteinuria in cats with ckd and might be a marker of renal hypoxia induced by low pcv. ace inhibitor therapy might reduce urinary vegf excretion because angiotensin ii causes constriction on efferent arterioles resulting in tubular hypoxia. fgf- is a phosphaturic hormone. fgf- concentrations increase with declining renal function in humans. the objectives of this study were to validate a method for fgf- quantification in feline plasma and to assess the association between fgf- concentration and plasma creatinine or phosphate concentration in cats with chronic kidney disease (ckd). non-azotemic and azotemic (plasma creatinine concentration (cr) . mg/dl) geriatric ( yrs) cats were recruited into the cross-sectional study from two london first opinion practices. cats were excluded from the study if they were fed a phosphate restricted diet, or had evidence of concurrent disease. the cats were categorized, using a modified iris staging system, into the following four groups: group (cr . mg/dl), group (cr . - . mg/dl), group (cr . - . mg/dl), group (cr . mg/dl). groups and were further subdivided based on the iris targets for plasma phosphate concentration (po ): group a (po . mg/dl), group b (po . mg/dl), group a (po mg/dl), group b (po mg/dl). fgf- concentrations were measured in feline edta plasma using a human intact fgf- elisa, validated by intraand inter-assay variability and assessment of dilutional parallelism. comparisons between groups were made using the kruskal-wallis test and mann-whitney u test, with statistical significance defined as p o . . bonferroni correction was applied where appropriate (statistical significance then determined as p o . ). results are reported as median [ th, th percentiles]. fgf- concentrations ! pg/ml (upper limit of quantification) were assigned the value of pg/ml. intra-and inter-assay variability of fgf- measurements were o . % and dilutional parallelism between feline samples and the calibration curve were demonstrated. plasma fgf- concentrations increased with increasing creatinine concentrations (group : [ , ] , n , group : [ , ] , n , group : [ , ], n , group : [ , ], n ). fgf- measurements were significantly different between all groups (p . to o . ) except between groups and (p . ). fgf- concentrations were significantly higher in cats with higher plasma phosphate concentrations (group a: [ , ] , n vs. group b: [ , ], n ; p . ) and (group a: [ , ] , n vs. group b: [ , ], n ; p . ). in conclusion, fgf- concentrations were higher in cats with more severe ckd or higher plasma phosphate concentrations as would be predicted from its known biological actions. further work is warranted to explore the role of fgf- in the development of renal secondary hyperparathyroidism by measuring parathyroid hormone (pth) and calcitriol in cats at different stages of ckd. progressive non-cardiogenic edema and lung dysfunction are common complications of acute kidney injury (aki) in people. pulmonary abnormalities have not been systematically reviewed in dogs with renal azotemia, but anecdotal reports of dogs with aki and concurrent non-cardiogenic pulmonary edema are suggestive of uremic pneumonopathy (up), a centrally-distributed pulmonary edema syndrome associated with kidney disease in people. we therefore hypothesized that pulmonary-associated clinical signs or thoracic radiograph abnormalities are more common in dogs with renal azotemia than in non-azotemic dogs, and that this association is more likely in dogs with aki than dogs with chronic renal failure (crf). our study objectives were ) to describe thoracic radiograph and lung histopathologic abnormalities in dogs with renal azotemia, ) to compare the occurrence of these findings in dogs with aki, crf, or non-systemic illness, and ) to determine if these abnormalities are associated with shorter survival times. records of dogs with renal azotemia evaluated from / / to / / were reviewed; dogs which could be classified as having aki or crf and which had complete thoracic radiograph studies available for review were included. dogs with primary intracranial disease and normal serum creatinine and a complete thoracic radiograph study were selected as controls. signalment, weight, presence of pulmonary-related clinical signs, azotemia duration and severity at time of radiography, and leptospirosis antibody titer were noted. alveolar, bronchial, interstitial, or nodular lesions were described using a -point scale, and lung tissue collected at time of necropsy was reviewed; both the radiologist and pathologist were blinded to final diagnoses. significance was p o . for all analyses. the final study population included aki, crf, and control dogs. crf dogs were older (p o . ) than aki and control dogs. pulmonary-related clinical signs were more commonly diagnosed at first evaluation in aki dogs ( / dogs, . %) than in crf ( / , . %; p . ) or control dogs ( / , . %; p o . ). presence of an alveolar pattern was the only radiographic finding which differed amongst groups (more common in aki [n , . %, p . ] and crf [n , %, p . ] dogs than in control dogs [n , . %]). there was no association between presence of an alveolar pattern and any other variable. alveolar mineralization was the most common lesion in aki dogs ( / dogs; . %), with concurrent alveolar space concretions or mineralization of vessels or bronchioles noted in dog each. necropsies had not been performed in any of the crf dogs, but mineralization was not seen in lung tissues from any control dogs (n ). neither pulmonary-associated clinical signs nor alveolar pattern were associated with median number of days from discharge until death in dogs with aki (p . and . , respectively) or crf (p . and . , respectively). in this group of dogs, presence and type of radiographic pulmonary abnormalities were associated with renal azotemia but not with median time until death. the association between and clinical relevance of alveolar mineralization in aki dogs were not determined, but both the radiographic and histopathologic abnormalities reported here differ from up in people. chronic kidney disease (ckd) is a common cause of morbidity and mortality in cats. the purpose of this study was to investigate the effects of chinese rhubarb (rheum officinale) supplementation on the progression of feline ckd. cats with stable iris stage ii or iii ckd and without comorbidity were included in the study. cats were divided into treatment groups and administered rhubarb extract (group , rubenal s , vetoquinol, mg tablet po q h), benazepril as a positive control (group , . mg/kg po q h), or both (group ). cats were fed a commercial renal specific diet and enteric phosphate binder as appropriate. body weight, laboratory data, and blood pressure were recorded every months for up to months. variables between groups at enrollment and within groups over visits were compared with anova and repeated measures ano-va, respectively. a treatment by visit interaction term was included in all repeated measures models. significance was set at p . . except for body weight there was no significant differences between treatment groups at enrollment. there was no significant change in body weight, hematocrit (hct), upc, or creatinine over time as compared to baseline within any group. there was no significant difference between groups over time in regards to change in weight, hct, upc, or creatinine. the treatment by time interaction was non-significant in all models. although there was no benefit associated with combination treatment, the results for rhubarb treatment alone were not different from benazepril treatment. azodyl, an encapsulated, enteric-coated probiotic/prebiotic nutraceutical, is marketed for reduction of azotemia (bun & creatinine) in dogs and cats. cat owners often sprinkle contents onto cat food to facilitate administration. however, exposure to air and stomach acid are thought to inactivate the lyophilized bacteria within the product. therefore, we examined the ability of foodsprinkled azodyl to reduce azotemia in cats with ckd. cats with ckd were enrolled in the study and randomized receive azodyl or placebo. owners were provided with - capsules of azodyl prior to enrollment to ensure compliance with administration. baseline blood samples were obtained month apart, and then & months after beginning therapy. clinicians and owners were masked as to medication assignment. we hypothesized that a % decrease in bun and/or creat in the azodyl group would be significant, and set a . . in order to maximize the probability of detecting a difference, we determined the % change as being the difference between the maximal baseline analyte concentration and minimal therapeutic concentration. we compared the % change between groups by mann-whitney u test. bun and creatinine did not differ between groups. based on these results, azodyl, applied by sprinkling onto food fails to reduce azotemia in cats with ckd. whether intact capsule administration reduces azotemia in cats with ckd remains unknown. lower urinary tract disease (lutd) occurs commonly in cats, and idiopathic cystitis (fic) and urolithiasis account for over % of cases in cats less than years of age. although several strategies have been recommended, a common recommendation is to induce dilute urine resulting in more frequent urination and to dilute calculogenic constituents. in addition to conventional therapy using modified diets, traditional chinese and western herbs have been recommended, although only one, chorieto, has published data. we evaluated commonly used herbal treatments recommended for use in cats with lutd including ( ) san ren tang, ( ) wei ling tang, and ( ) alisma. we hypothesized that these chinese herbal preparations would induce increased urine volume and decreased urine saturation for calcium oxalate and struvite. six healthy, spayed female, adult cats were evaluated in a placebocontrolled, randomized, cross-over design study. cats were randomized to of treatments including placebo (p), san ren tang (srt), wei ling tang (wlt), or alisma (a). treatment was for weeks each with a week washout period between treatments. at end of each treatment period, a -hour urine sample was collected using modified litter boxes. urine volume and biochemistries were measured, and urine saturation for struvite and calcium oxalate was estimated using equil . b. analysis of variance (anova) was used to analyze data statistically if distributed normally and kruskal-wallis was used to analyze data statistically if data were not distributed normally. a p o . was considered significant. body weights were not different between treatments. no differences were found in -hour urinary analyte excretions, -hour urine volume, urine ph, or -hour urinary saturation for calcium oxalate or struvite between treatments (table) . urolithiasis is a multifactorial disease, frequent and recurrent in dogs in the worldwide, in which breed, sex, age, diet, some anatomical abnormalities, urinary tract infection, urine ph and some geographical and hereditary features in the populations studied have been implicated as risk factors. the effective long-term management of urolithiasis depends on identification and control of the pathophysiological mechanisms involved, which, in turn, depend on accurate knowledge of the mineral composition of the uroliths. the aim of this study was to determine for first occasion the main epidemiological data of canine urolithiasis in mexico. this study was developed with dogs with urolithiasis from of the states of the country. chemical composition of the uroliths was determined by stereoscopic microscopy, infrared spectroscopy, scanning electron microscopy and x-ray microanalysis. urolithiasis affected nearly the same number of males and females; with ages ranging from two months to years with a median age of years. adult animals were the most affected. breeds more affected were schnauzer miniature, poodle, dalmatian, yorkshire terrier, scottish terrier, chihuahua and bichon frisee´. uroliths were found in the lower urinary tract in . % of the cases. mineral composition of the uroliths was: struvite . %, followed by calcium oxalate . %, purines . %, silicate . %, others . %, mixed . % and compound uroliths . %. struvite uroliths affected females in most cases, whereas calcium oxalate, purines and silicate uroliths, were mainly observed in males. our results are similar to studies developed in other countries and continents, though we found a higher frequency of uroliths containing silicate, either pure, mixed or compounds uroliths ( . %); in mexico city the frequency reached %. this high frequency may be due to high consumption of silicate in home-made food or in the groundwater derived from aquifers. acknowledgments: this work has been partially supported by a project of waltham foundation in mexico and the consejo nacional de ciencia y tecnologı´a (conacyt) of mexico. voiding urohydropropulsion is a non-invasive method for removing small urocystoliths from the dog, most commonly used in females due to the relatively wider and shorter urethra. this procedure is typically performed under general anesthesia to allow complete relaxation of the urethra, however, anesthesia results in longer procedure times and difficult endotracheal tube stabilization due to the vertical positioning of animals, especially in larger dogs. the aim of this study was to devise a novel injectable sedation protocol for urohydropropulsion when cystoscopy was not concurrently required. an intravenous catheter was placed, and a combination of medetomidine ( to mg/kg iv) and hydromorphone ( . to . mg/kg iv) was administered, with the addition of ketamine ( mg/ kg iv) in fractious animals; atipamezole (double volume of medetomidine, administered im) was used as a reversal agent upon procedure completion. this protocol was considered in cardiovascularly healthy, non-diabetic dogs without evidence of urinary obstruction. monitoring equipment included electrocardiography, blood pressure measurement, and pulse oximetry, and supplemental flowby oxygen was provided. two dogs received the proposed sedation protocol in order to perform urohydropropulsion. dog one was a year old female spayed shih tzu cross, and dog was a year old female spayed standard poodle. ultrasonography revealed a moderate number of urocystoliths present in both dogs, measuring up to mm in dog and . mm in dog . urohydropropulsion was performed and resulted in retrieval of urocystoliths in dog , and approximately urocystoliths in dog . repeat ultrasonography revealed no uroliths present after urohydropropulsion in both dogs. the time from administration of sedation to administration of reversal agent was minutes for dog , and . minutes for dog . records were obtained from dogs that had traditional general anesthetic protocols for urohydropropulsion with cystoscopy for confirmation of urocystolith removal, performed within the last years, and the average anesthetic time was minutes. subsequent to the use of medetomine-based sedation protocols for the above dogs, cystoscopy was performed in a year old neutered male golden retriever with prostatomegaly. medetomidine ( ug/kg iv) and butorphanol ( . mg/kg iv) were administered; atipamezole (double volume of medetomidine, administered im) was used as a reversal agent upon procedure completion. this sedation allowed adequate immobilization for cystoscopy of the urethra and urinary bladder, and endoscopic biopsying of the prostatic urethra and urinary bladder. the time from administration of sedation to administration of reversal agent was minutes for this dog. in conclusion, a novel sedative protocol for urohydropropulsion is proposed which allows for an appropriate level of sedation along with a short procedure time and rapid recovery. this sedation protocol may also be useful for certain cystoscopic procedures. analysis may be delayed for a variety of reasons, including the need for sample batching within the laboratory or shipping to an outsourced location. therefore, it is important to know how storage of the sample may affect enzyme activity. we hypothesized that urinary nag and ggt activity would be affected differently in samples stored by refrigeration vs. freezing. thirty-four canine urine samples submitted to the clinical pathology laboratory at kansas state university were included. samples were collected from clinical patients with a variety of medical/surgical disorders and were selected based on the day of the week and a minimum volume of ml. a complete urinalysis was performed on each sample; however there were no exclusion criteria based on urinalysis results. nag and ggt activity in the urine supernatant was assessed by colorimetric assay. aliquots of each supernatant were refrigerated for days and frozen at À c for and days at which time enzyme activity was re-assessed. compared to baseline values, enzyme activity for both nag and ggt were stable after days of refrigeration, however there were significant (p o . ) declines in ggt and nag activity when urine supernatants were frozen for and days. treatment for canine urinary tract infections (uti) typically consists of - days of antimicrobial drugs in primary care veterinary practice. compliance with this drug regimen can be difficult for some clients. enrofloxacin is a veterinary approved fluoroquinolone antimicrobial and is useful for treatment of canine uti. fluoroquinolones are often used in human medicine to treat uncomplicated utis in women and can be prescribed for as little as days. the primary objective of this study was to determine if dogs with naturally occurring uncomplicated uti have equivalent microbiologic cure with a high dose short duration protocol of enrofloxacin, compared to a standard antimicrobial protocol. client-owned adult dogs with naturally occurring, uncomplicated uti were prospectively enrolled in a multi-center clinical trial and assigned to of groups in a randomized blinded manner. group received treatment with - mg/kg oral enrofloxacin once daily for consecutive days. group dogs were treated with . - mg/kg oral amoxicillin-clavulante twice daily for days. both groups had urinalyses and urine cultures submitted on day , , and . at the time of this interim analysis, thirty-six dogs have completed the trial. bacteriological cure was achieved in dogs ( %) treated with enrofloxacin and dogs ( %) treated with amoxicillinclavulante, respectively. these data suggest that the high-dose, short-duration enrofloxacin protocol was equally effective to the standard protocol in treating uncomplicated canine uti in the sample patient population. and may represent a viable alternative therapeutic regimen for similar patients. azotemia is frequent in dogs with dmvd (nicolle et al; jvim ; : - ) and could result from renal hemodynamic alterations. renal resistive index (ri) allows assessment of renal vascular resistance. the aim of this prospective study was to assess ri in dogs with different dmvd stages. fifty-five dogs with dvmd were used (isachc class (n ), (n ), and (n )). physical examination, renal ultrasonography and echo-doppler examinations were performed in awake dogs by trained observers. plasma creatinine, urea and nt-probnp were measured. statistical analyses were performed using a general linear model. whereas ri of renal and arcuate arteries were unaffected by isachc class, left interlobar ri increased (p o . ) from . ae . (mean ae sd) in class to . ae . in class . left interlobar ri was also higher (p o . ) in azotemic ( . ae . ) than in non azotemic ( . ae . ) dogs. similar findings were observed for right interlobar ri. a positive effect of nt-probnp (p . ), urea (p o . ), creatinine (p . ), urea-to-creatinine ratio (p o . ), left atrium-to-aorta ratio (p o . ), regurgitation fraction (p . ), systolic pulmonary arterial pressure (p o . ) and shortening fraction (p . ) on ri was also observed. in conclusion, interlobar ri increases with the severity of dmvd and azotemia. a cause-effect relationship remains however to be established. antibodies against alpha-enolase are associated with immunemediated nephritis in people. it was previously shown that vaccinated cats commonly develop antibodies against alpha-enolase. the purpose of this study was to assess for associations between alphaenolase antibodies and azotemia in privately-owned cats. clinically stable privately owned cats ! years of age, with and without azotemia (creatinine mg/dl), and with an available vaccine history for ! years were recruited for the study. sera were assayed for creatinine concentrations and alpha-enolase antibodies by use of previously validated techniques. results from cats with and without azotemia were compared by student's -tailed t test or fisher's exact test with significance defined as p o . . median ages were years (range: - ) and years (range: - ) for cats with (n ) and without azotemia (n ), respectively. there was no significant difference in vaccine events (number, type, or route of administration) between groups. azotemic cats ( . %) were more likely than normal cats ( . %) to be positive for antibodies against alpha-enolase (p . ). in addition, alpha-enolase antibody concentrations were greater (p . ) in azotemic cats (mean % elisa . %) than cats with normal creatinine concentrations (mean %elisa . %). results of this study suggest that antibodies against alpha-enolase in cats may be associated with renal disease. additional prospective evaluation in a larger number of cats is indicated. aki is used in human medicine as a predictor of mortality based on the akin (acute kidney injury network) scoring system which utilizes relative increases in creatinine to determine stage. with this scheme, mortality has been shown to increase as the stage of kidney injury (indicated by akin score) increases. accordingly, we hypothesized that this system would improve predicting prognosis in dogs and cats. we retrospectively evaluated dogs and cats ( ) ( ) ) that had ! creatinine measurements within days, and whose first creatinine was o . mg/dl. patients were categorized as: level (no aki); level (second creatinine value o . mg/dl, but creatinine increased ! . mg/dl); or level (second creatinine . mg/dl with a creatinine increase ! . mg/dl). thirty and day survival for each level was compared to level . adjusted odds ratio (or) in dogs for day survival was . for level (ci %, . - . ) and . (ci %, . - . ) for level ; or for day survival was . for level (ci %, . - . ) and . (ci %, . - . ) for level . for cats, or at days was . (ci %, . - . ) for level and . (ci %, . - . ) for level ; or for day survival was . (ci %, . - . ) for level and . (ci %, . - . ) for level . thus, detecting increasing stage of aki helps predict mortality in dogs and cats. abstract n/u- feline urate urolithiasis: cases ( - . j dear , r shiraki , a ruby , j westropp . william r pritchard veterinary medical teaching hospital, university of california, davis, ca, gerald v. ling urinary stone analysis laboratory, university of california, davis, ca and the department of veterinary medicine and epidemiology, university of california, davis, ca. feline urate urolithiasis accounts for % of the feline stones our laboratory analyzes each year; little information is known about this disease, particularly the incidence of those cats with hepatopathies. the objective of the study was to characterize the signalment, clinicopathologic data, and diagnostic imaging of cats with this disease as well as the salts of uric acid present. a retrospective analysis of feline urate uroliths submitted to the stone lab between january -december were included. from these data, primary veterinarians were solicited to submit records. furthermore, all records from cats with urate uroliths from the vmth were analyzed separately. records were received from the primary care veterinarians. sixteen cases were identified from the vmth. median values for the cbc and chemistry panels available were within the reference ranges provided, with only a few outliers present. of the cats with radiographic reports, ( %) had visible evidence of uroliths. two external cases had confirmed pss; five cases from the vmth had a pss. cats with urate uroliths and pss were younger than cats without a documented hepatopathy ( years vs. years). the siamese breed was overrepresented. all stones were ammonium hydrogen urate. the pathogensis of urate uroliths in cats is poorly understood. most cats were not completely evaluated for pss, however, there were few clinicopathologic parameters which indicated hepatopathies were present. further studies are warranted to evaluate genetics and purine metabolism in cats with urate uroliths to help tailor proper management and breeding strategies. -indoxyl and p-cresyl sulfate (is, and cs, respectively), small protein-bound molecules derived from gastrointestinal protein metabolism, are among the most important uremic solutes affecting morbidity and mortality in human chronic kidney disease (ckd). in the blood stream, these compounds are predominantly bound to protein, but their debilitating effects on prognosis and quality of life in ckd appear to be driven by the free fraction. the objectives of the present study were to assess the normal, physiological levels of is and cs in healthy cats and to evaluate the correlation of the respective free and protein-bound levels. blood samples were taken from clinically healthy adult cats enrolled at five participating veterinary practices in germany. after centrifugation, the serum was deep frozen until transport on dry ice to the analytical laboratory. serum creatinine and urea levels were quantified by vettest s (idexx laboratories, inc.). total and free is and cs, respectively, were quantified by turbulent flow chromatography coupled with a tandem mass spectrometry detector. statistical analysis of the results comprised i) a descriptive report of the median with upper and lower bounds of the % confidence interval for reference values of is and cs, ii) a calculation of various pearson correlation coefficients r, also tested with reference to the null hypothesis of no relationship, and iii) wilcoxon-mann-whitney utest for an estimation of the effect of hemolysis on serum is or cs levels. six animals with serum creatinine or urea levels outside the reference range were excluded from the calculation of reference values. median levels of is in cat serum were . mg/l with upper and lower bound % confidence intervals at . and . mg/l, respectively. the corresponding median levels of cs were . mg/l (median) and . vs . mg/l (upper vs lower bound levels, respectively). these values showed a low, non-significant correlation with serum creatinine or urea levels. however, is and cs serum levels were moderately correlated (total levels r . , p o ). their respective free levels constituted about % of the total serum levels (r ! . , p o . ). non-hemolytic samples tended to yield lower values than hemolytic samples. due to the low number of hemolytic samples (n ) , the group difference could, however, not be statistically confirmed. the results indicate that it is sufficient to determine total levels of either is or cs in serum while studying the effects of therapeutic or dietetic interventions on the evolution of these parameters in feline ckd. reference values are provided for orientation towards clinically relevant changes. disrupted urothelial differentiation has been implicated in the pathogenesis of feline idiopathic cystitis (fic). studies of cultured human urothelium have shown that abnormalities in urothelial differentiation and repair may be mediated by persistent -hydroxy-prostaglandin dehydrogenase (pgdh) activity and subsequent metabolism of cytoprotective prostaglandins. the goal of this study was to confirm persistent pgdh expression in fic bladders compared to desmoplakin i ii expression, a marker of urothelial differentiation. urinary bladder biopsy specimens were obtained by cystotomy from symptomatic cats with chronic fic. cats with a history of another major disease, previous cystotomy, or recent treatment with corticosteroids, nsaids, antihistamines, antidepressants, or glycosaminoglycans were excluded. urinary bladder tissue specimens were also obtained from untreated clinically normal specific-pathogen-free cats. tissue specimens were fixed in buffered % formalin and embedded in paraffin. tissue sections were deparaffinized and subjected to citrate buffer microwave antigen retrieval. tissues were stained for pgdh using a rabbit anti-pgdh antibody, an isotype negative control or goat anti-desmoplakin i ii and developed using the avidin-biotin peroxidase complex method. all fic ( / ) and normal ( / ) cat bladder samples showed similar staining of urothelial cytoplasm for pgdh. however, desmoplakin i ii staining, found on the luminal cell surface in / normal tissues, was disrupted in / fic bladder samples. desmoplakin i ii staining confirmed altered urothelial differentiation in fic cats. however, pgdh expression remained intact in fic samples. we hypothesize that pgdh expression in fic may contribute to its pathophysiology due to breakdown of prostaglandins essential for urothelial healing. additional studies will explore this hypothesis. the university of tennessee college of veterinary medicine's picture archiving and communication system was searched over a month period for cats that had undergone both abdominal radiographs and ultrasound during the same visit. one hundred and three cats were identified (age range o to yrs; median yrs). kidney size was determined based on radiographic and ultrasound findings. of the included cats, . % had two normal sized kidneys, . % had one small and one normal, . % had one large and one normal, . % had two small, . % had two large, and . % had one small and one large kidney. the presence of mineralization, uroliths and hydronephrosis was also noted. medical records were reviewed for clinical chemistry data and historical information concerning previous urinary disease. no significant differences were found between kidney size and renal function, kidney size and the presence of uroliths, renal mineralization and function or the presence of uroliths and function. the presence of uroliths was significantly associated with hydronephrosis. of the cats with at least one large kidney, ( %) had hydronephrosis. of the cats with current or previously diagnosed uroliths, urinary tract infections or other uropathies, ( . %) had at least one small kidney. small kidneys were commonly found in older cats, however, this correlation was not statistically significant. based on these findings, small kidneys are more likely to be the result of urinary disease as opposed to being either congenital or due to aging. this study aimed to evaluate ife, which has been advocated for treatment of lipid-soluble drug intoxication, in the treatment of clinically-occurring canine ivermectin toxicosis. one australian shepherd and two miniature australian shepherds were included. all three dogs were homozygous for the mdr- gene mutation. two dogs roamed on horse ranches where ivermectin-based deworming products had recently been used. ivermectin was administered to the third dog ( mg/kg po). all three dogs exhibited tremors, ptyalism, and cns depression, which progressed over several hours to stupor in two dogs, and to a comatose state requiring mechanical ventilation in the remaining dog. a % formulation of ife (liposyn ii, hospira) was administered as a bolus ( . ml/kg) followed by a slow iv infusion ( . - ml/kg over minutes). no change was observed in the neurologic status of any patient. lipemia visible upon blood sampling persisted for hours in one dog. no other adverse effects were noted. serum ivermectin levels confirmed ivermectin exposure in each case. in this study, ife administration did not result in clinical benefit in cases of ivermectin toxicosis. brain ivermectin concentrations in mdr mutant/mutant genotype dogs may be too high to be overcome by ife. additionally, these dogs may lack p-glycoprotein-mediated biliary clearance mechanisms needed for optimal ife function. further investigation is needed to determine the utility and optimal dosing of ife in canine toxicoses, to characterize its safety, and to determine how mdr- status may alter the efficacy of ife in treatment of canine ivermectin intoxication. rufinamide is a recently approved antiepileptic drug used for the treatment of seizure disorders in human patients. rufinamide is administered at a dose of mg/kg divided twice daily to achieve therapeutic concentrations of mg/ml. the objective of this study was to determine the pharmacokinetic properties and short-term adverse effects of single-dose oral rufinamide in healthy dogs in preparation for a possible clinical trial evaluating the efficacy of rufinamide in the treatment of canine epilepsy. six healthy adult dogs were included. the pharmacokinetics of rufinamide were calculated following administration of a single mean oral dose of . mg/kg (range . - . mg/kg), extrapolated from the dose used in human patients. dogs were monitored by repeat physical examinations, electrocardiograms and blood pressure assessments during the course of the study. plasma rufinamide concentrations were determined using high-performance liquid chromatography. pharmacokinetic data were analyzed using winnonlin version . . no adverse effects were observed. the mean terminal half-life was . /À . hours. the mean maximum plasma concentration was . /À . mg/ml and the mean time to maximum plasma concentration was . /À . hours. mean clearance was . /À . l/hr. auc inf was . /À . mgÃh/ml. results of this study suggest that rufinamide given orally at mg/ kg twice daily in healthy dogs should result in a plasma concentration and half-life sufficient to achieve the therapeutic level extrapolated from humans without short-term adverse effects. further investigation into the efficacy and long-term safety of rufinamide in the treatment of canine epilepsy is warranted. the aims of this study were to investigate the abg for (i) the prevalence of skull abnormalities; (ii) the prevalence of sm; (iii) an association between lateral ventricular size, cerebellar size and sm; and (iv) associations between sm, skull abnormalities, csf pleocytosis and clinical signs. seventy-six abgs, recruited as part of a larger epidemiological and genetic study, underwent brain and spinal mri evaluation ( . t general electric signa hdx, milwaukee, wi). all dogs were evaluated neurologically, recording deficits and the presence of spinal pain. sequences acquired included t w, t w pre-and postcontrast, and t w flair, sagittal and transverse. cervical spinal cord central canal (cc) and or syrinx size and its percent area of spinal cord was measured using osirix s . the presence of chari-like malformation (cm) was assessed by recording the presence of caudal cerebellar deviation and/or foramenal vermal herniation. lateral ventricle and cerebellar volume was expressed as a percent of the cerebrum and intracranial volume qa respectively. forty-five dogs underwent atlanto-occipital cerebrospinal fluid tap at the time of mri and the white blood cell (wbc) count was recorded. student's t-tests were used to compare the measured variables between groups with and without skull abnormalities, spinal pain and neurological signs. the mean age of the males ( intact) and the females ( intact) was . months (range - ; median months). neurological deficits and neck pain were noted in ( %) and ( . %) of dogs respectively; dogs ( . %) exhibited both. cerebellar deviation and vermal herniation were present in ( . %) and ( . %) dogs respectively; twenty-three dogs ( . %) had both. mean height of the cc was . mm ( - . mm). forty ( . %) ccs were greater than mm in height; the mean length of these lesions was . vertebrae ( . - ). mean csf wbc count was . /ml ( - ). syrinx height and extent were significantly higher in dogs with neurological signs (size p . ; extent p . ). there were no significant differences in syrinx sizes and extent in dogs with or without skull abnormalities or spinal pain. there were no associations of syrinx height or extent with csf wbc count or age of dog. intact females had a significantly lower syrinx extent than intact males (p . ). there were no significant differences in presence of spinal pain or neurological signs between dogs with or without skull abnormalities. there was a significant negative association of ventricular percentage and cerebellar percentage (p o . ). there was a significant association of ventricular percentage with syrinx percentage (p . ) and height (p . ). this study suggests that sm and cm are prevalent in abgs. syrinx size and extent are associated with neurological signs and ventriculomegaly is associated with both small cerebellar size and large syrinx size. however, sm may not be associated with cm as defined by cerebellar herniation and deviation and is not associated with csf inflammation. the power tissue resection device (ptrd) is a hand-piece comprised of an outer cannula with motor driven vacuum-assisted inner cutting blade. this device was designed and is marketed for human neurosurgical brain/spinal cord tumor resection. the purpose of this study is to describe the use of the ptrd for intervertebral disc fenestration and to compare the effectiveness of manual fenestration to that of the ptrd. fifteen cadaveric lumbar spines were randomly placed into three study groups: group was the control group on which no fenestrations were performed, group was the manual fenestration group and group was the ptrd fenestration group. the effectiveness of fenestration via both manual and ptrd was assessed by calculating the ratio of remaining nuclear weight post fenestration to total nuclear volume. discs with lower ratios were more effectively fenestrated. results showed a smaller ratio of post fenestration remaining nuclear weight to nuclear volume following fenestration with the ptrd ( . ae . ) as compared to manual fenestration ( . ae . ). these results did not show statistical significance. when fenestrated samples were compared to control samples ( . ae . ), there was a statistically significant reduction in ratios. in conclusion, the ptrd is easy to use and is as effective as the manual technique for canine intervertebral disc fenestration. according to the human who classification gliomatosis cerebri (gc) is a rare astrocytic tumor affecting at least three lobes of the brain with extensive infiltration, but relative preservation of brain architecture. gc has not been reported to occur as a hereditary disease, neither in man nor in animals. here, we report the temporally clustered occurrence of gc in a family of bearded collies. a years old female bearded collie with forebrain signs was presented. differentials included inflammatory/ infectious, metabolic/ toxic, and neoplastic diseases. within a time period of months, offspring of this bitch were presented with similar clinical signs. two dogs were full siblings ( males). the remaining female dog originated from a match with a different male dog. mri was performed in all dogs and revealed a diffuse and extensive intra-axial lesion with moderate mass effect and midline shift. the ill defined lesion showed mainly a white matter distribution with hyperintense signal in t -w and flair images and iso-to hypointense signal in t -w images without contrast enhancement. the lesion was bilateral in all cases, continued along the white matter extending partially into the gray matter with contact to the brain surface. neuropathology revealed a diffuse and extensive infiltration of the brain and spinal cord by a neoplastic glial cell population involving white and gray matter of both hemispheres, thalamus, brainstem and cerebellum in all dogs. based on the cell morphology and immunoexpression of glial fibrillary astrocytic protein by neoplastic cells diagnosis of gc was made. this is the first report of familial occurrence of gc, which is likely the result of a germ-line mutation. several human hereditary cancer syndromes are associated with cns tumors including amongst others the li-fraumeni cancer family syndrome (p mutation), neurofibromatosis (type and ) (neurofibromin, merlin mutation), and tuberous sclerosis (hamartin, tuberin mutation). furthermore, familial clustering of human gliomas unassociated to the known inherited cancer syndromes has been described. in the dog, hereditary cns tumors are not known. the exact mode of inheritance and putative gene mutations of gc in this bearded collie family are currently under investigation. preliminary results are consistent with a monogenic autosomal dominant mode of inheritance, although a recessive inheritance cannot be completely ruled out at this time. mutations in the tp gene were not found following amplification and sequencing of exons - in affected dogs. previously presented at the ecvn annual meeting in cambridge, uk. the gm gangliosidoses are characterized by a deficiency of bhexosaminidase. there are two isoforms: hex a composed of an a and b subunit encoded by hexa and hexb genes respectively and hex b with two b subunits. hex a requires an activator encoded by gm a. two japanese chin dogs with confirmed gm gangliosidosis showed elevated total hexosaminidase and normal hexosaminidase a activity, a pattern associated with the ab variant in humans and consistent with prior reports in the breed. this study was performed to identify the mutation responsible using resequencing with an applied biosystems xl dna analyzer as previously described (awano ). mutations in gm a cause the ab variant in humans, but resequencing gm a revealed no mutation that could account for the disease. resequencing hexa and hexb revealed a c. g a mutation in hexa which was homozygous in both affected dogs. sixty-five normal japanese chin dogs were screened for the mutant allele; were homozygous for the ancestral allele and heterozygous. this mutation predicts a p. e k substitution affecting one of two primary active-site amino acids that participate in the hydrolysis of gm ganglioside. substitution of a lysine residue at this site is likely to eliminate subunit a enzymatic activity. the apparently normal levels of hexosaminidase a activity in affected dog samples may be a result of b subunit overexpression. human hex b possesses low levels activity against the artificial substrate used to assess hex a activity, but specificity of activity of the canine enzyme is not known. previously presented at the american society for neurochemistry: additional data in this abstract. phenytoin (pht) is the intravenous drug of choice in humans for seizure emergencies following benzodiazapines. iv fosphenytoin (fos) is a pht pro-drug which causes less administration related adverse events. while the short half-life of pht is not suitable for chronic oral therapy in dogs, iv fos has not been studied. two dogs received mg/kg phenytoin equivalent (pe) and two dogs received mg/kg pe of fosphenytoin intravenously at a rate of mg pe/min. blood for plasma levels were drawn at time-points over hours; total and unbound drug levels were measured by hplc. vital signs including ekg, blood pressure, and neurological examination were monitored. the half-life of metabolism of fos to pht was $ min, with % of fos metabolized to pht by minutes. eighty to % of pht was protein-bound during the first minutes after dosing, compared to - % in humans. the elimination half-life for total pht ranged from . - . hours and for unbound pht ranged from . - . hours. dogs receiving mg/kg pe intravenously achieved unbound pht plasma maximum concentrations of . - . ug/ml at minutes, consistent with human loading dose levels. adverse events observed in some dogs included vomiting, mild ataxia, and short lived tremors, the severity of which appeared dose dependent. all dogs were clinically normal within minutes of all doses. a mg/kg pe dose of iv fos appears adequate for production of pht levels predicted to be effective for the treatment of canine seizure emergencies. further studies in clinical canine patients are warranted. acquired myasthenia gravis (mg) is caused by antibodymediated inactivation of the acetylcholine receptor on the neuromuscular endplate causing focal, regional or generalized muscle weakness. many medical treatments have been reported; however, responses to therapy and outcomes are unpredictable and death often results from aspiration pneumonia. therapeutic apheresis is an extracorporeal procedure that separates blood into its components for removal or specific alteration prior to return to the patient. therapeutic plasma exchange (tpe) is an apheresis treatment in which plasma (containing pathologic antibodies) is removed and exchanged with donor plasma. tpe is used routinely to treat mg in human patients with severe disease or disease unresponsive to conventional therapy. we report the successful use of tpe to treat large breed dogs with confirmed mg (aceytlcholine receptor antibody concentration: . and . nm/l, respectively; normal concentration: o . nm/ l) that was severe and not adequately responsive to traditional therapies. both dogs were non-ambulatory, recumbent, and demonstrated megaesophagus and aspiration pneumonia. three tpe treatments ( plasma exchange each) were performed over and days, respectively, in each dog without complication. both dogs became ambulatory within days of starting tpe treatment with subsequent resolution of regurgitation and megaesophagus. pyridostigmine was continued during tpe sessions and discontinued in both dogs within - months. both dogs remain asymptomatic and have had no recurrence of mg during and months of follow-up, respectively. tpe is a viable treatment option for dogs with mg that have severe disease, life-threatening complications or that remain unresponsive to traditional therapies. tpe may alleviate clinical signs more rapidly, and improve long-term outcomes when compared to historical experiences in patients with comparable disease. clinical findings, clinicopathologic data, imaging features, and treatment of canine spinal meningiomas have been described in the veterinary literature, but histological characteristics and tumor grading have less commonly been reported. the aims of this retrospective case series were to describe the clinical, imaging, and histologic features of seven canine spinal meningiomas including a cervical spinal cystic meningioma that had imaging and intraoperative features of a subarachnoid cyst. medical records from dogs with a histopathological diagnosis of spinal cord meningioma presented to the veterinary teaching hospital between and were reviewed. signalment, presenting clinical signs, physical and neurologic examination, clinicopathologic data, surgery reports and available images were reviewed. all meningiomas were histologically classified and graded following the international who human classification for cns tumors. seven dogs were included, males and females. median age at presentation was . years (range, . - . years), and median weight was kg (range, - kg) . median time between onset of clinical signs and diagnosis was days (range, days - year). cerebrospinal fluid (csf) analysis was performed in dogs, showing increased protein concentration in cases, and being normal in the other . spinal radiographs revealed vertebral canal widening in one case. myelography ( / ) showed intradural/extramedullary lesions in three cases, one of them consistent with a csf-filled subarachnoid cavity, and an extradural lesion in one case. magnetic resonance imaging (mri) was performed in all cases and revealed mild to marked hyperintensity on t w and precontrast t w images and homogeneous contrast enhancing (ce) intradural/extramedullary masses ( cervical and thoracic) in six cases, with one of these showing an additional intramedullary ce pattern. a dural tail was identified in two dogs. one dog had a fluid-filled subarachnoid enlargement located dorsally to the spinal cord. this lesion was hyperintense on t w, hypointense on t w and flair images, and did not enhance. it was diagnosed as a spinal subarachnoid cyst, but the histopathological study of the surgically resected mass revealed a grade i cystic meningioma. five other cases underwent cytoreductive surgery, two transitional meningiomas (grade i) that survived (alive at the time of writing) and months; and three anaplastic meningiomas (grade iii) that survived - . months before neurological deterioration and euthanasia. another anaplastic meningioma was euthanized right after diagnosis. there are few reports grading canine spinal meningiomas, with most being grade i or ii. of the few grade iii tumors reported, only one had been treated surgically and was euthanized days later because of neurological deterioration. we report four grade iii (anaplastic) meningiomas, three of which surgically treated and with longer survival times. finally, cystic meningioma should be considered in the differential diagnosis of cases with imaging features consistent with arachnoid cyst because of their similar appearance, making histopathological analysis essential for a definitive diagnosis. head trauma is a common veterinary emergency, but few prognostic indicators have been studied in dogs, making it challenging for clinicians to counsel clients about the odds of recovery. a recent meta-analysis showed that higher plasma glucose, lower plasma ph and lower hemoglobin at admission were associated with increased risk of death in human head trauma. the goal of this retrospective study was to investigate the association between admission point of care blood gas parameters and survival to discharge in dogs with head trauma. fifty one dogs presenting to the cornell university hospital for animals with head trauma from to that had a blood gas analysis done within hour of presentation were eligible for inclusion. parameters assessed included glucose, base excess (be), anion gap (ag), ph, hemoglobin, and sodium. biochemical data were found to be normally distributed using the kolmogorov-smirnov test. t-tests or welch tests were used to compare parameters between survivors (s,n ) and non-survivors (ns, n ). of glucose, be, ag, ph, hemoglobin, and sodium, only mean glucose (s mg/dl, ns . mg/dl, p . ) was significantly different between groups, although there was a trend for a difference in mean be (s À . , ns À , , p . ). logistic regression analysis showed that of the parameters, only be was independently associated with outcome (odds ratio . , % ci . - . , p . ). these results suggest that two easily measured biochemical parameters (glucose and be) may yield useful prognostic information in dogs with head trauma, but further studies are needed to further elucidate these findings. type i intervertebral disc disease (ivdd) commonly affects chondrodystrophic dogs. neurological recovery and outcome following surgical decompression may be unpredictable due to suspected ischemic neuronal injury. hyperlactatemia has been associated with spinal cord injury in humans and experimental animals. the purpose of the study was ) to determine the relationship between serum and csf lactate levels and ) to compare lactate levels with neurological outcome following decompressive surgery in dogs with ivdd. healthy, chondrodystrophic dogs diagnosed with ivdd localized to the t -l spinal cord were included. serum lactate levels were obtained at: anaesthetic induction, skin incision, muscle dissection, and extubation. in patients with hyperlacatemia at extubation, additional samples were obtained. csf was analyzed for lactate concentration. neurological status was recorded at presentation and multiple times during the recovery period. dogs were included in the study ( - years old). / dogs had normal lactate levels throughout the study. / dogs had serum hyperlactatemia prior to anaesthetic induction; / dogs returned to normal during anaesthesia and / dogs had continued hyperlactatemia until the end of the observation period. neurological status of the dogs varied similarly between all groups. in / dogs where csf lactate levels were measured, initial serum levels were lower than csf lactate levels; in / dogs where csf and serum were collected simultaneously, serum lactate concentration was consistently lower than csf lactate. no association between presenting neurological status or neurological outcome and serum or csf lactate concentration was made. neither serum nor csf lactate concentration is useful for predicting neurological outcome in dogs with ivdd. chiari-like malformation (cm) has been associated with syringomyelia (sm) in cavalier king charles spaniel (ckcs) and is postulated to result from a mismatch between the volume of the caudal cranial fossa and the brain parenchyma contained within. the objective of this study was to assess the role of cerebellar volume in caudal cranial fossa overcrowding and syringomyelia. three dimensional models were created using t -weighted transverse magnetic resonance images in the commercial software package mimics s . volumes of cerebellar parenchyma were analyzed as percentages of caudal cranial fossa volume (cerebellar caudal cranial fossa percentage) and total brain parenchyma volume (cerebellar brain percentage). data was assessed for normality and the appropriate statistical test was used to compare means/medians between groups. forty-five small breed dogs (sb), ckcs and labradors (ld) were compared. as sm is thought to be a late onset disease process, two subgroups were formed for comparison: ckcs younger than years with sm (group ) and ckcs older than years without sm (group ). ckcs had a larger cerebellar caudal cranial fossa percentage than the other groups . %] vs. sb . % [ . - . %] and ld . % [ . - . %]; p o . ). the cerebellar brain percentage was also larger in ckcs compared to the other groups (ckcs . % [ . - . %] vs. sb . % [ . - . %] and ld . % [ . - . %]; p o . ). group had a significantly larger cerebellar caudal cranial fossa percentage than group ( . % ae . vs. . % ae . , p . ) and a significantly larger cerebellar brain percentage ( . % ae . vs. . % ae . , p . ). our findings show that the ckcs has a relatively larger cerebellum than small breed dogs and labradors and there is an association between increased cerebellar volume and sm in ckcs. chiari-like malformation (cm) is nearly omnipresent in the cavalier king charles spaniel (ckcs) breed. the mis-match of the caudal cranial fossa and the parenchyma within is thought to lead to syringomyelia (sm). there is currently a lack of information if the morphological changes seen in ckcs with cm are progressive or non-progressive. in this retrospective study we used established measurements of cerebral volumes, foramen magnum height and cerebellar herniation length to assess if there is a significant difference between subsequent magnetic resonance (mr) imaging of the brain of the same dog. electronic patient records were reviewed for ckcs with cm which had two separate mri scans, which were a minimum of months apart. ckcs with diseases affecting measurements were excluded. for the volumetric measurements three-dimensional models were created using t -weighted transverse mr images in the medical imaging software (mimics v . , materialise n.v, ) . volumes of the caudal cranial fossa parenchyma were analyzed as percentages of caudal cranial fossa volume and caudal cranial fossa volume was analyzed as a percentage of total cranial cavity volume. the volume of the ventricular system was recorded as a percentage of total parenchymal volume. data was assessed for normality and the appropriate statistical test was used to compare means/medians. twelve ckcs were included with a median scan interval of . months ( - months). the size of the foramen magnum increased significantly between the first and second scan ( . ae . cm vs. . ae . cm; p . ), as did the length of cerebellar herniation ( . ae . cm vs. . ae . cm; p . ) and the caudal cranial fossa percentage ( . % [ . - . %] vs. . % [ . - . %]; p . ). there was no significant difference noted between the two time points in any of the other volumetric measurements ( this work could suggest that overcrowding of the caudal cranial fossa in conjunction with the movements of cerebrospinal fluid and cerebellar tissue secondary to pulse pressures created during the cardiac cycle causes pressures on the occipital bone. this leads to a resorption of the bone and therefore an increase in caudal cranial fossa and foramen magnum size allowing cerebellar herniation length to increase. the cord dorsum potential (cdp) is a stationary potential arising in dorsal horn interneurons after stimulation of sensory nerves. cdps have been recorded in normal anesthetized dogs previously, and normal latency values have been determined for tibial and radial nerves. this study was undertaken to determine whether cdps could be reliably recorded from the caudal nerves in normal dogs, thus allowing electrophysiological assessment of the cauda equina, and whether neuromuscular blockade improved recording quality. ten adult dogs weighing from . to . kg were anesthetized and cord dorsum recordings were compared before and after administration of atracurium. recording needles were placed onto the dorsal lamina at intervertebral sites from l /s to l / . stimulations were made on the lateral aspect of the caudal vertebrae approximately - cm from the tail base. recordings from stimulations were averaged. cdps were recorded successfully in all dogs. onset latency varied from . to . ms. the cdp was largest when recorded closest to the site of entry of the stimulated nerve into the cord, as determined by post-mortem examination immediately after testing in dogs. administration of atracurium did decrease muscle artifact, and in some cases helped isolate the origin of the cdp. these data show that cdps can be readily assessed from the caudal nerves of anesthetized dogs, with or without atracurium. cord dorsum potentials from caudal nerves may add important information about the integrity of the cauda equina in dogs with suspected degenerative lumbosacral stenosis. canine intracranial glial tumors and many human brain tumors express heat shock proteins (hsps) associated with their degree of malignancy. the up-regulation of hsps during tumor cell growth helps keep tumor proteins stable and therefore makes them a reasonable target for therapy. ki expression and ec have been strong indicators of cell proliferation and dedifferentiation, respectively.the aims of this study were to determine (i) if canine meningiomas express hsp and/or hsp ; (ii) whether the expression of the hsps was associated with ki and/or e-cadherin (ec) expression; and (iii) whether peritumoral edema was associated with hsp, ki and/or ec expression. forty-one formalin-fixed, paraffin-embedded canine intracranial meningiomas underwent immunohistochemical staining using anti-hsp , or antibodies. these tumor samples were also immunohistochemically stained for ki and ec expression. canine mammary carcinoma and squamous cell carcinoma tissues served as the control samples, as both have previously been shown to express hsps. skin was used as control for ki and ec. four non-overlapping high power fields of each stained sample were selected and cell staining was analyzed using a semi-quantitative method for hsps and ki ; a qualitative assessment was used for ec. all analyses were performed using sas v . (cary, nc). descriptive statistics of staining percentages were calculated for all tumors tested. simple pearson's correlation was used to test for correlations of ec area with hsp areas and ki- percent positive cells and of ec intensity with hsp intensities and ki- percent positive cells. all hypothesis tests were sided and the significance level was a . . thirteen meningiomas had mr images quantitatively evaluated for peritumoral edema using t flair sequences. the edema index (ei) was evaluated for an association with hsp , hsp , ec and ki expression. hsp was expressed in % (mean . % of cells; range - %), hsp in % (mean . % of cells; range - %) and ec in % of meningiomas. there was no association demonstrated between either hsp expression variable and ec or ki- expression. there was also no association between the ec expression variables and ki- . however, there was a significant negative association between hsp extent (p . ) and area (p . ) with ei. in conclusion, hsp and expression was demonstrated in canine intracranial meningiomas but was not associated with ki- or ec expression. this study suggests that hsps may not have a significant role in the maintenance of canine meningiomas and so do not represent a novel treatment target for this group of tumors unlike canine glial cell tumors. however, hsp may be involved in the pathogenesis of peritumoral edema in meningiomas and warrants further investigation. an extended release (xr) formulation of levetiracetam, a second generation antiepileptic drug, was recently approved for human use on a once daily basis. although levetiracetam is clinically effective for seizure control in dogs, it requires a three times daily administration. the potential benefits of the xr formulation include reduced daily dosing leading to improved compliance and relatively constant plasma concentrations. the aim of this study was to compare the pharmacokinetics of levetiracetam xr tablets with immediate release (ir) tablets following single dosing in dogs. five clinically and neurologically normal mixed breed dogs were used in a cross-over design. all dogs (mean body weight . kg; range . - . ) had normal hematology, serum chemistry and urinalyses. following a hour fast, each dog was administered oral ir levetiracetam ( mg; mean dose . mg/kg; range . - . ). heparinized blood for drug analysis was taken from each dog prior to administration and . , . , . , , , and hours after. blood was immediately centrifuged and supernatant plasma was stored at À c until analysis. after a day wash-out period, each dog was administered mg oral xr levetiracetam and blood samples were taken at identical timings. plasma samples were thawed at room temperature before preparation by solid phase extraction for hplc analysis. reverse phase chromatographic separation was performed. levetiracetam and an internal standard were detected using ultraviolet spectroscopy at nm. concentrations of levetiracetam were determined by peak area comparison to the internal standard. mean data were fit to a one compartment pharmacokinetic model with first order elimination and absorption and included a lag-phase for xr formulation. no adverse clinical effects were noted in any of the dogs. the auc associated with xr was hr ug/ml, a . fold increase over that with ir ( . hr ug/ml). the absorption half-life was . hr with xr and . hr with ir, a . fold difference. the elimination halflife was . hr with xr and . hr with ir, a . fold difference. the tmax associated with xr . hr and . hr with ir, a . fold difference. the cmax associated with xr was . mg/ml and . mg/ml with ir, a . fold difference. the plasma concentration of ir levetiracetam was not detectable at hr after administration whereas it was greater than mg/ml at hr after xr administration. based on the auc data, there is an approximately fold increase in bioavailability of the xr compared to the ir formulation. the cmax was approximately times greater following xr administration and a high plasma level in excess of the suggested canine therapeutic concentration ( mg/ml) for at least hours. although specific dosing recommendations cannot be made from this data, the favorable pharmacokinetics of xr over ir suggests that single, daily administration could be efficacious. thoracic and lumbar vertebrae are frequently affected by fractures and or luxations in dogs following trauma. surgical repair is part of the emergency treatment described for this disorder but does not guarantee improvement of the associated clinical signs. multiple surgical repair techniques have been described but have not been compared in terms of their success and the factors associated with a positive outcome. the aims of this study were to retrospectively evaluate the effect of different types of vertebral repair, injury type and injury location on outcome in dogs with thoracolumbar (tl) and lumbosacral (ls) spinal trauma. medical records were searched for dogs with radiographic evidence of a tl or ls vertebral fracture and or luxation ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ; signalment, body weight and duration of disease were recorded. dogs were retrospectively scored neurologically ( - ; normal to plegic with absent pain perception) on admission and at re-evaluation following surgery. lesion location was classed as t -l and l -s ; dogs were evaluated as one group and as two separate groups with respect to outcome. a subset of lesions were classed as cord compression or not based on advanced imaging. three repair techniques were evaluated (i) pins and polymethylmethacrylate (pmma); (ii) screws and pmma; and (iii) spinal stapling. regression analysis was applied to test for an association between the type of surgery and a successful outcome (non-painful and ambulatory). simple bivariate analyses were performed to investigate for variables predictive of a successful outcome. fifty-nine dogs were included. twenty-eight dogs were classed as t -l and were l -s . there were dogs with fractures and with luxations; dogs had both. thirty-one of dogs evaluated had spinal cord compression. ten dogs were repaired with im pins and pmma, dogs with screws and pmma and dogs with spinal stapling. overall, there was a . % success rate; there was no significant difference in outcome between the anatomic sites (p . ). all dogs initially graded as - pre-operation were classed as a successful outcome after at least one week following surgery; % of dogs initially graded as (plegic with pain perception) were classed as successful recovery. one dog ( . %) initially as graded as (plegic with no pain perception) had a successful outcome. a low admission score was statistically predictive of a successful outcome (p o . ). surgery type was not associated with a successful recovery (p . ). signalment, body weight, location of injury, injury type (fracture, luxation or both), presence of compression, and duration of disease did not predict outcome. from this study, the successful recovery of dogs following surgical fixation is high and is only dependent on the neurological score at the time of admission. the choice of surgical technique does not seem to influence outcome although a prospective study comparing two surgery types is warranted to further investigate this issue the results of which can be confounded by surgeon experience and variable follow-up. cranial thoracic intervertebral disc disease (ivdd) is extremely rare due to the presence of the intercapital ligament, although anecdotic data suggest german shepherd dogs (gsd) can share some predisposition for this disorder. the objective of the study was to retrospectively evaluate through mri if cranial thoracic ivdd is significantly more common in gsd compare to other large breed dogs. a search was done through database of the ontario veterinary college. any gsd were a spinal mri including t -t spine was performed was recruited. a group of large-breed non-gsd was used as a control. in the midsaggital t wi plane, three variables were assessed and graded for each intervertebral disc space t -t : spinal cord compression (scc), disc degeneration (dd), and herniation. wilcoxon sign rank test was used to assess if scores were different between groups. exact conditional logistic regression was used to determine whether any intervertebral disc space was a risk factor. gsd and large breed non-gsds were recruited. the gsd group had significantly higher scores than the non-gsd for scc, and herniation. regarding the individual intervertebral discs, in the gsd group t - , t - , t - discs had significantly an increased risk for scc, and t - for herniation. the results of this study show that gsd have a higher risk of cranial thoracic disc ivdd than other large breed dogs. that risk was higher in discs t -t , t - , and t - , particularly in t - . genetic and/or conformational factors, such as weakness of the intercapital ligament, may predispose gsd to this lesion. diskospondylitis is a common disease of the canine spine; however, few reports of mr imaging findings in dogs are available. the purpose of this study was to describe the signalment, clinical and mr imaging features in affected dogs. twenty-three dogs with a diagnosis of diskospondylitis based on clinical signs, mr imaging, and urine, blood, csf and/or intervertebral disk cultures were included. large breed dogs ( kg) accounted for of the cases. the mean age was . years with males and females equally represented. most dogs ( / ) were ambulatory with varying degrees of pain and paresis. mr imaging characteristics of sites were reviewed. on t w images, vertebral endplates were of mixed signal intensity ( / ) while the vertebral body was hypointense ( / ). the intervertebral disk space was hyperintense on t w ( / ) and stir ( / ) images and mixed signal intensity ( / ) on t w images. paravertebral soft tissue hyperintensities were noted on / t w and / stir images. contrast enhancement occurred at / endplates and / intervertebral disk spaces. only / vertebral bodies and / parvertebral soft tissues contrast enhanced. intramedullary spinal cord t w hyperintensity was noted at / sites. spinal cord or cauda equina compression occurred at / sites. based on the spearman correlation coefficient, a significant direct correlation was found between the degree of spinal cord or cauda equina compression and the patient's neurologic status (p . ). the incidence and severity of spinal cord compression in canine diskospondylitis may have prognostic value and may have been previously underestimated using other imaging modalities. hemilaminectomy and pediculectomy are both well described and commonly utilized techniques to access the spinal canal. these procedures are most often performed to approach a compressive lesion, such as intervertebral disc disease and neoplasia, the goal being adequate visualization of the spinal canal and access to the offending lesion. a proposed benefit of pediculectomy is preservation of the articular facets and thus better maintaining stability of the vertebral column, but at the cost of reduced access to the spinal canal. the purpose of this study was to describe standardized anatomical limits of each technique and report any observed differences that could be considered during presurgical planning. ten canine cadavers had both procedures performed on opposite sides to access the t - , t -l , and l - spinal canal. measurements were obtained after performing a computed tomography study of the spine and recorded from the transverse slice most representative of the defect. the surgical technique, vertebral site, and side of vertebral column were compared with the mean spinal canal and defect height using a covariate model. dorsal and ventral remnant lamina heights were also compared. the height of the defect relative to the spinal canal was - % with hemilaminectomy and - % with pediculectomy. the observed difference in defect height of - % (p o . ) and varied with spinal canal height. dorsal remnant lamina height was . - . % of spinal canal height with hemilaminectomy and - % with pediculectomy. ventral remnant lamina height ranged from - % and . - . %, respectively, though the difference was not statistically significant. while a larger defect is expected with a hemilaminectomy procedure, our results demonstrate that this difference increases with increasing spinal canal height. interestingly, the proportion of exposed spinal canal decreases with increasing canal height for both procedures. the difference in defect height between techniques was due to greater removal of the dorsal spinal canal, possibly making the hemilaminectomy technique better suited for more dorsal lesions, while no statistically difference in access to the ventral canal is observed. no effect of vertebral site was detected. of note was the involvement of articular facets in half of the pediculectomy defects, involving an average of % of the articular facet height. this result questions the suggested benefit for the vertebral stability, but further biomechanical studies would be required. low level laser therapy (lllt) is a treatment used in human and veterinary medicine for a variety of clinical syndromes. some uses in human medicine include acute pain associated with osteoarthritis, rheumatoid arthritis, tendonitis, tmj disorders, chronic joint disorders, and wound healing. research is currently on-going to determine the adequate wavelengths to promote effective treatment results with lllt in these conditions. it is purported that lllt acts via the mitochondria to increase cellular metabolism promoting wound healing and a decrease in pain and inflammation. in this study, we hypothesized that dogs treated with lllt in conjunction with hemilaminectomy would display quicker recovery times regardless of the presence or absence of deep pain sensation. seventeen dogs ( dachshunds, chihuahuas, french bulldogs, lhasa ahpsos, and each of a pembroke welsch corgi, and a miniature poodle) were selected and divided into two groups. the dogs ranged in age from to years old, weighed between and pounds, and underwent hemilaminectamies after acute onset of paraplegia secondary to intervertebral disc disease (surgically confirmed). one group received laser treatments on days through of hospitalization. the second group did not receive lllt, but followed the same peri-operative medication protocol. the laser used in this study was an erchonia laser model pl ( nm). the hertz setting was similar for each patient using the previously established protocol for intervertebral disc disease (ivdd) with pulse rate ranging from hz to hz. all dogs received advanced imaging pre-operatively with myelogram or mri. results of the study revealed that treatment with lllt of nm wavelength did not shorten or improve recovery times for dogs with acute onset paraplegia secondary to ivdd after hemilaminectomy procedures. dogs that showed recovery to ambulation at the two week recheck were consistently dogs that were deep pain positive on presentation. a lengthened recovery time or no recovery was seen in the majority of those dogs with absent deep pain on presentation as has been revealed historically in past studies. lllt did not appear to have an effect on this result. however, there are few data describing normal glucose uptake of the canine brain for comparison with suspected or confirmed disease. thus the purpose of this study was to assess the normal distribution of fdg uptake of canine brain structures using a high-resolution research tomography-pet and t-magnetic resonance imaging (mri) fusion system. fdg-pet and t -weighted mr imaging of the brain were performed on healthy laboratory beagle dogs. acquired pet and mr images were automatically co-registered by the image analysis software. on mr images, regions of interest (roi) were manually drawn over intracranial structures, including gross structures (whole brain, telencephalon, diencephalon, mesencephalon, dorsal metencephalon, ventral metencephalon and myelencephalon). a standard uptake value (suv) and relative suv ratio (rsuv suv of roi/suv of whole brain) were calculated for each roi. t-mr images compensated the low anatomical resolution of pet qj;by proving good spatial and contrast resolution for the identification of the clinically relevant brain anatomy. among gross structures, mesencephalon and ventral metencephalon had the highest (suv: . ae . ; rsuv: . ae . ) and the lowest (suv: . ae . ; rsuv: . ae . ) fdg uptake respectively. when suvs were calculated on detailed regions, rostral colliculus and corpus callosum had the highest (suv: . ae . ; rsuv: . ae . ) and the lowest (suv: . ae . ; rsuv: . ae . ) value respectively. these data acquired from normal dog brain will be used in clinical neurology to investigate various intracranial diseases such as inflammation, neoplasm and behavioral disorders. degenerative lumbosacral stenosis (dlss) is a multifactorial condition affecting predominantly large breed dogs. the combination of stenosis and compressive neuropathy cause lumbar pain, lameness and neurologic dysfunction. previous reports describe urinary and fecal incontinence in severely affected dogs. the objectives of this retrospective case series were to describe the clinical signs associated with dysuria and eventual diagnosis of dlss in dogs, and to describe factors associated with regained micturition following prompt diagnosis and treatment. medical records from the university of georgia and the university of missouri between and of dogs were reviewed. inclusion required observation of dysuria, urine retention, absence of structural lower urinary tract disease and concurrent presumptive diagnosis of dlss. dysuria was defined as inability to initiate or sustain a urine stream. urine residual volume was evaluated postvoiding. dysuria was further evaluated using urethral contrast studies, urodynamic testing (urethral profilometry ( ) and cystometry ( )), ultrasonography ( ), and urine culture ( ). presumptive diagnosis of dlss was based on imaging using plain radiography and epidurography ( ), computed tomography ( ) or magnetic resonance imaging ( ). breeds represented included the german shepherd dog (n ), golden retriever (n ), burnese mountain dog (n ), and each labrador retriever, weimaraner, rottweiler and mixed-breed. all dogs were male. were intact at onset of clinical signs. median body weight was . kg (range . - ) and median age was years (range - ). median duration of clinical signs prior to admission was months (range . - ). other pertinent presenting clinical signs included dyschezia ( ), fecal incontinence ( ), general proprioceptive ataxia ( ), weakness ( ), and difficulty rising ( ). physical examination findings included pelvic limb muscle atrophy ( ) and prostatomegaly ( ). abnormal neurologic examination findings included postural reaction deficits ( ), hyporeflexia ( ), decreased tail tone ( ) and lumbosacral hyperesthesia ( ). neurologic examination was normal in dogs. dorsal laminectomy was performed and diagnosis confirmed in dogs; recovery was monitored for a median of . months (range . - ). three of the dogs ( %) regained normal micturition within . - . months of surgery. though not statistically significant, dogs that regained micturition tended to have a shorter duration of clinical signs (median . months, range . - ) versus dogs that remained dysuric (median months, range - ). two of the dogs that regained micturition were neutered at the onset of clinical signs, but only of dogs that remained dysuric was neutered. signs improved in all dogs with postural reaction deficits and decreased tail tone. hyperesthesia resolved in of dogs ( %) and fecal continence returned in of dogs ( %). these findings suggest that following prompt diagnosis and surgical decompression, normal micturition could be regained in dlss affected dogs presenting with signs of dysuria. glycogen storage disease type ia (gsdia; von gierke disease) is an inherited metabolic disorder resulting from a deficiency of glucose -phosphatase-a (g pase). previous reports indicate that clinical manifestations of gsdia occur only in individuals with homozygous expression of a p.i l mutation. heterozygote dogs (het) have been previously reported to exhibit an overall normal outward phenotype. the purpose of this report is to briefly describe some differences that have been observed between het and homozygous wild type (wt) dogs. a colony of dogs at the university of florida contains a mix of affected, wt, and het individuals. in the course of studies designed to determine the effectiveness of gene therapy for correction of gsdia in dogs, both wt and het dogs have been utilized as controls. available information about body weights, clinical pathology tests, fasting studies, and liver biopsies was retrieved from records for both wt and het dogs and compared. although birth weights are similar, het dogs have a slower average rate of weight gain than wt dogs and this difference is especially prominent during the first few months of life (figure ) . in contrast to affected dogs, both wt and het dogs are able to maintain normal blood glucose concentrations for up to - hours of fasting, however, after longer fasts of - hours, het dogs have lower glucose and higher lactate concentrations (table ). in addition, liver biopsy samples from het dogs had greater apparent levels of glycogen suggested by pas staining than did samples from wt dogs, and this correlated with the results of proton magnetic resonance spectroscopy which demonstrated . times greater glycogen content in a liver biopsy sample from a het dog compared to a sample from a wt dog. together, these findings suggest that the level of g pase activity in heterozygote dogs does not provide a completely normal physiological, biochemical, or histological phenotype as previously reported. the glucokinase gene (gck) encodes an enzyme involved in cellular glucose-sensing mechanisms in pancreatic beta cells and hepatocytes. gck mrna is present in feline pancreas but the gene is not expressed in feline liver. hepatic gck expression is abundant in omnivores so its absence may reflect an evolutionary adaptation of strict carnivores, like feline species. we hypothesized speciesspecific features in the gck hepatic promoter may underlie the gene expression pattern observed in cats. the putative feline gck (fgck) promoter region was located using bioinformatic software to identify homology with human gck (hgck). genomic dna from a dsh cat was subjected to direct sequencing using a series of pcr reactions with speciesspecific primers. dna clones thus obtained were aligned to generate the feline sequence. direct sequencing yielded . kb of genomic dna sequence with high homology with sequences (acbe , acbe ) archived in the feline genome project. the feline sequence had six regions homologous with non-coding regions of hgck; four of these conserved regions are upstream of the putative fgck start. a . kb segment immediately upstream of feline hepatic exon is not present in hgck. the . kb insert is the reverse complement of a conserved sequence located downstream of exon in feline and human sequences. in conclusion, the putative hepatic promoter of fgck shares extensive homology with the hgck promoter but contains a . kb insert not found in hgck. functional studies are needed to confirm the role of the unique insert in regulation of fgck gene expression. deuterium oxide (d o) dilution has been proposed for quantifying body water content, but remains difficult to perform routinely. the objective of this study was to assess if the volume of distribution (vd) of creatinine could be proposed as an alternative in dogs for such a measurement. creatinine and d o vd were measured before (c) and after induction (o) of obesity (by giving an hypercaloric diet ( kcal/ kg) for months) in six healthy adult beagle dogs. creatinine ( mg/kg) and d o ( mg/kg) were simultaneously injected by bolus iv. blood was collected before administration and then at , , , , , , , , and min post-injection (creatinine), and , , , , , , and min (d o) . plasma concentrations of both markers were determined. vd was calculated using pharmacokinetic equations. the body weight increased from . ae . (c) (mean ae sd) to . ae . kg (o). d o vd decreased from ae (c) to ae (o) ml/kg. similarly, creatinine vd decreased from ae (c) to ae (o) ml/kg. the individual difference between creatinine and d o vd (expressed in % of d o vd) ranged from À . to . % (c) and from À . to À . % (o). in conclusion, creatinine vd provides a good estimate of d o vd in both normal and obese conditions. a wk double blinded study was conducted comparing the affect of two foods on mobility in dogs. all work was approved by an iacuc. healthy beagle dogs ( - years old, mixed gender) were used. affected (a) and non-affected (na) dogs were identified based on orthopedic examination and radiography as having or not having evidence of naturally occurring joint pathology (presence of osteophytes, dysplasia, effusion, pain on manipulation etc) in one or more joints. a and na dogs were evenly distributed between two locations. foods had nutrient profiles adequate for maintenance according to the aafco official publication. the test food contained greater amounts of methionine, manganese, carnitine, vit. e,, vit. c, alpha linolenic acid (ala), and eicosapentaenoic (epa) acid: the food provided mg n fatty acids and mg n fatty acids per kcal. all dogs were fed the control food for wks followed by a wk feeding period where a and na dogs consumed the test food and a and na dogs the control. blood and urine were collected at weeks , , and and analyzed for serum fatty acids and urine thromboxane:creatinine ratios were determined. evaluators in this study were different than those making the original diagnosis and so were blinded as to treatment and diagnosis. orthopedic exams were performed by two veterinary surgeons at each site on weeks , , and . the same two evaluators examined the same dogs throughout. the data was evaluated for the difference between a and na dogs and between foods with age, gender and location as covariates. body weight, disease status, age and gender were blocked. analysis included anova repeated measures mixed procedure (sas version . ) to determine treatment effects over time.serum epa was greater and arachidonic acid lower at weeks , and in the test food fed dogs (p o . ). urine thromboxane:creatinine ratios were decreased in the a dogs fed the test food compared to the a dogs fed the control food at wks (p o . ). lameness score was significantly improved (p o . ) within and between groups of dogs fed the test food. a significantly greater proportion of a dogs fed the test food had improvement in total het (n ) blood glucose (mg/dl) (ae ) (ae ) blood lactate (mmol/l) . (ae . ) . (ae . ) joint score, lameness, functional disability and overall assessment score at wks compared to a dogs fed the control food. % of a dogs had an improved overall assessment score on the test food after wks and at wks compared to % at wks and % at wks of a dogs consuming the control food. this study shows that a food with moderate amounts of added linolenic acid and epa can have a positive impact on systemic inflammation and mobility in - weeks. a similar abstract will be presented at the orthopedic research society meeting in january to an audience largely of orthopedic researchers interested in human orthopedics. fat is an important dietary component, serving both as a source of energy and as a supplier of essential fatty acids (fa). medium-chain triglycerides (mct) contain intermediate length fa that do not rely on l-carnitine for transport across the inner mitochondrial membrane, bypassing this rate-limiting step in fa oxidation. longchain (n- ) polyunsaturated fatty acids (pufa) from fish oil (fo), and in particular eicosanoids derived from eicosapentaenoic acid (epa), may protect against excessive inflammatory reactions, which may be exacerbated by eicosanoids derived from (n- ) arachidonic acid (aa). this study investigated the effects of adding mct:fo and l-carnitine to a control diet (prescription diet s k/d s ) on lean body mass, and serum fa and metabolites. forty healthy beagles ( . to . y) were fed one of three foods (n to dogs each) for mo. the study protocol was reviewed and approved by iacuc, hill's pet nutrition, inc. all foods were complete, balanced, and sufficient for maintenance of adult dogs; and had similar concentrations of moisture, protein, and fat (approx. . %, . %, . %, respectively). composition of serum fa was determined by gas chromatography of fa methyl esters. metabolomic profiles of serum samples were determined from extracted supernatants that were split and run on gc/ms and lc/ ms/ms platforms, for identification and relative quantification of small metabolites. body composition was determined by dual energy x-ray absorptiometry. serum concentrations of lauric and myristic fa increased; epa and dha increased in a dose-dependent manner; and aa decreased in dogs fed treatment food (proc-mixed procedure in sas; all p . ) when compared to dogs fed treatment foods or . serum concentrations of acetylcarnitine and succinylcarnitine increased, indicating lcarnitine incorporation, in dogs fed treatment foods and . thus, a diet enriched with mct:fo significantly altered serum fa composition, enriching (n- ) pufa and lowering aa concentrations. there was no change in lean body mass for any of the diets compared to baseline values, and no difference between treatments, showing that all three treatment foods met protein requirements. ten owned dogs, obese for more than months (body condition score [bcs] of ; fat mass [fm] . ae . %) were studied. these dogs had their weight reduced by % (bcs ; fm . ae . %; p o . ) being designated weight reduced (wr) group and then were fed to maintain constant body weight during days (bcs . ; p . ), designated maintenance (main) group. a control (ct) group of beagles was also included (bcs . ; fm . ae . %; p o . ). in all groups the glucose postprandial response test was performed after hours of fasting. blood samples were taken prefeeding and after , , , , , , , , , and minutes of the consumption of cooked rice enough to the ingestion of g of starch/kg body weight. tnf-a and il- were dosed in milliplex tm map panel, insulin and leptin by radioimmunoassay. statistical analysis included paired or non-paired t-tests and wilcoxon (p o . ). the regimen normalized meal glucose response, the area under the curve (auc) of glucose for wr was lower than for obese (p o . ) and similar to main and ct (p . ). insulin secretion did not normalize immediately, as obese and wr exhibited similar auc of insulin and higher values than for ct (p o . ). main, however, presented similar auc of insulin than ct, with lower values than obese and wr (p o . ), suggesting that dogs require some time to adapt their metabolism. leptin, tnf-a, and il- presented significant reductions after weight loss (p o . ), without differences between wr, main and ct (p . ), suggesting an improvement of the pro-inflammatory state consequent to obesity. studying food base excess (be) modification, methionine intoxication was described. in a basal kibble dog diet (be meq/kg; . g/kg of s) two dosages of ammonium sulphate and methionine was added, resulting in diets with be of meq/kg ( . g/kg of s) and À meq/kg ( . g/kg of s), or be of meq/kg ( . g/kg of s) and meq/kg ( . g/kg of s), respectively. a  factorial plus a control diet design, resulting in five treatments, and adult health beagle dogs were used, in a completed randomized design with six dogs per diet. a -d adaptation phase followed -d of total urine collection (in bottles with mg of thymol). urine were pooled by dog and analyzed for density, volume and ph. food macroelements were determined by standards methods (aoac, ) and used for be calculation. dog's acid-basic status was studied by blood gas analysis of venous blood, at : h (pre feeding) and hours after meal. a dose-dependent reduction of urinary ph was verified for both compounds (p o . ). blood bicarbonate (r . ; p o . ), and blood base excess (r . ; p o . ) were highly correlated with food be. acidemia and reduced blood be were verified in diets with be close to zero (higher dose of both compounds, or . g/kg of s), resulting in daily or each other day vomiting episodes in the dogs. ataxia, seizures, and vomiting were previously describe in dogs fed g/kg of methionine, but our results suggest that a much lower value ( . g/kg) was toxic and that the safe upper limit should be between this value and . g/kg (the lower evaluated dose). in people with chronic kidney disease and heart failure, obesity is associated with longer survival times. this association, called the "obesity paradox," also has been recognized in dogs and cats with heart failure. excess weight appears to modulate the serious deleterious effects of muscle loss in these diseases. the purpose of this study was to determine the effects of body condition and body weight changes in dogs with naturally-occurring chronic kidney disease (ckd). dogs diagnosed with ! iris stage ii ckd between and at iowa state university and tufts cummings school of veterinary medicine were eligible for the study. dogs o year of age and those with acute renal failure or suspected congenital renal diseases were excluded. medical records were reviewed using a standardized data form, and data were collected for initial body weight and body condition score (bcs, - scale), clinicopathologic values, changes in body weight and bcs, comorbidities, and treatments. dogs were classified as underweight (bcs - ), moderate weight (bcs - ), or overweight (bcs - ). a change in body weight was defined as . kg. survival times were determined for all dogs that were discharged from the hospital and lived day. associations between survival and bcs or body weight changes were analyzed using cox proportional hazards models. one hundred two dogs were enrolled in the study. at the time of diagnosis, dogs were classified as iris stage ii, dogs were stage iii and dogs were stage iv. median body weight at baseline was . kg (range, . - . kg). for dogs with body condition scores recorded (n ), were underweight ( %), were moderate ( %), and were overweight ( %). for dogs that had at least two body weights recorded over the course of their disease, gained weight, lost weight, and had no change in weight. changes in body weight were not associated with survival; however, bcs at the time of diagnosis was significantly associated with survival. dogs classified as underweight had a significantly shorter survival time compared to both moderate (p o . ) and overweight dogs (p o . ). these results suggest that body condition is an important consideration in dogs with acquired chronic kidney disease. further studies are warranted to evaluate the relationship between obesity and longer survival in dogs with ckd. protein restriction is the cornerstone of dietary management of kidney disease. the national research council recommends % crude protein and the american association of feed control officials (aafco) recommends a minimum of % crude protein for maintenance for healthy adult cats. protein requirement is unknown for adult cats with kidney disease. most commercially produced cat foods for adult maintenance contains % or more crude protein on a dry matter basis. a typical therapeutic food for cats with kidney disease contains about % crude protein. the objective of the present study was to investigate whether dietary crude protein at . % would be adequate for the maintenance for adult cats with impaired kidney function. seven adult cats, female and male, with age ranging from to . years old (mean: . years) were used in the study. all cats had elevated serum creatinine concentration ( . mg/dl, range: . - . mg/dl) and reduced glomerula filtration rate (mean: % reduction; range: - % reduction) during the study. they did not have other systematic diseases, e.g., hyperthyroidism, at the beginning of the study. cats were fed an expanded dry food made with ingredients commonly used in commercial dry cat foods. the food contained . % crude protein (chemical analysis) and kcal/kg (calculated) on a dry matter basis, or . g protein/ kcal. each essential amino acid in the food was at least % of that recommended by aafco. other nutrients in the food also exceeded aafco's recommendations for maintenance for adult cats. cats were fed the food for weeks. lean body mass (dual x-ray absorptionmetry; hologic, hologic, inc, ma) and serum albumin concentration were measured periodically to monitor protein status of cats. the average lean body mass (mean ae sd) was . ae . kg, . ae . kg, . ae . kg, and . ae . kg in weeks , , , and of the study, respectively. paired t-test did not detect statistical difference (p . ) when comparing the lean body mass in weeks versus weeks , , and , respectively. serum albumin concentration were within the normal reference range during the study (mean ae sd: . ae . %, . ae . %, . ae . %, and . ae . % in weeks , , , and , respectively) . these data show that . % dietary crude protein in a dry food with kcal/kg on a dry matter basis, or . g protein/ kcal, is adequate for maintenance for cats with impaired kidney function. in humans, several disease conditions exist that involve abnormal patterns of polyunsaturated fatty acids and similar abnormalities may be present in companion animals. indeed there have been reports of decreased plasma arachidonic acid and reduced delta- desaturase activities in dogs with atopy and other skin disorders. the present study investigated serum fatty acid profiles in dogs and cats presented to the texas a&m university veterinary teaching hospital, clinical pathology laboratory over the past one year period. results were compared with normative data generated among dogs and cats from earlier feeding studies. sera used were residual samples submitted to the laboratory for other diagnostic procedures and stored frozen for no more than months after collection. the samples were grouped according to presenting disorders involving liver, kidney, digestive, and cardiac diseases. total lipids were extracted using chloroform:methanol ( : v/v) and fatty acid methyl esters were prepared for capillary gas chromatography. relative percentage distribution of individual serum fatty acids for each animal were then compared with average normative serum phospholipid fatty acid values (dogs, n ; cats, n ) by calculating the ratio of the value in the diseased individual to the normal mean value and used as an index of normalcy. normalcy ratios were then plotted on a logarithmic scale with normal at . . the ratio was then compared to changes greater than , and standard deviations of the normal mean values. in this way a graphical presentation of resultant values was obtained. although the animals had been fed various commercial diets and some home-prepared foods, a number of noteworthy patterns emerged from this analysis. dogs showed increased linoleic acid, decreased arachidonic acid, increased total monounsaturated and decreased saturated fatty acids at p o . ; oleic acid was increased at p o . . remarkably, these findings were similar for all canine disease categories evaluated (n , heart; n , kidney; n , liver, and n digestive disorders). in cats, a slight decrease in arachidonic acid and large decrease in : was observed but only in heart disorders. by contrast, modest elevations of arachidonate were observed in kidney, liver, and digestive disease groups but at p o . . sample sizes of the feline sera were considerably smaller (range of - per group). a limitation of this analysis is that variability of normal data may exist depending on diet fed making comparisons less reliable however, these preliminary data suggest that metabolic diseases of dogs may depress plasma arachidonic acid independent of diet fed suggesting either reduced conversion from linoleic acid or increased utilization of arachidonate for eicosanoid production during times of metabolic stress. conversely, in cats, increases in arachidonic acid may be associated with diet arachidonate or other mechanisms. additional studies to verify these findings are warranted. the objective of this study was to determine whether or not lalanyl-l-glutamine (ala-gln) supplementation in dogs with parvoviral enteritis improves the survival rate and ameliorates clinical signs without side effects. this randomized, double-blinded, placebo-controlled clinical trial included client-owned dogs. the dogs were randomly assigned into two groups and administered ala-gln solution (dipeptiven; . g/kg) or an equivalent volume of placebo orally twice a day. all of the dogs (ala-gln group [n ] and placebo group [n ]) received standard treatment while hospitalized and were monitored daily according to a clinical scoring system and diagnostic evaluation for days. among the dogs, (ala-gln-treated group [n ] and placebo group [n ]) were vaccinated and (ala-gln-treated group [n ] and placebo group [n ]) were not vaccinated. the population consisted of purebreds and mixed breed dogs, with a mean age of . ae . weeks. the survival data were compared statistically by means of a log-rank test for the kaplan-meier survival curves. the clinical scores of ala-gln-treated dogs improved significantly relative to the placebo group. there was a significant difference between the two groups in the survival distribution (p . ); specifically, of the ala-gln-treated dogs ( . %) died, whereas of the dogs in the placebo group ( . %) died. no side effects were associated with the administration of ala-gln. these results suggest that the oral administration of ala-gln is effective in improving clinical signs and survival rate in dogs with parvoviral enteritis. bleeding disorders, thrombocytopenia and alterations in platelet function have been documented in humans receiving lipid-containing parenteral nutrition formulations. despite a lack of evidence in the veterinary literature, it is believed that parenteral lipids are contraindicated in critical illness when the development of bleeding disorders is likely. the objective of this study was to determine if there is an in vitro effect on platelet function and thromboelastography (teg) in normal dogs with varying concentrations of a % soybean oil emulsion (intralipid s ). twelve clinically healthy dogs were used for this study. whole blood platelet aggregation, using adp and collagen agonists, was measured using multiple electrode aggregometry in hirudinated blood with final lipid concentrations of , , , and mg/ml. the teg parameters r, k, a-angle, and maximum amplitude (ma) were evaluated from citrated whole blood with equivalent final lipid concentrations as platelet aggregation. there was no significant difference between groups with collageninduced platelet aggregation. there was a significant increase in the area under the curve (auc) with adp-induced aggregation at a lipid concentration of mg/ml (p . ). the ma was significantly reduced at both the mg/ml (p o . ) and mg/ml (p o . ) lipid concentration. there was no statistical difference between groups evaluating the other teg parameters. while platelet aggregation appeared enhanced at the highest concentration evaluated, this concentration is not clinically relevant. the reduction in ma seems discordant but both fibrinogen and platelets contribute to the ma. therefore the higher lipid concentrations may be interfering with fibrinogen kinetics or fibrinogenplatelet interaction. in vivo studies are indicated to determine if any of these changes are clinically significant. rosiglitazone is a peroxisome proliferator-activated receptor gamma (pparg) agonist and an fda-approved anti-diabetic agent in humans that has been investigated for its ability to reduce tumor cell growth. specifically, the combination of rosiglitazone and carboplatin has demonstrated enhanced tumor control. the purpose of this study was to determine the peak plasma concentrations and side effect profile of rosiglitazone after oral administration in dogs with spontaneously occurring cancer. all dogs received carboplatin intravenously concurrently with oral rosiglitazone. ten cancer-bearing dogs with normal pre-treatment hepatic and renal function were enrolled. complete pre-treatment hematological and biochemical parameters were available in ten dogs and post-treatment parameters in nine dogs. peak plasma concentrations varied with dose and ranged from . - . ng/ml and occurred between minutes and hours post administration and rapidly declined after the peak. the dose limiting toxicity was hepatic at a dose of mg/m . there was one grade iii, two grade i alt, and one grade iii ast elevations noted. no changes in total bilirubin, alkaline phosphatase, or ggt values were noted. blood glucose values remained within normal limits. mild, self-limiting gastrointestinal and hematologic toxicities were observed when rosiglitazone was administered in combination with carboplatin. based on this study, the recommended dose of rosiglitazone in cancer-bearing dogs with normal hepatic function is mg/m orally once daily. side effects of the combination appear similar to side effects noted with carboplatin alone. further study is needed to determine efficacy of this combination and if more frequent dosing is required to maintain plasma concentrations. carboplatin has shown little activity as a single agent for the treatment of canine transitional cell carcinoma (tcc). however, gemcitabine has shown synergism with carboplatin in human cell lines. the purpose of this study was to evaluate the activity of gemcitabine against canine tcc cell lines alone or in combination with carboplatin. we hypothesized that gemcitabine in combination with carboplatin would have synergistic effects in vitro. the results of this study could provide a rationale for treatment of canine tcc with the combination of these drugs. tcc cell lines tcc-kiss, tcc-knapp-js, tcc-axa, tcc-hxc, and tcc-sh were treated with gemcitabine, carboplatin, or the combination. cell proliferation was assessed using cyquant assay, cell cycle was evaluated using propidium iodide staining, and apoptosis was assessed by measuring caspase- / activation. synergy was quantified by combination index analysis using compusyn software. treatment of canine tcc cell lines with carboplatin or gemcitabine decreased cell proliferation, induced cell cycle arrest, and apoptosis. when tcc cell lines were treated with gemcitabine and carboplatin in combination at a therapeutically relevant concentration (gemcitabine o um, carboplatin o um), a significant decrease in cell proliferation was observed compared to gemcitabine or carboplatin alone, and the drug combination was synergistic in of cell lines, and additive in the remaining lines. gemcitabine exhibits biologic activity against canine tcc cell lines and carboplatin combined with gemcitabine exhibits synergistic activity at biologically relevant concentrations. our results support further evaluation of these drugs in dogs with tcc to determine the clinical efficacy of this combination. metronomic chemotherapy has been shown in murine models and humans to improve tumor control by inhibiting tumor angiogenesis and suppressing regulatory t cells (treg). treg are a subset of t lymphocytes demonstrated to be increased in humans and dogs with cancer and are thought to suppress cellular immune responses against tumors. the purpose of this study was to determine whether metronomic cyclophosphamide therapy depletes treg and/or exhibits antiangiogenic activity in dogs with soft tissue sarcoma. client owned dogs with histologically confirmed grade i or ii soft tissue sarcoma were administered cyclophosphamide at . mg/m or mg/m orally once daily for days. whole blood and tumor biopsies were obtained on days , , and . flow cytometric analysis of blood was performed to assess changes in t lymphocyte subsets, including cd and cd cells as well as cd foxp treg. tumor microvessel density (mvd) was assessed by performing immunohistochemistry for cd . five dogs were enrolled in the . mg/m /day dose cohort and six dogs were enrolled in the . mg/m /day dose cohort. in patients that received cyclophosphamide at . mg/m /day, the mean number of treg decreased from day to but there was no change in the mean percentage of treg or mvd. for patients that received . mg/m /day, both the mean number and percent of treg as well as mvd decreased over the day time period. cyclophosphamide at . mg/m /day or greater selectively depletes treg and inhibits angiogenesis in dogs with soft tissue sarcoma. arsenic trioxide (ato) is used to treat leukemias, multiple myeloma, and relapsed lymphoid malignancies in humans; its use has not been explored in veterinary oncology. prior therapy with glucocorticoids decreases likelihood and duration of remission for dogs with lymphoma treated with chemotherapy. we hypothesized that ato will re-sensitize glucocorticoid-resistant canine lymphoma cells to glucocorticoid-induced apoptotic death. the osw canine lymphoma cell line was cultured with um dexamethasone. remaining viable dividing cells were considered resistant. resistant cells were exposed to . um and . um of ato without dexamethasone, after which cells were washed and re-exposed to um dexamethasone. after , and hours of dexamethasone exposure, cells were counted using trypan blue stain. apoptosis was assessed by tunel assays on cytospin preparations collected at , , and hours from ato-exposed and control groups. statistical analysis was performed using way anova and tukey's test. the proportion of dead cells increased over time in both . um and . um ato exposed groups. the proportion of dead cells was greater for . um ato (p o . ) and . um (p o . ) groups compared to control. apoptosis increased with increasing ato concentration and duration of dexamethasone exposure compared to control. these results support the effectiveness of ato at re-sensitizing glucocorticoid-resistant canine lymphoma cells to apoptotic death following re-exposure to glucocorticoids. ongoing gene expression studies aim to elucidate this mechanism. additional studies to determine if this effect is seen with other chemotherapeutic agents are warranted. lymphoma is the most common hematopoietic tumor of dogs. protein disturbances may be associated with this disease including monoclonal gammopathies in a low percentage of cases. serum protein electrophoresis (spe) is routinely used to aid diagnosis of various canine diseases including lymphoma when total protein concentration is elevated. the purpose of this study was to compare spe changes in lymphoma patients without elevated total proteins with a population of healthy dogs. agarose gel electrophoresis was performed on residual serum from healthy control dogs and untreated dogs with multicentric lymphoma (stage iii -v) after measuring total protein (tp) using the biuret method. densitometric traces of the protein bands were obtained using computer software (totallab ) and the albumin, alpha- , alpha- , beta- and gamma globulin subfractions were identified by visual inspection. the total protein concentration, the number of subfractions and the relative and absolute protein subfraction concentrations were then compared statistically between the two populations. in lymphoma dogs, tp, absolute albumin, beta- and gamma globulin concentrations and both relative and absolute concentrations of the alpha- globulins were significantly lower however relative and absolute alpha- globulin concentrations were significantly elevated. no monoclonal gammopathies were identified in any of the dogs and not every patient with lymphoma had the above changes in their electrophoretogram. this study has demonstrated that significant changes occur in the albumin and globulin fractions of canine lymphoma patients despite no obvious increase in tp. further investigation is required to identify the proteins responsible for these changes. it is well known that immunophenotype has a prognostic value for the outcome of canine lymphoma, with t-cell lymphomas having a worse prognosis than b-cell lymphomas. the recent advent of flowcytometric techniques allowed easy detection of many different markers on lymphoma cells and therefore, not only distinguish between t and b cells, but also estimate possible aberration on immunophenotype. in human oncology, although some controversy persists, it seems that non-hodgkins lymphoma and acute leukemia carrying aberrations have a worse prognosis. the aim of this study was to evaluate the role of immunophenotype aberration in canine high-grade lymphoma considering outcome and time span to achieve complete response under chemotherapy. samples of bone marrow, blood and lymph node suspensions from twentythree dogs were evaluated with flow-cytometry. eleven dogs had aberrant expression of neoplastic lymphocytes and twelve were non-aberrant. the most common aberrations found were: positivity to cd , biphenotypes, double expression of tantigens (cd , cd ), diminished expression of cd . all dogs were treated with a chop-based protocol. there was a significant difference for the time to achieve response to chemotherapy (partial or complete). / non aberrant lymphomas went into cr or pr after the first treatment (l-asparaginase), while aberrant lympho-mas needed more than treatment to reach cr or pr. there was a trend for a prolonged disease free interval with non-aberrant versus aberrant, although it was not statistically significant. aberration of immunophenotype may be a prognostic factor for canine lymphomas, but further studies with larger groups are needed. class ii major histocompatibility expression is a significant and independent predictor of prognosis in human b cell lymphoma. low class ii mhc is consistently associated with poorer outcome. the mechanism underlying this relationship is not clear, but one hypothesis is that high class ii mhc allows for better antigen presentation and tumor-specific immune responses. in the this study, we investigated whether that class ii mhc expression in canine b cell lymphoma was associated with remission and survival times. a total of patients were categorized by level of class ii mhc,expression of cd and cell size for on neoplastic b cells. multivariable cox-proportional hazard analysis was used investigate this research question using a randomly selected subset of the data, and the predictive ability of this model was validated on the remaining / of patient data. results suggested that low class ii mhc expression was associated with decreased times to relapse and death as is seen in human b cell lymphoma, and that large neoplastic cells were associated with decreased survival time. cd expression was not associated with patient outcomes. these findings have implications for the use of dogs to model human lymphomas, for the study of tumor vaccines, and for prediction of mortality in dogs with b cell lymphoma with a high level of specificity. one of the reasons for the failure of canine lymphoma treatment is related to the resistance of tumor cells against chemotherapy drugs. the major form of this resistance is provide by multidrug resistance abc transporters. abc transporters proteins comprise a large superfamily of transmembrane proteins, atp-dependent, that extrude a large variety of drugs from the cells. multidrug resistance phenotype in cancer cells is associated with overexpression of these transmembrane proteins. abcg , also known as bcrp, is a residue half-transporter protein that protect hematopoetic stem cells against toxic compounds. the aim of this study was to investigate the expression of bcrp (abcg ) in canine multicentric lymphoma. samples were collected by fine needle aspiration of an enlarged lymph nodes, from dogs with multicentric lymphoma (stage iii to v) at diagnosis, and normal lymph nodes (control). dogs that were previously treated with prednisone or chemotherapy were excluded from the study. quantitative rt-pcr was used to measure the mrna expression level of bcrp and flnb expression as a endogenous reference canine gene. a widely range expression value for abcg expression was found for canine multicentric lymphoma. high gene expression was observed in % ( / ) canine lymphoma, but % of dogs had a lower expression when compared with normal lymph node. gene expression was not associated with clinical staging, complete or partial remission, relapse and survival time. in conclusion, abcg was expressed in canine lymph node and canine multicentric lymphoma at the diagnosis, and it was not correlated with clinical response. osteosarcoma (osa), the most frequent primary malignant bone tumor of dogs, is both locally aggressive and highly metastatic. prognostic factors for canine osa include tumor location, distant metastatic disease, and serum alkaline phosphatase (alp) concentration. an increased serum alp concentration is associated with poor prognosis; however the mechanisms underlying this phenomenon are currently unclear. during normal bone development alp may be used as a marker for osteoblasts. additionally, alp is a downstream target of activated canonical wnt/b-catenin signaling. therefore, we hypothesized that increased serum alp would be associated with increased expression of b-catenin in canine osa. the goals of this study were: ( ) characterize and compare cellular alp expression in osa tissue from patients with normal and high serum alp; and ( ) assess b-catenin expression in those same patient populations. we used frozen osa samples collected from patients with either high alp (n ) or normal alp (n ). total rna was isolated from the frozen tissue, converted to cdna, and analyzed using quantitative reverse-transcriptase polymerase chain reaction (qrt-pcr) with either target gene alp (aim ), or target gene b-catenin (aim ). additionally, b-catenin expression was analyzed by western blot. qpcr data for bcatenin and alp expression were normalized to s, and relative expression was calculated by the ddct method. the relative expression of cellular alp was higher in high serum alp samples compared to normal serum alp samples: . ae . (mean relative expression ae standard deviation; p o . ). further, the relative expression of b-catenin was also increased; b-catenin expression of high serum alp samples relative to low serum alp samples was . ae . (p o . ), which is also seen by western blot. this study begins to clarify the mechanism behind high serum alp in canine osa, and suggests the wnt signaling pathway may be active in this population of patients. further work will focus on elucidating the role active wnt signaling plays in the biology of osa. in the future, serum alp status of osa patients may help identify patients that would benefit from therapies targeting this pathway. accurate assessment of abdominal lymph node status is of vital importance for appropriate treatment planning and determining prognosis in dogs with apocrine gland adenocarcinoma of the anal sac (agaas). pretreatment knowledge of lymph node status is helpful for determining prognosis and planning the optimal extent of lymphadenectomy. in addition, pretreatment knowledge of lymph node status may help in selecting patients who might benefit from adjuvant chemotherapy and radiation therapy. abdominal ultrasound is currently the most commonly employed test to screen for abdominal lymphadenopathy in dogs with agaas. imaging studies in people indicate that magnetic resonance imaging ( to determine and compare the plasma concentration of cyclophosphamide and its metabolite -ohcp, within the plasma of lymphoma bearing dogs being treated with either oral or intravenous cyclophosphamide. in this prospective study, patients were randomly assigned to either receive oral or intravenous cyclophosphamide, at a dose of mg/m . based on a priori power calculation eight patients per treatment group were enrolled. plasma was obtained at times , , , minutes, and then at , , , , hours post administration for evaluation of -ohcp concentrations by liquid chromatography-dual mass spectrometry (lc/ms/ms). average values were obtained for both cyclophosphamide and -ohcp concentrations within the plasma of both groups. the following values were obtained, half life (hl), time to maximum concentration (tmax), maximum concentration (cmax), and area under the curve (auc). the mann-whitney statistical test was used to compare the groups. the auc for cyclophosphamide was statistically significant (p o . ) when compared between the two groups. the auc for -ohcp was not statistically significant between the groups. the difference between cmax for cyclophosphamide and -ohcp was statistically significantly (p o . ) between the groups. although the auc for cyclophosphamide was statistically significant between the two groups, the auc for the active metabolite -ohcp was not different when administered intravenously or orally. thus drug exposure to the active metabolite of cyclophosphamide is the same when administered intravenously or orally. previously the percentage of successful intraosseous (io) catheter insertions, insertion times, and ''ease of use'' scores using the ez-io g power driver by a wide spectrum of novice participants in feline cadavers were evaluated. novice users' mean io catheter insertion time using the ez-io g driver was also compared to the mean iv catheter insertion time in normovolemic feline and canine patients presented to the western college of veterinary medicine (wcvm) small animal hospital. novice users included wcvm personnel ( technicians, veterinary students, interns, residents, clinicians). after watching a -minute ez-io g training video, each participant inserted io catheters using the ez-io g driver. site (proximal humerus or trochanteric fossa of the femur) and side of cat (right or left) were randomized for each attempt for each participant. a gauge x mm long needle and a gauge x mm needle were used for io catheter insertion in the humerus and femur, respectively. participants then graded the ''ease of use'' of the ez-io g device on a visual analog scale (vas) that was converted to a -point scale. twenty-six iv catheter insertions in normovolemic feline and canine patients performed by wcvm small animal hospital personnel ( technicians, veterinary students, intern, resident, clinicians) were then timed and compared to the mean io catheter insertion time in feline cadavers by study participants using the ez-io g device. the io catheter was inserted correctly on every attempt by % ( / ) of participants. no difference was found between participant groups for mean io catheter placement confirmation percentage (p . ). percentage of io catheter ''slippage off the bone'' at the time of placement did not vary across participant groups (p . ). mean io catheter insertion times were all less than seconds and did not differ significantly as a function of attempt number (p . ) or as a function of participant group (p . ). participants rated the ez-io g 's ''ease of use'' favorably and subjective scores did not differ across participant groups with varying levels of clinical experience (p . ). compared to the mean insertion time for iv catheterization ( sec), mean io catheter insertion by participants using the ez-io g ( sec) was significantly faster (p . ). regardless of their level of clinical experience, participants rated the ez-io g device favorably in terms of its ''ease of use'' and their willingness to use the device in the future. regardless of their level of clinical experience, study participants successfully placed io catheters using the ez-io g device and did so significantly faster than the reported iv catheter insertion time in normovolemic feline and canine patients in the wcvm small animal hospital. intraosseous catheterization using the ez-io g has the potential to provide very rapid vascular access and is a skill that can be easily learned. previously presented at the western college of veterinary medicine undergraduate poster competition. multicavitary effusion is a common cause of presentation for dogs to emergency medical centers. the goal of this study was to identify common underlying causes of multicavitary effusion as well as determine their relative importance. a retrospective analysis of cases of multicavitary effusion admitted to the icu of a tertiary referral center (ontario veterinary college) from to was performed. twenty-three different breeds, with golden and labrador retrievers ( . % and %, respectively) being most commonly seen, were included in the study. ages ranged from to years with a median age of years and a mean of . years. . % of cases were males ( / cases). most common presenting signs included lethargy ( . %), anorexia ( . %), vomiting ( %) and dyspnea ( %). cavitary effusion was detected by either ultrasonography (pericardial, pleural or abdominal) or radiographs (pleural). bicavitary effusion was present in cases ( . %) whereas cases ( . %) had tricavitary effusion. neoplasia was found to be the most common underlying cause overall ( . %), with hemangiosarcoma being the leading type ( . % of neoplasia cases), followed by congestive heart failure ( . %), gastrointestinal lymphangectasia ( . %), peritonitis/pancreatitis ( . %), cirrhotic liver disease ( . %) and acute renal failure ( . %). in cases ( . %), no underlying cause could be found. of these, ( . % of all cases) were diagnosed as having idiopathic pericardial effusion. taken together, these findings suggest a strong association between multicavitary effusion and diseases carrying a guarded prognosis in dogs. infection control practices in veterinary clinics and hospitals are becoming increasingly important, with rising client expectations, growing concern about the spread of antimicrobial-resistant pathogens, and the potential for zoonotic transmission of disease. surgical patients are at increased risk of developing infections, and can serve as sources of these pathogens for other animals and people with whom they have contact within and outside the clinic. taking all reasonable precautions to reduce the risk of surgical site infections, beginning with preoperative preparation of the surgeon and patient, is therefore an important part of any infection control program. while guidelines are available for preoperative preparation procedures, there has been no objective investigation of compliance with these guidelines in veterinary practices. the objectives of this pilot study were to describe a range of preoperative hand scrub and surgical site preparation practices in veterinary clinics, and to determine if there were any areas that consistently require improvement. observation of preparation practices was performed in each of ten clinics over - days using - small wireless surveillance cameras. data was coded for surgical patients, and surgeons performing a total of hand scrubs. patient hair removal was most commonly performed after induction of the animal ( / , %) and using clippers ( / , %) . steps in surgical site aseptic preparation ranged from - . contact time with soap ranged from - s (mean s, median s), and with alcohol from - s (mean s, median . s). application of alcohol or antiseptic using a ''cleanest to dirtiest'' pattern was infrequent ( / ( %) and / ( %), respectively). potential contamination of the surgical site occurred most frequently when the animal was moved to the surgery table after initial preparation ( / , %). preoperative alcohol hand rub was used in / facilities, but soap and water hand scrub was still more commonly used even at these clinics. proximal-to-distal scrubbing was noted in / ( %) of soap and water scrubs. contact time during surgeon hand preparation ranged from - s (mean s, median s) for soap and water and from - s (mean s, median s) for alcohol-based hand rub. approximately % of the variation in contact time was due to inter-surgeon variation. no significant changes in practices were identified over the course of the observation period. some preoperative preparation practices were fairly consistent between clinics in this study, while others varied considerably. contact times with preparatory solutions were often far shorter than recommended, and there was a high frequency of non-sterile contact with the surgical site during movement of patients to the surgical suite. the camera system used to perform this study did not have a significant time-dependent effect on the behavior of participants, and could be useful for performing similar field-based observational studies in the future. this prospective randomized study compared the percentage of successful intraosseous (io) catheter insertions, insertion times, and ''ease of use'' scores using the ez-io g power driver to manual io catheterization in feline cadavers. the io catheter insertion time in cadavers using the ez-io g device was also compared to iv catheter insertion time in normovolemic feline and canine patients. after a purposely limited training period, a preclinical veterinary student was timed and video-recorded as she performed io catheter placements in feline cadavers ( io insertions by placing an illinois needle manually and io insertions using the ez-io g ). order of technique (manual or ez-io g ), site of io placement (proximal humerus or trochanteric fossa of the femur), and side of cat (right or left) were randomized for each attempt. when using the ez-io g , a gauge x mm long needle and a gauge x mm needle were used for io catheter insertion in the humerus and femur, respectively. after each attempt, the student graded the ''ease of use'' of each technique on a visual analog scale (vas) that was converted to a -point scale. twenty-six iv catheter insertions in normovolemic feline and canine patients performed by western college of veterinary medicine (wcvm) small animal hospital personnel ( technicians, veterinary students, intern, resident, clinicians) were then timed and compared to the student's mean io catheter insertion time using the ez-io g .median io catheter insertion times for the techniques were significantly different (manual io technique sec; ez-io g sec) (p o . ); the manual method took seconds longer ( % confidence interval of to seconds) than the ez-io g method. insertion time was more variable for the manual technique than for the ez-io g . percentage of catheter ''slippage off the bone'' and extravasation around the inserted catheter were significantly higher for placement of the manual io catheter compared with placement of the ez-io g catheter (p o . ). student's subjective ratings were more favorable and more consistent for the ez-io g technique compared to the manual technique for io catheter insertion. compared to the mean insertion time for iv catheterization in the wcvm small animal hospital, io catheter insertion by the student using the ez-io g was significantly faster (iv catheter sec; ez-io g io catheter sec) (p o . ). intraosseous catheter insertion using the ez-io g can be said to be significantly faster, less traumatic, more user-friendly, and as effective as io catheter placement using the manual technique. vascular access via io catheter insertion using the ez-io g device may be suggested to be faster than iv catheter insertion. previously presented at the western college of veterinary medicine undergraduate poster competition. computed tomography (ct) has been widely investigated and applied as a means for non-invasive quantitative bone mineral determination in human medicine. the aim of this study was to assess age-related changes and anatomic variation in bone mineral density (bmd) using quantitative ct in normal cats. seventeen normal cats were included in this study and divided into the following age groups: o year (n ); - years (n ); and years (n ). a computed tomographic scan of each vertebra from the th thoracic to the th lumbar spine, and the pelvis, was performed with a bone-density phantom ( , , and mg/cm , calcium hydroxyapatite, cirs phantom s ). on the central transverse section, the elliptical region of interest (roi) was drawn to measure the mean hounsfield unit value. those values were converted to equivalent bmd by use of the bone-density phantom and linear regression analysis (r . ). the mean bmd value of the thoracic vertebrae ( . ae . mg/cm ) was significantly higher than of the lumbar vertebrae ( ae . mg/cm ). the maximum bmd occurred at the t , t , and l levels in all age groups. there was a statistically significant difference in the mean bmd value among the age groups at the t (p o . ), t (p o . ), and l levels (p . ), respectively. in addition, there was no significant difference between the mean bmd value of the left and right iliac bodies ( . ae . mg/cm and . ae . mg/cm , respectively). the present study suggests that age-related changes and anatomic variation in bmd values should be considered when assessing bmd using quantitative ct in cats with bone disorders. dynamic contrast-enhanced computed tomography (dce-ct) is a rapid and widely available method of cerebral perfusion imaging. however, there is no established reference value of cerebral blood flow (cbf) measured by dce-ct according to a dog's age. the purpose of this study was to identify the correlation between regional cbf and aging in clinically normal dogs using dce-ct. fourteen dogs with no evidence of hemodynamic disorders and central nervous system dysfunction were included in this study. dogs were assigned to the following age groups: o year (group ); - years (group ); and o years (group ). dce-ct scans were performed at the level of the third ventricle and mesencephalic aqueduct. cbf in the gray and white matter was calculated using stroketool-ct s software. the overall mean ae standard deviation quantitative estimate for regional cbf in clinically normal dogs was . ae . ml/min/ g, . ae . ml/min/ g, and . ae . ml/min/ g in groups , , and , respectively. there was no significant regional cbf difference between the right and left sides of the brain in each group. also, a statistically significant difference in the regional cbf was observed between groups and (p o . ). thus, aging affects the regional cbf in normal dogs and the values should be considered assessing the results of dce-ct. according to several clinical behavior guidelines, ''toileting'' type inappropriate urination (i.e. large amounts of urine deposited in horizontal surfaces) can arise in cats suffering from a medical problem (typically lower urinary tract disease). by contrast, ''spraying'' type behaviour (i.e. possibly smaller amounts of urine deposited on vertical areas) is more typically associated with anxiety brought about by a threat to local resources, arising from either a change in the physical environment or threat to these resources from another cat. however, there is some evidence that ''sprayers'' may also be presented with a medical problem, which might be linked to the disease (e.g. painful voiding associated with crystalluria may lead to a standing posture being adopted and small amounts eliminated at a given time). this might be associated with an apprehensive state or simply a co-morbid state. as part of a larger research project aimed at investigating behavioral and physical aspects of cats presented with inappropriate urination, owners of ''spraying'' and ''toileting'' cats with appropriate control subjects from the same households were recruited throughout local media coverage and the internet. the case-control dyads were brought by the owners to the veterinary hospital of the university of sa˜o paulo, at the same time, for a medical work-up (i.e. physical examination, complete blood count, biochemical profile, urinalysis, urine culture and abdominal ultrasound). no significant differences between the ''sprayers'' and ''toileters'' regarding the occurrence of medical problems were found. both groups had a similar proportion of cats affected by medical illnesses (sprayers: . %, toileters: . %; chi , p . ), directly or indirectly relating to the urinary system (e.g. diabetes, chronic kidney disease). in both groups, control cats also had a relatively high occurrence of medical concerns ( . % and . %, respectively for each control group). these results emphasize the importance of careful medical evaluation of cats presented for a urinary housesoiling problem. the relatively high prevalence of medical concerns among apparently healthy cats in multi-cat households may have arisen, at least in part, as a result of an inability/failure of owners to monitor individuals, thus allowing some early signs to pass unnoticed. the way in which medical and behavioral elements are linked (if at all) remain unknown but deserve further investigation. considered as a semi-social species, domestic cats appear to be highly sensitive to the effects of social stress, especially when living in high density populations. cats are capable of adapting to live ingroup; nonetheless, they do not appreciate living in close proximity with others as result of an environment lacking of great opportunities of escaping and hiding. this study aimed at testing the following hypotheses: (a) owners' perceived quality of life affects cats' global levels of stress; (b) cats' global levels of stress are influenced by cats' personality; (c) cats' living style (single housing versus large group housing) does affect stress levels in cats. to our knowledge, this is the first study investigating stress levels of domestic owned cats, under natural conditions, throughout measurement of faecal glucocorticoids metabolites concentration, and taking into consideration cat personality, cat living style and owner's subjective life quality. in this study, adrenocortical activity, as a valuable physiological indicator of emotional stress, was evaluated throughout the measurement of faecal glucocorticoids metabolites in fourteen single and sixteen in-group housed cats. cat personality as well as owners life quality was evaluated by self reported questionnaires given to the owners to answer. significant differences in mean glucocorticoids metabolites concentrations (mgcm) between the two populations (i.e. single versus in-group cats) were not detected (random effect model, p . ). however, when mgcm were taken as a function of cat personality, there were differences regarding single catstimid cats showed higher levels in comparison to easy-going (random effect model, p . ) and bossy (random effect model, p . ) cats. as to owner subjective life quality, a direct association between the scores given by the owners to the social dimension and mgcm was found for single cats only (i.e. the better the owner felt itself social wise the higher the mgcm of the cat; random effect model, p . ). social stratification may compensate the stress resulting from spatial restriction in large in-group living cats. other underexplored factors such as feline personality and owner life style seem to play an equally important role in domestic cats' day to day levels of stress, especially in the cats kept as single pets. in dogs, raas activation is a major feature of congestive heart failure (chf). benazepril (fortekor s ) is a potent ace inhibitor with well-documented effectiveness in canine chf. although ace activity (ace a ) has been used in preclinical studies as a surrogate marker of efficacy, some authors have reported a poor correlation between plasma ace a and changes in angiotensin ii (aii) or aldosterone (al). the purpose of this study was to investigate the effect of benazepril on canine plasma renin activity/concentration (pra/prc), angiotensin i (ai), aii, al, and fractional excretion of potassium (ufek), sodium (ufena) and aldosterone (ufeal). sixteen beagle dogs were fed a low-sodium diet and dosed with placebo or benazepril tablets ( mg po, q h) for days. blood and urine samples were collected on day (d ) and day (d ) over -hour periods. data were analyzed by repeated measures anova of baseline corrected values, and anova of auc hours . compared with placebo, benazepril induced a significant increase in pra and ai at d (p-value [pra] : . , p-value [ai] : . ) and d (p-value [pra] : . , p-value [ai] o . ). no differences in prc were noticed. based on auc hours, aii levels were % lower in the benazepril group at d (p-value [aii] : . ). ufeal and al decreased by up to % and % at d and d , respectively, though differences did not reach statistical significance. benazepril markedly influences raas dynamics in dogs. decreased exposure to aii and al are likely to be the key events required to counteract pathological remodeling of the heart in chf. this study compared two intravenous anesthetic agents, alfaxalone (alf) (alfaxan s , jurox pty. ltd.) and propofol (ppf) (rapinovet s , schering plough animal health) and their effects on spontaneous ventilation after induction of anesthesia in dogs at various doses. this randomized, crossover, dose-escalation study used six dogs in weight and gender-matched pairs ( m- f). for each drug, each dog was dosed incrementally at , , , and times the labeled anesthetic induction dose rate (alf mg/kg, ppf . mg/kg) or until a dose was reached that rendered the dog apneic. a minimum of three days was allowed between doses. for each dose administration, the entire calculated dose was delivered constantly over min. the primary variable was apnea, defined as an absence of spontaneous ventilation for minute. apneic dogs were manually ventilated with oxygen until they resumed adequate spontaneous ventilation. once the apneic dose was determined for an individual dog for one drug, the dog began incremental doses with the alternate drug. for each anesthetic episode times were recorded from completion of induction dose to; removal of endotracheal tube, dog lifting head, dog attaining sternal recumbency and dog standing. pulse rate, respiratory rate, spo and etco were each measured every min. within-dog comparisons were made using the paired student's t-test. for both alf and ppf all dogs respired voluntarily at the labeled ( x) dose. for ppf at and x doses, and dogs respired voluntarily respectively. for alf at , and x doses, all , and dog respired voluntarily respectively. for all six dogs to become apneic required x dose of ppf and x dose of alf. the mean no observable adverse effect dose (noael) expressed as a multiple of the labeled dose was higher for alf ( . x) than for ppf ( . x) (p . ). there were no significant differences between times to extubation, head lift or attaining sternal recumbency after alf and ppf at , and x doses. at the x dose, dogs took longer to stand after alf ( . ae . min) than ppf ( . ae . min). we concluded that based on anaesthetic duration, the manufacturer's labeled dose rates of mg/kg for alf and . mg/kg for ppf were equivalent. however, based on the dose escalation, the number of dogs becoming apneic at each dose-multiple is consistent with ppf having a narrower safety margin, i.e., ppf caused more respiratory depression than alf. parenteral levetiracetam (lev) has been shown to rapidly attain therapeutic levels in dogs when given iv or im, and has been used offlabel for the treatment of seizure emergencies. the purpose of this study was to determine the safety and pharmacokinetics of subcutaneously administered levetiracetam in healthy dogs. potential application of these results would be use of sq lev instead of or in addition to rectal diazepam for the treatment of cluster seizures at home. lev was administered sq between the shoulder blades to healthy, purpose-bred hound dogs, at a dose of mg/kg (undiluted). blood samples were collected at , and minutes after lev administration via jugular venipuncture. plasma lev concentrations were measured by high pressure liquid chromatography. none of the dogs became sedated, nor was there pain evident on palpation of the injection site. mean (standard deviation) lev concentration was . ( . ), . ( . ) and . ( . ) mg/ml at , and minutes, respectively. administration of sq lev was well tolerated, and exceeded the suggested therapeutic range ( - mg/ml) within minutes of administration, and remained above the range for at least hours. these data indicate that sq lev administration may be an alternative for the at-home treatment of cluster seizures in dogs, and prospective studies in epileptic dogs are warranted. the purpose of this study was to assess the effects of cyp inhibitors (ketoconazole, chloramphenicol, fluoxetine, trimethoprim, cimetidine, and medetomidine) in varying combinations on the bioavailability of oral methadone in healthy greyhound dogs. the iacuc approved this study. cyp inhibitors were administered po for hours prior to methadone administration. methadone hydrochloride was administered po at a targeted dose of mg/kg. blood was obtained for the determination of methadone plasma concentrations by mass spectrometry. the area under the curve (auc) of methadone for each treatment group was compared statistically to the auc of methadone administered without inhibitors using the mann-whitney rank sum test. significant increases (p o . ) in the methadone auc occurred in all treatment groups which included chloramphenicol, including chloramphenicol as the only inhibitor. the magnitude of increase was at least fold. mean concentrations of methadone exceeded ng/ml for at least hours in all groups administered concurrent chloramphenicol. no significant increases in the auc occurred in any of the groups which did not include chloramphenicol. in conclusion, chloramphenicol significantly inhibits the metabolism of methadone in greyhound dogs. as a result, the oral bioavailability of methadone is significantly increased and plasma concentrations are achieved that are reported to be effective in humans for - hours after a single oral administration. doxycycline hyclate is used frequently in small animals, horses and exotic animals for treatment of a wide variety of infections. because doxycycline hyclate tablets may not be suitable for oral administration in some animals, particularly horses and cats, it has been compounded into liquid suspensions. the commercially available doxycycline calcium mg/ml oral suspension, vibramycin s (pfizer) is not suitable for use in animals due to its low concentration and flavoring that animals find unpalatable. because of the known inherent instability of doxycline in aqueous vehicles under storage, this study was conducted to determine the potency of two formulations stored in dark and light conditions. a high pressure liquid chromatography (hplc) assay with uv absorption at nm was developed for analyzing doxycycline in formulations, in comparison to a reference standard from the united states pharmacopeia (usp). doxycycline hyclate mg tablets were first tested for potency. the tablets were then crushed and mixed with a pharmaceutical vehicle to make two concentrations: . mg/ml and . mg/ml. the vehicle used was a : mixture of a vehicle for oral solution (ora-sweet, usp-nf) and vehicle for oral suspension (ora-plus, usp-nf). the suspensions were prepared in replicates of . each replicate was divided, with one aliquot stored at room temperature in lighted conditions, and the other aliquot stored at room temperature in the dark. doxycycline was extracted from the formulations and measured by hplc at day , , , , , , and . each replicate was tested and the potency reported as the percent doxycycline relative to the usp reference standard. on day , , , and , the potency of each formulation was within - % of the reference standard (range . - %). this value is within the accepted range cited in usp o on pharmaceutical compounding-non-sterile preparations. however, starting at day , the potency declined dramatically and remained low for the tests performed on day and . the potency on day , , and was below % of the reference standard (range - %). there was also a noticeable change in the quality of the formulation starting on day , and a change in the color of the formulation to a dark brown. these results indicate that when doxycycline hyclate tablets are compounded as a suspension in an aqueous vehicle as described in this study, at . and . mg/ml under the storage conditions used in this study, potency of the formulation cannot be assured beyond days. we recommend a beyond-use-day (bud) of days for formulations prepared and stored at room temperature in light or dark conditions. therapeutic options for multidrug resistance (mdr) escherichia coli urinary tract infections (uti) are limited. fosfomycin (fos) tromethamine is an oral, broad-spectrum, cell-wall active, bactericidal drug approved for treatment of uncomplicated uti in humans. the purpose of this study was to determine time dependency of fos and the disposition of fos tromethamine in dogs. using a randomized, double crossover design, client-owned dogs received fos sodium iv ( mg/kg) and fos tromethamine (po, mg/kg) either with (n ) or without food (n ). serum and urine were collected for hr; fos was quantitated with a bioassay (atcc e. coli , serum or atcc proteus vulgaris , urine). in-vitro killing curves (cell counts through hours) were performed at (control), . , , , , and x mic for mdr e. coli canine fos susceptible (e-test s ) uropathogens. killing curves indicated fos to be time dependent. after iv administration, clearance (mlÃkg/hr), volume of distribution (l/kg), elimination half-life (hl; hr) and mean residence time (mrt, hr) were (mean ae sd): ae , . ae . , . ae . , . ae . and . ae . , respectively. for po, c max , hl and mrt were ae , . ae . and . ae . , respectively. serum fos exceeded the mic reported for multidrug resistant (mdr) e. coli ( . mg/ml) for hr (iv; . mg/ml) and hr (po, mg/ml diminazene is an aromatic diamidine, anti-protozoal drug that has shown promise in a small number of cases of cytauxzoonosis. in a noncontrolled case series, of cats with clinical cytauxzoonosis given mg/ kg of diminazene aceturate survived infection. dosage frequency was two intramuscular injections given one week apart. commercial formulations contain the diminazene diaceturate salt. the active base is diminazene with the salt consisting of two aceturate molecules. currently there is no data available on the pharmacokinetics of either diminazene compound in cats. the objective of this study was to determine the pharmacokinetics of diminazene diaceturate in healthy cats. four purpose bred cats with normal physical examination, cbc, chemistry and urinalysis were used. a powdered commercial drug formulation (veriben s , ceva sanet animale) was freshly reconstituted with sterile water to a concentration of mg/ml prior to administration and sterile filtered solution. heparinized blood samples were collected just before (hour ) or . , , , , , , , , , , , , and hours after intramuscular administration of mg/kg ( . mg/kg of diminazene base) diminazene diaceturate. the plasma was separated by centrifugation within minutes of collection and frozen (À c) until analysis. concentrations of diminazene were measured by hplc analysis using uv absorption and ion-pairing conditions. the pharmacokinetic profile was analyzed using a simple one-compartment model. in these cats, diminazene had a mean terminal half life (t / ) of . ( /- . ) hrs and mean peak plasma concentration (c max ) . ( /À . ) mg/ml. the mean residence time (mrt) of diminazene was . hrs ( /À . ). systemic clearance (cl/f) was . ( /À . ) l/kg/hr. the volume of distribution per fraction absorbed (vd/f) was . ( /À . ) l/kg. a single intramuscular dose of diminazene diaceturate was well tolerated by all cats. without knowing the concentration required to inhibit or kill cytauxzoon felis, it is not yet possible to make suggestions regarding optimum dosing schedules for this drug. additional toxicology data and studies to assess clinical efficacy for the treatment of cytauxzoonosis are indicated before routine clinical use can be considered. meloxicam has been shown to accumulate in areas of inflammation in both the rat and human. the objective of this study was to investigate the concentration of meloxicam in synovial fluid of inflamed joints versus that of non-inflamed joints in dogs. eight male dogs were treated with . mg/kg of meloxicam on day one and . mg/kg of meloxicam on day two. all treatments were administered orally. on day three reversible acute synovitis was induced in one stifle by aseptic, intra-articular administration of ml sodium urate crystal suspension ( mg/ml). in four dogs synovitis was induced in the l stifle and in four dogs the same procedure was used in the r stifle. in each dog the stifle without induction of synovitis served as the ''normal'' joint sample. a synovial fluid sample was collected from both the r and l stifle of each dog. sample collection occurred eight hours after administration of sodium urate and twenty four hours after the last administration of meloxicam. synovial meloxicam concentration was analysed using high performance liquid chromatography-mass spectrometry (hplc/ ms-ms). the concentration of meloxicam in the inflamed versus non-inflamed joint in each dog was compared using the paired t-test. the results indicate that meloxicam preferentially accumulates in inflamed joints in the dog as meloxicam concentrations are statistically significantly higher in inflamed joints than in non-inflamed joints. no national surveillance system exists for monitoring emergent resistance in companion animals. however, e. coli resistance is an increasing therapeutic and public health concern in these in dogs and cats. the purpose of this study was to describe current resistance patterns of canine and feline pathogenic e. coli throughout the united states and identify risk factors of antimicrobial resistance. isolates (n ) of clinical e. coli collected from dogs or cats from may through may located in different regions. susceptibility was determined to drugs ( drug classes) by broth microdilution methods. pharmacodyamaic statistics were described regionally. phenotypes were determined and type of resistance was based on the number of drug classes to which resistance was expressed: none (ndr), single (sdr) and multi (mdr). the majority of isolates were from urinary tract ( . %) and dogs ( . %). the proportion of resistance type for each drug was: ndr ( . %), sdr ( . %) and mdr ( . %). the proportion of mdr was greatest in the southwest ( . %) and least in the northwest ( . %) (p o . ). for all regions, the proportion of resistance was: cephalothin (cph, . %) amoxicillin-clavulanic acid (amx, . %), ampicillin (amp, . %), tricarcillin-clavulanic acid (tcx, . %), doxycyline (dxy, . %) cefoxitin (cfx, . %), cefpodoxime (cpx, . %), chloramphenicol (chp, . %), enrofloxacin (enr, . %) , ciprofloxacin (cif, . %), trimethoprim-sulfamethoxazole (tmx, . %), ceftazidime(cfz, . %), gentamicin (gtm, . %), cefotaxime (cft, . %) meropenem ( . %) (p o . ). the mic exceeded the resistant breakpoint for amp, amx, cpx, cph, cif, cfx, dxy and enr whereas mic did not surpass the susceptible breakpoint. beta-lactams ( . %) was the most and aminoglycosides the least ( . %) sdr. the drug class most frequently involved in mdr was beta-lactams ( . %) and least, gen ( . %). resistance differs regionally, being greatest in the southwest. cph is the most and meropenam is the drug least associated with resistance; these patterns are consistent with current drugs used by veterinarians. the fluoroquinolones (fqs) are common choices for treatment of e. coli urinary tract infections (utis) in animals and humans. nd generation drugs approved in animals include enrofloxacin (enr), marbofloxacin (mar), orbifloxacin (orb); human drugs include ciprofloxacin (cip). rd and th generation fq for humans include moxifloxacin (mox), gatifloxacin (gat) and ofloxacin, (ofl]), its lisoform levofloxacin (lev). for animals, pradofloxacin (pra) is approved for use in europe. the purpose of this study was to assess the in vitro activity of st (naladixic acid [nal] through th generation fqs (n ) toward dog or cat e.coli uropathogens (n ). isolates were subjected to susceptibility testing to drugs classes ( drugs). isolate phenotypes included no (ndr; n ), single (sdr; n ) or multidrug (to more than drug classes; mdr; n ) resistance (including enr resistant [enr r -mdr; n ] or enr susceptible (enr s -mdr, n ). the minimum inhibition concentrations (mics) were determined for each isolates using broth microdilution (e. coli atcc s served as a negative control). mic statistics were generated for each drug among phenotypes. the overall potency (mic ) for all enr susceptible isolates (ndr, sdr and enr s -mdr) was gat pra, mox, mar, lev, cip sar, orb, ofl enr nal. each e. coli isolate expressing ndr or sdr was susceptible to all fq. however, isolates expressing resistance to st or nd generation fq were also resistance to later generation drugs. glucocorticoids (gc) are standard therapy for allergic asthma but do not reverse the underlying type i hypersensitivity. allergenspecific immunotherapy (asit), a process of ''desensitization'', is potentially curative but requires identification of offending allergens. the purpose of this study was to determine if oral or inhaled gc administered at routinely used dosages would interfere with allergen identification. we hypothesized that oral but not inhaled gc would interfere with accurate identification of allergen-specific ige using skin and serum testing in experimentally asthmatic cats. asthma was induced in eighteen cats using bermuda grass allergen (bga). cats (n /group) were randomized to receive oral gc ( mg prednisolone q hr po), inhaled gc ( ug budesonide q hr) or placebo (gelatin capsule q hr po) for one month. intradermal skin testing (idst) and bga-specific ige amounts were measured prior to, during (weeks one and four) and every two weeks after treatment until both tests were positive. a paired t test was used to compare serum ige among groups pre-and post-treatment (p o . significant). idst reactivity was eliminated in / cats on oral gc, / on inhaled gc, and / placebo-treated cats. within two weeks after stopping treatment, idst was again positive in all cats. contrary to our hypothesis, serum ige reactivity to bga was not significantly diminished by any treatment. in conclusion, a two week withdrawal from gcs is adequate for idst identification of allergen but no withdrawal is required prior to serum ige testing to identify the sensitizing allergens. previously in people, increasing severity of asthma is associated with low serum concentrations of -hydroxyvitamin d ( -oh-d). -oh-d is thought to ameliorate lower airway inflammation primarily by decreasing the production of pro-inflammatory mediators, and by increasing the production of the anti-inflammatory cytokine il- . in people, serum -oh-d concentration is associated with sunlight exposure as well as dietary intake. cats do not rely on sunlight for vitamin d synthesis; all vitamin d comes from dietary intake. cats have a naturally occurring lower airway disease syndrome (lad) that shares many features with human asthma. the goal of this study was to evaluate serum -oh-d concentrations in cats with lad. cats with naturally developing lad were enrolled. criteria for a diagnosis of lad included a history of cough, wheeze or respiratory distress, radiographic evidence of a bronchial pattern and hyperinflation, negative heartworm antigen and antibody test, and a resolution of clinical signs in response to glucocorticoids. dietary history was obtained. -oh-d concentrations were determined on serum samples by a commercial laboratory. twelve cats with lad were enrolled. all cats ate commercial cat food. the median -oh-d concentration was nmol/l with a range of - nmol/l which is within the reported reference range of - nmol/l. in contrast to human asthma, lower airway disease in cats is not associated with low serum concentrations of -oh-d. interstitial lung diseases (ild) are uncommon in dogs, with the most commonly recognized ild idiopathic pulmonary fibrosis (''westie fibrosis''). in human medicine, ild represent a large umbrella of pulmonary diseases, with ipf only a subset. other, more treatable, ilds are also identified, and may respond to either the removal of a stimulus (hypersensitivity) or steroid therapy. the goal of this report is to describe the clinical course, including outcome, computed tomography and histopathology of dogs affected with an ild. the computed tomography (ct) log was reviewed for dogs that underwent thoracic ct scanning for evaluation of respiratory signs, and had changes consistent with ild as the primary abnormality, including the presence of diffuse disease in all lobes, and at least of the following: reticulation, ground glass opacity, consolidation, or traction bronchiectasis. survival time from ct date was calculated. the presence of moderate pulmonary hypertension [phtn] ( mmhg) as estimated by tricuspid regurgitant jet, was also reported and survival times were compared with a mann-whitney rank sum with p o . considered significant. thirteen dogs were identified. terriers and chihuahuas were the most commonly affected breeds. two dogs were adolescents, the remaining dogs ranged from - years, with a median of years. histopathology results (n ), including moderate to severe interstitial fibrosis ( ) alveolar proteinosis with fibrosis ( ), and interstitial eosinophilic pneumonia ( ). one had suspected cryptogenic organizing pneumonia and had a good response to glucocorticoids. eight dogs died of respiratory failure, with a median post ct survival time of days (range - ), two dogs died of non-pulmonary disease, dogs had severe lower respiratory infections as puppies with persistent respiratory signs, and both are still alive at years since diagnosis, terrier is alive at months and was lost to follow up. dogs had phtn, with a median survival of days ( - ), while the dogs without had a survival of days (range - ), [p . ]. interstitial lung disease in dogs is not just idiopathic pulmonary fibrosis. following respiratory infection, young dogs may develop an ild with a relatively indolent course and rare ild is steroid responsive. ct is useful to identify ild but further research correlated with echocardiography and histopathology is advised to use it to prognosticate. idiopathic pulmonary fibrosis (ipf) is an interstitial pulmonary disease, mainly described in west highland white terriers (whwt). identification of molecular pathways important in the pathogenesis of ipf would improve our understanding of this disease and may help identify therapeutic targets. the aim of the present study was to investigate gene expression in lungs of whwt with ipf using oligonucleotide microarray. total rna was extracted from post-mortem pulmonary samples from five whwt with ipf and five control dogs (ctrl) without pulmonary disease. the rna was pooled from each group (ipf and ctrl) and analysed using the canine specific affymetrix microarray technology. genes with a minimum of a two-fold difference in expression between the two groups were selected for further analysis. the most significant biological functions for these genes were identified using ingenuity pathways analysis. more than genes were identified as having greater than twofold difference in expression. the significant biological functions associated with these genes were related to cellular movement, cellular proliferation and apoptosis. most notable among these were genes encoding the leukocyte chemotactic proteins: ccl (fold change . ), ccl ( . ) and il ( . ); the proteins involved in fibroblast migration; and the matrix metalloproteinases (mmps) involved in matrix degradation: mmp (À . ), mmp (- . ), mmp (À . ). this study has identified genes which may be important in pathogenesis of ipf, e.g. proteins involved in leukocytes chemotaxis, fibroblast recruitment and activation, regulation of apoptosis, and extracellular-matrix turn-over. however, real-time quantitative rt-pcr studies are needed to confirm these results before any definitive conclusions can be drawn. idiopathic pulmonary fibrosis (ipf) is an interstitial disease, mainly described in west highland white terriers (whwt). defini-tive diagnosis ultimately relies on lung histopathology. identification of specific biomarkers would be very helpful. expression microarray is a powerful screening tool to study local gene expression in a disease state. the aim of the present study was to measure gene expression profiles in lungs of whwt with ipf to identify potential blood or bronchoalveolar lavage fluid (balf) biomarkers. total rna was extracted from post-mortem pulmonary samples from five whwt with histopathologically confirmed ipf and five control dogs (ctrl) without pulmonary disease. the rna was pooled from each group (ipf and ctrl) and analysed using the canine specific affymetrix microarray technology. ipa-biomarkers analysis (ingenuity system) was used to filter and prioritize biomarkers candidates using the three following criteria: a minimum of a two-fold difference in expression between ipf and ctrl; expression of the gene in lung tissue; possible detection of the protein in blood or in balf. fifty-four molecules met all the criteria. based on difference in expression, promising proteins included ccl (fold change . ), a -actinin ( . ), ccl ( . ), serum amyloid a ( . ), il ( . ), plunc (À . ), mmp (À . ). some are well-known biomarkers of ipf in humans either for diagnosis (mmp , il ) or prognosis (ccl ). these results provide novel potential biomarkers of canine ipf. measurement of these proteins in blood and balf of healthy dogs, dogs with ipf and with other respiratory diseases is needed to assess their use as biomarkers of canine ipf. heliox is a mixture of helium and oxygen that has been used therapeutically in human medicine for treatment of airway obstruction. helium's low density and other physical properties have been shown to reduce the work of breathing by limiting turbulence. the purpose of this study was, therefore, to evaluate respiratory parameters in response to inhaled heliox in dogs with meso-and brachycephalic conformation. eleven healthy dogs were recruited, five were mesocephalic and six were brachycephalic. flow-volume loops were collected using commercial software (buxcor) while breathing : helium: oxygen (heliox) and : nitrogen:oxygen (nitrox) in a randomized order via a low dead-space face mask. due to the intrinsic gas properties, gas flow rates and volumes were corrected in-vitro by a conversion factor for the effect of helium on the pneumotachograph. respiratory rate, tidal volume (ml), minute ventilation (l), inspiratory time (ti), expiratory time (te), peak inspiratory flow (pif) and peak expiratory flow (pef) were recorded while breathing heliox or nitrox. values were compared using a paired sample t-test, with p o . considered significant. all dogs cooperated with testing. there was no significant difference in respiratory rate, tidal volume, minute ventilation, inspiratory or expiratory times, or peak inspiratory flow. peak expiratory flow was significantly higher (p . ) while breathing heliox than when breathing nitrox in brachycephalics but not in mesocephalics (p . ). heliox is well-tolerated in healthy dogs and results in an increased expiratory flow rate in brachycephalic dogs. further investigation of heliox is warranted in dogs with airway obstruction. of this prospective multicentric study is to assess the effects that surgical correction has on the severity of clinical signs and levels of acute phase proteins (c-reactive protein [crp] , haptoglobin [hp]) and cardiac troponin i (ctni). thirty three brachycephalic dogs with boas were included and evaluated before and, approximately two months, after surgical correction. the most common components of boas found were elongated soft palate ( / ; %), stenotic nares ( / ; %) and everted laryngeal saccules ( / ; . %). staphylectomy was performed by means of two different surgical techniques: laser (n ) or electrical scalpel (n ). there were significant differences between dogs depending on the surgical technique used, with a higher reduction of respiratory signs (p o . ) and a better postsurgical improvement (p o . ) with the use of laser. the levels of crp, hp and ctni were categorized into normal or elevated. before surgical treatment three ( . %), six ( . %) and thirteen ( . %) dogs had elevated values of crp, hp and ctni, respectively. two months after surgical correction, five ( . %), eleven ( . %) and fourteen ( . %) dogs had elevated values of crp, hp and ctni, respectively. there were no statistical differences between values of crp and ctni before and after surgical correction but the levels of hp increased significantly after surgical treatment (p o . ), probably due to postsurgical treatment with corticosteroids. as previously suggested by others, there was a statistically significant reduction of respiratory and gastrointestinal signs in dogs with boas submitted to surgical correction (p o , ). according to the results obtained in the present study, the determination of crp, hp and ctni before and two months after surgical treatment do not have a prognostic value in dogs with boas. even though, near half of the dogs studied had elevated levels of ctni ( . %) that persisted after surgical treatment ( . %), suggesting some degree of myocardial damage is present. further studies are needed considering the influence of breed and age. to the authors' knowledge, this is the first description of crp, hp and ctni determination in dogs with boas. overweight and obesity are common conditions that lead to alterations in respiratory mechanics, airway resistance, pattern of breathing and gas exchange in humans. the objective of the present study was to investigate if there are significant differences on respiratory parameters and arterial gas analysis of obese and overweight cats, in conscious state and under general anesthesia. twenty nine adult cats were arranged in three groups: obese (n ), overweight (n ) and with ideal body score index (bsi) (n ). mean of bsi in the groups were: , (obese), , (overweight) and , (ideal bsi). cats did not had respiratory, cardiac or others systemic diseases. the respiratory parameters were evaluated with a ventilometer equipment coupled to facemasks in conscious cats and directly to the endotracheal tube in anesthetized cats under spontaneous respiration. the anesthesia was performed with propofol ( ae , ml/kg) and the cats were maintained in the same anesthetic plan. the three groups were compared by analysis of variance followed by tukey's test and conscious and anesthetized cats were compared by student's t test, with a % significance level. there were not observed differences on the respiratory parameters evaluated on ventilometry (tidal volume, expiratory and inspiratory times and peak pressures, respiratory rate and partial pressure of end tidal co (petco )) and on arterial gas parameters (pao e paco ) in the three groups. the pao of cats with ideal bsi was , ae , mmhg, although was not significantly different (p , ) from overweight ( , ae , mmhg) and obese cats ( , ae , mmhg). comparison of anesthetized to conscious cats, it was detected decreases in tidal volume, expiratory and inspiratory times and peak pressures and increase in petco in respiratory rate in the anesthetized cats. only petco , inspiratory time and respiratory rate in overweight cats did not differ in anesthetized cats. these results suggest that obesity and overweight did not result in impairment of respiratory function in cats and propofol induced respiratory depression. osteosarcoma (osa) is the most common bone tumor in dogs, however, little is known regarding the mechanisms underlying malignant transformation in these tumors. breeds such as rottweilers and greyhounds are at higher risk for developing osa, suggesting that heritable factors play a role in this disease. mirnas have tumor/tissue specific roles in regulating gene expression and dysregulated mirna expression is found frequently in cancer. we hypothesize that canine osa is characterized by a unique mirna expression profile(s) with dysregulation of some mirnas being associated with specific breeds. mirna expression profiling of primary osa tumors from greyhounds and rottweilers was performed using the nanostring technologies ncounter mirna expression assay kit, interrogating the mirna expression profile of human mirnas, of whose mature sequences are % conserved between human and dog. mirnas were differentially expressed in greyhound versus rottweiler tumors (p o . ), suggesting that breed-specific dysregulation of mirnas may contribute to the development and progression of spontaneous osa. hierarchical clustering revealed distinct mirna expression signatures in greyhound osa tumors as compared to rottweilers. based on these preliminary results, we are evaluating a larger cohort of osa tumor samples including greyhounds, rottweilers, golden retrievers, and a mixed population of other breeds. statistical analysis will be performed to determine the association of mirna transcript levels with specific breeds and overall outcome. characterization of mirna expression in canine osa will facilitate our understanding the biology of this disease and has the potential to identify targets for therapeutic intervention. originally combination therapies using drugs with documented single-agent activity and lack of overlapping toxicities could potentially improve outcome. the hypothesis intended to be tested is that palladia s can be safely administered concurrently with a standard weekly protocol of vinblastine (vbl), at dosages known to have activity against mast cell tumors. dogs with histologically confirmed measurable mast cell tumors were evaluated for eligibility to enter a standard phase i dose-finding trial ( cohort), at a starting dose of . mg/m iv vbl (weekly for a total of treatments) and . mg/kg po palladia s eod, concurrently. dose escalation of palladia s was scheduled in . mg/kg increments until mtd was established or fda label dose completed ( . mg/kg). safety evaluation was performed weekly throughout the week study period. dose-limiting toxicities were described following established vcog-ctcae(v . ) criteria. while antitumor response is not a primary endpoint of phase i trials, activity was documented prior to vbl treatments - , and monthly thereafter, based on recist criteria. nine dogs have been enrolled; cohort is filled and approaching completion of the evaluation period. hematologic dose limiting toxicity led to de-escalations of vbl. the current safe combination appears to include vbl at . mg/m every other week and palladia s at . mg/kg eod. response was seen in all but one dog. without head to head trials comparing efficacy of bi-weekly vbl combined with palladia s and vbl alone, choice of therapy should remain at the clinician's discretion. originally prostate specific membrane antigen (psma) is a transmembrane protein expressed by tumor-associated neovasculature, but not normal blood vessels. based upon its selective expression in endothelial cells associated with cancer, psma may serve as a conserved angiogenic target shared by macroscopic solid tumors of various histologies. to investigate the feasibility of targeting a homogenous population of psma-expressing endothelial cells as a novel anticancer strategy, we have investigated psma expression in several canine hemangiosarcoma (chsa) cell lines, and subsequently developed self-assembling nanoparticles containing diagnostic (near infrared dyes) and therapeutic (doxorubicin) cargo which selectively bind to psma by means of the a aptamer, a commercially-available oligonucleotide. the expression of psma by chsa cells was confirmed transcriptionally and translationally by real time pcr and immunohistochemistry, respectively. selective binding and endocytosis of a decorated nanoparticles was studied by fluorescent microscopy. the ability of a decorated nanoparticles encapsulating doxorubicin to exert in vitro cytotoxic effects in chsa cells was assessed by colony forming assays. using a chsa xenograft murine tumor model, clinically-relevant anticancer effects of a decorated nanoparticles encapsulating doxorubicin were tested. all chsa cell lines expressed psma mrna and protein. a decorated nanoparticles were selectively endocytosed by psma-expressing cells, and when these nanoparticles encapsulated doxorubicin, significant cytotoxic effects were exerted in vitro. finally, a decorated nanoparticles encapsulating doxorubicin significantly reduced the size of macroscopic chsa tumor burdens in transplanted mice. diagnostic and therapeutic nanoparticles can be targeted to psma-expressing endothelial cells, and chsa provides a comparative model for the future study of nanoparticle therapeutics. canine transitional cell carcinoma (tcc) is the most common tumor of the urinary tract, and is similar to human invasive tcc in histopathologic characteristics, molecular features, sites of metastasis, and response to medical therapy. prevalence is increasing, and novel therapies and strategies are needed to effectively treat this aggressive form of cancer in both species. personalized medicine techniques intend to improve treatment outcome by using patient tumor profiling to identify potential and individualized therapeutic targets. a genomic algorithm has been developed termed ''coexpression extrapolation'', or coxen, that aims to use expression microarray data to predict drug activity in patient tcc samples. the utility of this predictive methodology has been established in other types of cancer in vitro, however its clinical utility has not yet been determined. validation studies of coxen in canine tcc cell lines were conducted. the goal was to determine the value of coxen in predicting baseline sensitivity of canine tcc to chemotherapy agents (gemcitabine, mitoxantrone, carboplatin, vinblastine and cisplatin) that would then be used in a proposed clinical trial. additionally, expression data from canine treatment-naı¨ve primary tumor samples were generated on an affymetrix array platform (canine genome v . ). both the expression data and tcc cell line data (antiproliferative effects, % growth inhibition or gc ) were used to establish a canine specific predictive coxen algorithm. coxen scores for canine tcc cell-line drug activity were then analyzed. scores predicted the activity of cisplatin, gemcitibine, and mitoxantrone in all cell lines, and of carboplatin in cell lines. because all of the cell lines were sensitive to vinblastine (gi o . mm), the coxen score was not predictive of its potency. interestingly, coxen fails to predict vinblastine response in human tcc cell line data as well. in concurrent work, comparative genomic studies to define and compare the gene expression signatures of tcc in dogs and humans provides further evidence that canine tcc is a valuable genomic model of the human disease. current studies involve testing the chemo-predictivity of this derived canine coxen algorithm in additional canine tcc cell lines. canine tcc offers an excellent model for in vitro and in vivo studies of the coxen approach. this preclinical work will be used to guide the feasibility of future coxen clinical trials in dogs and humans with tcc. a small molecule complex (aminoact) isolated from bovine milk is a natural peptide mixture with multi-kinase inhibitory effects against epidermal growth factor receptor (egfr) and insulin-like growth factor receptor- (igfr- ). ingestion of aminoact in people with cancer results in lower serum tnf-alpha, an increase in antioxidant superoxide dismutase (sod) enzyme activity, and subjects' blood serum causes apopsotis in cancer cell lines. this study was designed to first assess safety and secondly the efficacy of three dosage levels of ax- in sustaining progression free survival (pfs) for dogs with refractory advanced and/or metastatic cancer. the prospective, open label study included dogs of different breeds with naturally occurring histologically confirmed malignancies. the first dogs received aminoact at g/m ; the second group of dogs subsequently received the same dosage mg of aminoact; and the third group of dogs subsequently received g/ m . each dog was treated orally daily for six weeks along with mg betaine hcl, that aids in peptide absorption. all patients were evaluated for toxicity using the vcog-ctcae and efficacy using the recist criteria via assessment of clinical parameters, blood work and client questionnaires. no toxicity other than mild, transient (grade i) nausea was noted, nor were there any changes in hemograms or biochemical profiles in any patient. dogs with tumors that were confirmed as responders ( % reduction in size) include pulmonary adenocarcinoma, mast cell tumor, trichoepithelioma and soft tissue sarcoma. it appears in limited studies that the response rate may be more durable at higher dosages. the response to aminoact is dose dependent and only transient mild toxicity was observed, which suggest the maximum effective dosage has not been reached. further clinical studies will be valuable in determining the effective dosage and response duration. treating cancer in dogs with aminoact offers a unique opportunity as a model for human cancer biology and translational cancer therapeutics. stereotactic radiation therapy (srt) combines patient immobilization, image guidance, and intensity modulated delivery to achieve ablative radiation doses within the tumor, while preferentially sparing surrounding normal tissues. the purpose of this study was to evaluate the efficacy of srt as a means of achieving local tumor control for canine nasal tumors. retrospective analysis was performed on dogs with a nasal tumor confirmed by histopathology and computed tomography, no previous surgical or radiation therapy, at least six months of follow-up, and completion of three fractions of srt at csu.srt was administered via the varian trilogy linear accelerator once daily for three consecutive days. the varian eclipse treatment planwas reviewed to determine the planned target volume (ptv) and dose to % of the ptv. kaplan-meir survival analysis was performed for disease free interval (dfi) and overall survival (os). sixteen patients with nasal tumors ( adenocarcinoma/carcinomas, squamous cell carcinomas, chondrosarcomas, osteosarcomas, and undifferentiated sarcoma) were treated with srt. a median dose of . gy was administered to % ptv with a median ptv of . cc. srt was well tolerated by the normal tissues with minimal, manageable side effects. to date, the median dfi is days, while the median os is days. based upon the initial clinical experience, stereotactic radiation therapy is an emerging modality in the management of canine nasal tumors. canine leptospirosis can vary from subclinical infection to illness that ranges from mild to severe, including death, depending on the susceptibility of the dog, virulence of the organism, and route and degree of infection. the objective of this study was to evaluate the ability of a canine leptospira bacterin to prevent infection and disease following challenge with virulent leptospira canicola, l. pomona, l. grippotyphosa, or l. icterohaemorrhagiae. groups of week-old beagles were vaccinated (day ) and boosted (day ) with placebo (n ) or the -way bacterin (n ! ) and subsequently challenged with each serovar. the results demonstrated that blood and various tissue samples from placebo-recipients became reliably infected, and the dogs developed typical clinical signs of leptospirosis including loss of appetite, ocular congestion, depression, dehydration, jaundice, hematuria, melena, vomiting, petechiae, and death. in addition, placebo-recipients developed kidney and liver dysfunction. in contrast, some vaccine-recipients became infected, but the organisms were cleared quickly from the blood. vaccinated dogs failed to develop severe clinical disease requiring medical intervention, and no animals died (p ! . ). a few of the vaccinated dogs developed clinical abnormalities, but the clinical signs remained mild and were self-limiting (p o . for each serovar). administration of the bacterin also prevented thrombocytopenia ( ciprofloxacin, a synthetic fluoroquinolone antimicrobic, is not fda-approved for veterinary use. however, due to recent availability of less expensive generic formulations, extra-label use of ciprofloxacin by veterinarians appears more common. although ciprofloxacin crystalluria and uroliths have been reported in humans, we are unaware of any published reports in dogs. this is surprising since mean urine ciprofloxacin concentration ( . mg/ml) in dogs following a modest iv dose ( mg/kg) was times higher than the solubility of ciprofloxacin in water ( . mg/ml). to identify the occurrence of ciprofloxacin uroliths in dogs, records from the minnesota urolith center were reviewed. between january and december , ciprofloxacin was identified in uroliths from dogs; uroliths were composed of % ciprofloxacin in , mixed uroliths containing ciprofloxacin were identified in , a shell of ciprofloxacin was observed in , and ciprofloxacin surface crystals were identified in . based on an experimental study in which % of human volunteers consuming mg of ciprofloxacin with nahco exhibited ciprofloxacin crystalluria (urine ph . ), while no volunteers consuming mg of ciprofloxacin and nh cl to acidify urine formed crystals; we postulated that ciprofloxacin uroliths could be dissolved in acidic urine. to test this hypothesis, canine uroliths composed of % ciprofloxacin from a single source ( -yr-old male, english bulldog receiving mg/kg of ciprofloxacin po, q hr to manage superficial pyoderma; turbulent flow chromatography/tandem mass spectrometry detected mg of ciprofloxacin/g of urolith) were incubated in urine at selected ph's and monitored for dissolution. urine obtained from multiple dogs not receiving fluoroquinolones, was pooled and divided into aliquots. aliquots were adjusted with hcl or naoh to a ph of , , , , or . aliquots were capped and preserved by refrigeration; ph was monitored and readjusted weekly. ten uroliths of approximately equal weight were randomly assigned to individual flasks containing mls of urine. flasks were constantly agitated and maintained at c. every hours, urine was discarded and replaced with mls of urine of identical ph until stone dissolution was complete. ciprofloxacin urolith dissolution times at each urine ph are reported below. ciprofloxacin uroliths are a newly recognized disease and a potential adverse effect of ciprofloxacin administration in dogs. in vitro dissolution of ciprofloxacin uroliths was achieved in canine urine, supporting the premise that in vivo dissolution is possible. urolith dissolution times were shortest at lower and higher ph's, which is consistent with the pka ( . and . ) of this amphiprotic antimicrobic (more soluble at ph below the acidic pka and above the alkaline pka). foods designed to promote struvite urolith dissolution may be designed for short term feeding facilitating rapid dissolution or may be formulated with a more moderate target urine ph to allow for dissolution and then life-long maintenance feeding minimizing recurrence. the purpose of this study was to compare the efficacy and rate of dissolution of a maintenance food with a struvite dissolution food. sixteen client-owned adult cats ( fs, mc) with naturally occurring struvite urocystoliths (mineral composition based on history, radiographs, urinalysis, urine culture and physical examination) were randomized to either a dry maintenance food (test) or a dry food known to dissolve struvite uroliths (control). the clinical care team and owner were blinded to treatment assignment. the test food was formulated to provide . % mg (dm), . % p, % protein, and a calculated target urine ph value (uph) of . - . . the control food was formulated to provide . % mg (dm), . % p, % protein, and a targeted urine ph of . - . . owners were advised to feed the assigned diet exclusively in an amount to maintain body condition. after diet assignment radiographs were performed at eight weekly intervals until there was no evidence of uroliths or until there was evidence that the uroliths were the same size or larger. a physical examination, complete blood count, serum chemistry profile, urinalysis and urine culture were repeated at the conclusion of the study. statistical analysis was by anova. all uroliths dissolved in all cats and both foods were palatable. radiographs of cats fed the control food indicated the uroliths dissolved in a significantly shorter time (mean ae std dev of . ae . weeks) compared to cats consuming the test food (mean . ae weeks; po . ).). cats in the control group finished the study at (n ), (n ) and weeks. cats in the test group finished the study , , (n ), , , , and weeks. all the minnesota urolith center occasionally receives uroliths for analysis that are immersed in formalin. results of quantitative analysis of these uroliths revealed that some submitted in formalin consisted of newberyite (magnesium hydrogen phosphate trihydrate). because newberyite is uncommonly found in uroliths formed by cats and dogs, we hypothesized that this mineral was an in vitro artifact caused by exposure of struvite (magnesium ammonium phosphate hexahydrate) to formalin. the purpose of this study was to determine if formalin alters the mineral composition of uroliths. urolith submissions containing stones of either % struvite (n dogs and cats), % calcium oxalate (n dogs and cats), % calcium phosphate apatite (n dogs and cats), % cystine (n dogs and cats), % ammonium urate (n dogs and cats), and % silica (n dogs) preserved by only air drying were tested. one urolith from each submission was quantitatively analyzed by polarized light microscopy or infrared spectroscopy. a subsequent urolith from the same submission was immersed in ml of % buffered formalin for hours at room temperature. uroliths were then air dried for minutes and the analysis was repeated. after exposure to formalin, portions of all struvite uroliths were transformed into newberyite. three ( dog and cats) of ammonium urate uroliths were completely dissolved. newberyite was not detected in any of the remaining uroliths. likewise quantitative mineral analysis of non-struvite uroliths remained unchanged. to avoid misdiagnosis of mineral composition, uroliths should not be immersed in formalin prior to analysis. we previously reported that transfusion to normal dogs of autologous erythrocyte concentrates (prbcs) that had been stored for days causes a profound inflammatory response ( x increase in leucocyte count and fibrinogen, x increase in c-reactive protein). we speculated that inflammation was due to cytokines produced during the storage period, and hypothesized that transfusion of fresh (f) prbcs would elicit less inflammation than would stored (s) prbcs. a whole blood unit was collected from healthy dogs (n ) for prbcs on day , then again on day . on day dogs received an autologous transfusion of prbcs stored for either days (s, n ) or days (f, n ). cbcs and in-tem thromboelastometry (ct:coagulation time, cft:clot formation time, a:alpha, mcf:maximum clot firmness) were evaluated on blood samples collected at (pre) and , , , , and hours after transfusion. fresh prbcs did not elicit any change in leucocytes, platelets, or thromboelastometry. stored prbcs elicited a degenerative left shift ( hr) followed by a regenerative left shift ( - hr), thrombocytopenia ( % decrease at hr), and marked hypocoagulability characterized by prolonged ct ( , , hr) and cft ( , hr), and decreased a ( , hr) and mcf ( , , hr). data are mean(sd). a: p o . between groups f and s by t test. b: p o . compared to '' '' by rm anova. transfusion of autologous stored prbcs elicits a greater inflammatory response than fresh prbcs, and results in hypocoagulability on thromboelastometry. clopidogrel is a potent antiplatelet drug that is gaining popularity in veterinary medicine for antithrombotic therapy. the parent molecule is an inactive prodrug that must be converted by hepatic isozymes to an active metabolite. the majority of the parent molecule is directed to the formation of inactive metabolites with only an extremely small proportion of parent molecule directed to the formation of the active metabolite. there are multiple hepatic isozymes responsible for the formation of the active metabolite. a non-specific hepatic isozyme inducer such as rifampin could increase the formation of the active metabolite of clopidogrel thereby increasing the pharmacodynamic response which may allow a reduced drug dose to achieve a clinical effect. we have previously presented data supporting the increased pharmacodynamic response of clopdiogrel after rifampin therapy. the goal of this study was to demonstrate an increased pharmacokinetic response of clopidogrel after rifampin induction of hepatic isozymes. six healthy, purpose-bred dogs were used for this study. the pharmacokinetics of clopidogrel were determined by measuring the parent molecule, primary inactive metabolite and active metabolite through lc/ms/ms. the pharmacodynamics of clopidogrel were determined by measuring collagen-induced whole blood aggregation. blood samples were collected prior to clopidogrel administration (baseline), after days of mg/kg clopidogrel po q hrs, and after days of mg/kg clopidogrel po q hrs mg/ kg po q hrs rifampin. given the absence of a known standard for the active metabolite, only a semi-quantitative assessment of active metabolite concentration can be made. there was no identifiable active metabolite peak noted at baseline or after clopidogrel treatment. however, with clopidogrel and rifampin combined administration there was an active metabolite peak identified in all dogs with a mean area of . ae . . the development of the active metabolite peak was associated with an increase in the pharmacodyamic response of clopidogrel in the dogs. this is the first study in any species to document the increased formation of the active metabolite of clopidogrel in response to a strong, non-specific hepatic isozyme inducer. this increased pharmacokinetic response was associated with an increased pharmacodynamic response of clopidogrel. this data provides supportive evidence to develop therapeutic protocols to improve the pharmacodynamic response to clopidogrel in dogs that may reduce dosing requirements or correct subtherapeutic pharmacodynamic response. critical illness-related corticosteroid insufficiency (circi) has been identified in humans, foals, dogs and cats with lower-thanexpected circulating cortisol concentrations, and/or by a blunted cortisol response to acth stimulation. our purpose was to determine if circi exists in critically ill horses. endogenous plasma acth and serum cortisol concentrations, and cortisol at t and t min after . mg/kg cosyntropin, were measured by radioimmunoassay from horses with colic or systemic illness on admission, and days , and of hospitilization. horses were divided into mild, moderate, or severe illness groups based on clinicopathologic data. inappropriately low cortisol was defined as endogenous cortisol o mean- sd achieved after administration of . mg/kg cosyntropin to normal horses ( o nmol/l). inadequate delta cortisol was defined as o mean delta cortisol in normal horses after . mg/kg cosyntropin ( o nmol/l). cortisol, acth and delta cortisol were compared using anova between groups, with p o . considered significant. fifty-eight horses classified as having mild ( ), moderate ( ) and severe ( ) disease at admission had survival rates of %, % and % respectively. admission acth and cortisol concentrations were highest in severely ill horses ( ae pg/ml, ae nmol/l) compared to moderate ( ae , ae ) and mildly ill horses ( . ae . , ae ). admission cortisol concentrations were higher overall in severely ill horses (p . ), but were low in % ( / ). admission delta cortisol was low in % ( / ) of severely ill horses, and was associated with marked adrenal hemorrhage in non-survivors. severely ill horses have high cortisol and acth, but low cortisol and delta cortisol may indicate circi secondary to adrenal hemorrhage. equine pituitary pars intermedia dysfunction (ppid) is a common endocrinopathy of aged horses that results from neurodegeneration of the dopaminergic periventricular neurons that innervate the intermediate lobe of the pituitary. factors that initiate spontaneous dopaminergic neurodegenerative disease remain elusive, however accumulation of misfolded a-synuclein protein and dysfunctional protein clearance have been implicated. misfolded protein accumulation occurs due to increased protein production or decreased clearance of damaged macromolecules through the process of autophagy. while have previously demonstrated that horses with ppid have increased asynuclein in the periventricular neurons compared to controls, it remains unknown whether the protein accumulates due to increased production or decreased clearance. we hypothesized that autophagy is decreased in the pituitary neurointermediate lobe from horses with ppid compared to controls. neurointermediate lobe pituitary tissue was from collected from horses with ppid (n ) and healthy horses (n , - years). realtime pcr was used to determine the relative expression of autophagy genes (mtor, beclin , atg , atg , atg , pink, lamp ) and a-synuclein relative gene expression from horses with ppid were compared to healthy horses by t-test following log transformation. a pearson coefficient of correlation was calculated comparing a-synuclein expression with autophagy gene expression. the expression of a-synuclein, autophagy-related genes (atg , beclin, lamp ), and mtor was greater in horses with ppid than in healthy horses. age was not correlated to a-synuclein or autophagy gene expression. there was a significant positive correlation between expression of a-synuclein and beclin , atg , atg , atg , and pink, but not mtor expression. accumulation of a-synuclein protein in horses with ppid may result from increased a-synuclein expression. autophagy genes are upregulated in horses with ppid, suggesting a compensatory response, although these findings need to be confirmed by demonstrating an increased functional response. asynuclein expression was positively correlated to expression of autophagy genes except mtor, suggesting a-synuclein may stimulate autophagy in an mtor independent manner. acvim forum session a efficacy of delayed antiviral therapy against ehv- challenge. lk maxwell , ll gilliam , n pusterla , r carmichael , rw eberle , jw ritchey , tc holbrook , t gull , gb rezabek , d mcfarlane , cg macallister . oklahoma state university, stillwater, ok. university of california, davis, ca. equine herpes virus type- (ehv- ) outbreaks are often not recognized until exposed horses are at immediate risk for developing equine herpes myeloencephalopathy (ehm). the objective of this study was to determine whether delayed therapy with the antiviral drugs valacyclovir or ganciclovir could protect those horses most at risk for ehm. eighteen aged ( years) mares were randomized to treatment: no therapy (control), oral valacyclovir therapy, or intravenous ganciclovir therapy. drug administration was initiated at the onset of the second febrile phase, between days - after ehv- inoculation (pi), and continued for one week. neurological examinations were performed prior to the study and for three weeks pi. one horse was excluded from the study for failure to become febrile. body temperature was significantly lower in the ganciclovirtherapy horses as compared to control horses on days - pi (p o . ), whereas valacyclovir-therapy horses did not differ from control horses. viremia in whole blood, as determined by pcr, was also lower in the ganciclovir-therapy horses on days - pi and on day pi in the valacyclovir-therapy horses (p o . ). although antiviral drug administration did not reduce the risk of ataxia (p . ) or nasal shedding, ganciclovir therapy did decrease the severity of ataxia (p o . ) as compared to valacyclovir-therapy and control horses, where / , / , and / horses, respectively, developed at least a two grade change in ataxia. in summary, ganciclovir administration provided better protection against ehm than did valacyclovir when therapy was initiated just prior to the onset of neurological disease. equine vaccination is amongst the most important method of prophylaxis against equine influenza virus (eiv), a pathogen in which continuous antigenic drift can lead to vaccine failure. a month duration of immunity (doi) challenge infection study was conducted using commercial inactivated vaccines containing different strains of a/equine/ /influenza virus's, including innovator tm , containing kentucky/ (pfizer animal health, new york, ny), and calvenza, containing a combination of ohio/ , kentucky/ , and newmarket (boehringer ingelheim vetmedica, st. joseph, ms) . the challenge virus strain was colorado/ , the most contemporary challenge strain currently in use. the study design was a blinded, randomized challenge trial. three groups of yearling ponies, with no history or serological evidence of eiv infection were established. each group received one of three treatments: vaccination with innovator tm ; vaccination with calvenza tm ; or injection with a saline placebo. each treatment was administered times, at intervals of month between the first two treatments, and months between the second and third treatments. all ponies were challenged by nasal nebulization of x eid influenza virus a/eq/ /colorado/ months after the third treatment. clinical signs of disease, including rectal temperature, nasal discharge, anorexia, coughing, and depression, were recorded daily for days prior to challenge infection, and days post-challenge. nasal shedding of eiv was measured on the same days, using a realtime pcr test procedure. eiv-specific antibody responses were measured by elisa. differences between groups were analyzed by non-parametric repeated measures anova, and differences were declared significant when p o . . all control group ponies demonstrated clinical signs of disease consistent with eiv infection post-challenge infection, including pyrexia, nasal discharge, inappetance and partial anorexia. these signs were significantly lower in both vaccine groups; mean body temperature was elevated ( . f) for days in controls, but only days in vaccine groups. nasal shedding of eiv was detected in all ponies in all groups: over the duration of the study the calvenza group shed significantly less virus than innovator and control. over time antibody titers were significantly higher in the calvenza than the innovator group, and both were significantly greater than controls. this study demonstrated that both current commercial inactivated eiv vaccines have a duration of clinical protection of at least months after a highly pathogenic challenge with a recent eiv isolate. both antibody responses and virological protection differed between the vaccines. formulation difference between the vaccines, including the eiv antigens employed, may have contributed to this performance difference. degenerative myelopathy (dm) may be homologous to a form of amyotrophic lateral sclerosis in humans which has excitotoxic and immunologic pathogeneses described. the aims of this study were to determine (i) presence or absence of abnormalities in concentrations of csf amino acid (aa) neurotransmitters (glutamate, glycine and gÀaminobutyric acid (gaba)) and cytokines in dogs with dm and if present (ii) investigate associations with disease severity. twenty-two dogs histopathologically confirmed for dm and dogs with suspected dm based on thorough diagnostic investigations and clinically normal age-matched control dogs were included in the study. the neurological severity of the dm dogs was graded ( - ) using an established scale. csf was evaluated for presence of glutamate, glycine and gaba by high performance liquid chromatography and for gm-csf, ifn-g, il- , il- , il- , il- , il- , il- , il- , il- , ip- , kc (keratinocyte chemoattractant), mcp- (monocyte chemotactic protein- ) and tnf-a using a commercially available, canine multiplex immunoassay (millipore, billerica, ma). all data analyses were performed using sas v . (cary, nc). analyte levels were compared between dm confirmed, dm suspected and control dogs by an analysis of variance (anova). spearman correlation was used to test for correlations of analyte levels and neurological grades. all hypothesis tests were -sided with a . . there were no significant differences between individual csf analytes in dm confirmed and dm suspected dogs. glutamate levels were not significantly different between dm affected (mean . mg/ ml; range . - . ; sd . ) and control dogs (mean . mg/ ml; range . - . ; sd . ). control dogs (mean . mg/ml; range . - . ; sd . ) had significantly higher levels of gaba (p o . ) than dm dogs (mean . mg/ml; range . - . ; sd . ). control dogs (mean . mg/ml; range . - . ; sd . ) also had significantly higher glycine concentrations (p o . ) than dm dogs (mean . mg/ml; range . - . ; sd . ). dm-affected dogs also had significantly higher levels of il- (p . ), kc (p o . ) and mcp- (p . ) than control dogs. neurotransmitter levels were not significantly associated with neurological grade. kc levels were significantly higher in the least affected dogs (p . ). there were no associations with disease severity and analyte concentrations. dm affected dogs have an imbalance of csf aa concentrations creating a relatively excitotoxic environment. reports in human als confirm an imbalance between csf excitatory and inhibitory aas suggesting a pathogenic role for excitotoxicity in als. it also appears that dm affected dogs have increases in csf cytokines and chemokines suggestive of an immunologic component to the pathogenesis as is similar to als. further prospective analysis of dm is warranted to evaluate the role of treatment on csf variables. the pathogenesis of neuropathic pain (np) and syringomyelia (sm) in association with chiari-like malformation (clm) in dogs has focused on the anatomical anomalies and secondary cerebrospinal fluid (csf) flow abnormalities. neuropathic pain in humans has been associated with abnormalities of neurotransmitters such as glutamate and serotonin as well as immunologic mechanisms. the aim of this study was to investigate the csf neurotransmitter and cytokine levels in brussels griffon dogs (bgs) with clm, sm and np. as part of an mri study investigating the prevalence of sm in bgs, atlanto-occipital csf was acquired from dogs and stored at - c until analysis. all dogs underwent a neurologic exam prior to mri; osirix s software was used to measure sm and the presence of cerebellar herniation and deviation were recorded. deproteinized csf samples were analysed for presence of serotonin (ng/ml), glutamate, glycine and gaba (mg/ml) by high performance liquid chromatography. all csf samples were evaluated simultaneously for gm-csf, ifn-g, il- , il- , il- , il- , il- , il- , il- , il- , ip- , kc, mcp- and tnf-a. a commercially available, canine multiplex immunoassay (millipore, billerica, ma) was used for the cytokine analysis (pg/ml). student's t-tests were used to compare the means of neurotransmitter and cytokine values between groups with and without skull abnormalities or spinal pain. simple pearson's correlation was used to test for correlations of neurotransmitter and cytokine values with syrinx dimensions and correlations of neurotransmitter with cytokine values. all hypothesis tests were -sided and the significance level was a . . np was detected in dogs ( %); sm was present dogs ( %); and cm was detected in dogs ( %). ifn-g levels were significantly lower in dogs with np than without (p . ). there were significant positive correlations between syrinx size and il- (p . ), kc (p . ) and mcp- (p . ). there were significant negative correlations between ifn-g and syrinx height (p . ) and extent (p . ). there was a significant negative correlation between il- and syrinx height (p . ). neurotransmitter levels were not associated with skull abnormalities or spinal pain, but there was a positive correlation of glycine with il- (p . ) and mcp- with glutamate (p . ) and serotonin (p . ). the size of the syrinx in bgs with sm is associated with several cytokine elevations but only a decrease of ifn-g was associated with np. based on this study it does not appear that excitotoxicity plays a role in either sm development or np. further work is justified on the role of the immune system in cm, sm and np. current knowledge about the conservative management of disk associated cervical spondylomyelopathy (da-csm) is rather limited and mainly based on retrospectively retrieved data. the goals of this study were to prospectively evaluate the evolution of clinical signs in dogs treated conservatively for da-csm. additionally, several potential prognostic parameters and the correlation of initial clinical signs with magnetic resonance imaging (mri) and transcranial magnetic stimulation (tms) were investigated. twenty-one dogs were included. after neurological evaluation, neurological status was graded from ( normal) to ( tetraplegia). all animals underwent low-field mri and tms with measurement of onset latencies and peak-to-peak amplitudes from the extensor carpi radialis and cranial tibial muscles. from the mr images, the following dimensions were calculated: remaining spinal cord area; compression ratio; vertebral occupying ratio of the spinal cord; canal height to body height ratio (cbr); canal height to body length ratio (cblr); and the canal compromise ratio. intraparenchymal intensity (isi) changes were graded from to . all dogs were reevaluated by the same person after , , , , and months. eight of dogs ( %) experienced a positive clinical evolution with improvement of clinical signs or stabilization of mild clinical signs. all dogs with a negative clinical evolution month after diagnosis experienced a further progression of clinical signs resulting in a poor outcome. the opposite was true for all dogs with a positive clinical evolution after month. outcome was further significantly associated by the remaining spinal cord area and the vertebral canal compromise ratio. prognosis was not significantly affected by clinical presentation or tms. progression of clinical signs, in unsuccessfully treated dogs, was generally characterized by a rapid and dramatic deterioration of neurological status. there were no significant correlations between clinical presentation, mri and tms. two dogs underwent necropsy and histopathological examination. this revealed in both cases chronic wallerian degeneration and segmental myelomalacia. the results of this study suggest that conservative treatment of da-csm is associated with a rather guarded prognosis. clinical evolution month after diagnosis and selected mri parameters can be considered as prognostic indicators. the lack of correlation between clinical presentation and outcome, medical imaging and electrophysiological evaluation is disturbing and warrants further investigation. a mri-guided stereotactic brain biopsy system has not been clinically evaluated in dogs. the purpose of this study was to determine the ability of the brainsight tm system to obtain histologically diagnostic samples and access the impact of this procedure on neurologic status for hours after the biopsy. five dogs with mri definable lesions in the brain have been enrolled. breeds included a pitbull mix, pembroke welsh corgi, french bulldog, border terrier and west highland white terrier. age ranged from - years. weight ranged from . - . kg.dogs presented with seizures (n ), ambulatory paresis(n ), unilateral blindness(n ) and head tilt(n ). one dog had a normal neurologic exam. lesions chosen for biopsy were in the olfactory and/or frontal lobes (n ), parietal lobe(n ), and pyriform lobe(n ). lesions were between - mm in diameter. all lesions were well-circumscribed and contrast enhancing except for one. histologic diagnosis of meningioma(n ) and granulomatous meningoencephalitis(n ) were made. the poorly-circumscribed, non-contrast enhancing frontal mass yielded non-specific necrosis. following biopsy, three dogs returned to pre-biopsy neurologic status within hours. the french bulldog took hours to return to previous neurologic status due to brachycephalic syndrome that required oxygen support. one dog had acute respiratory arrest hours post-biopsy. necropsy is pending. these results suggest that this mri-guided biopsy system can provide an accurate histologic diagnosis of brain lesions. biopsies of poorly-circumscribed and non-contrast enhancing brain lesions may be less diagnostic. further evaluation is on-going to determine the true diagnostic yield and complication rate of this procedure. concurrent malformations of the craniocervical junction are commonly identified in humans with chiari type i malformation. recent evidence suggests such craniocervical junction abnormalities (cjas) also occur in dogs suspected of having chiari-like malformation (clm). the purpose of this study was to objectively describe morphometric features of the craniocervical junction region of dogs with suspected clm and to investigate for associations between these features and the occurrence of other malformations in this region. magnetic resonance (mr) and computed tomographic (ct) images from dogs with clm were evaluated. three regions of neural tissue compression were assessed: cerebellar compression (cc); ventral compression at the c /c articulation, termed ''medullary kinking'' (mk); and dorsal compression (dc) at the c /c articulation. a compression index (ci) was calculated for all abnormal regions for each dog. multiple logistic regression analysis was performed (p o . ) to ascertain whether ci values for the different regions of compression were associated with the incidence of other craniocervical junction abnormalities. % of dogs had mk and % of dogs had dc. % of dogs also had evidence of atlanto-occipital overlapping (aoo medical infrared imaging (mii) is a non-invasive diagnostic imaging technique that measures skin surface temperature and generates thermal pattern maps based on predetermined color scales. because skin temperature, dependent on regional perfusion, is under direct control of the sympathetic nervous system, mii provides information about the function of the autonomic nervous system. because of recent advances in technology and lack of sedation needed to image patients, mii has potential use as a screening test for a variety of conditions that may result in autonomic dysregulation like chiari-like malformation in dogs (clm). the purposes of this study were to establish a mii protocol for dogs suspected of having clm, to identify thermal imaging patterns for various regions of interest (roi), to evaluate changes in thermal patterns and compare the results to those of mri findings, considered the standard for diagnosing clm in dogs. one hundred and five cavalier king charles spaniel dogs with clinical signs attributable to clm and confirmed clm with mri were evaluated with a complete blood count and chemistry profile, examination by a board certified surgeon/neurologist, multidetector ct scan of the craniocervical junction, whole body mri and mii. the protocol for thermal imaging included cranial and caudal views of the body, full lateral right and left body views, dorsal views of the head and body, and right and left lateral views of the head. thermal patterns were assessed with custom image recognition software. after each dog was imaged awake, general anesthesia was administered and the dogs re-imaged using the same protocol. mri findings in dogs with severe or moderate cerebellar compression and cerebellar herniation were compared with mii results. the top of head and front of head roi were . % and . % successful in identifying dogs with clm. based on these preliminary findings, mii may be a viable screening tool to detect clm in dogs. medical infrared imaging (mii) is an imaging technique that measures skin surface temperature derived from cutaneous perfusion and generates thermal pattern maps based on color scales. mii has been used as a test for a variety of conditions that cause autonomic dysregulation resulting in altered cutaneous perfusion. acute thoracolumbar intervertebral disk disease (tlivdd) is common in dogs. the purpose of this study was to: ) determine the success of mii in identifying dogs with tlivdd, ) compare the mii localization with mri results and surgical findings ) determine if the mii pattern returns to that of normal dogs following decompression surgery. small breed chondodystrophic dogs with tlivdd confirmed with mri and dogs with no tlivdd were evaluated with mri and mii. regions correlating with the intervertebral disk spaces were analyzed for average temperatures and thermographic patterns. thermal patterns were assessed with computer recognition pattern analysis (crpa) software. dogs were re-evaluated weeks after surgery using the same protocol. when analyzing temperature averages over a region, no significant difference was found between control and affected dogs. crpa was % successful in differentiating normal from affected dogs. crpa was % successful in identifying the intervertebral disk space when compared with mri and surgical findings. based on these findings, mii may be a viable screening tool to detect tlivdd in dogs. microglia physiologically shows regional topographical differences in immunophenotype and function within the central nervous system indicating the endowment for a prompt response to pathological stimuli such as trauma. spinal cord injuries (sci) consist of a primary injury encompassing the mechanical impact and the ''secondary wave'' of injury occurring minutes to weeks later and comprising various consecutive effects such as increased production of free radicals, excessive release of excitatory neurotransmitters and inflammatory reactions. activated microglia has the potential to perform some of these reactions, their contribution to the secondary wave is therefore controversially discussed. it has to be considered a double-edged sword as both, beneficial and deleterious effects have been attributed to these cells. the purpose of the presented study was to assess microglial involvement, particularly in the ''secondary wave'' following sci. microglia from dogs with sci was isolated and characterized ex vivo in terms of morphology, immunophenotype, and function by flow cytometry. the results were compared to region-specific findings obtained from healthy control dogs (n ). the histopathological exam confirmed the diagnosis of sci in the cervical (n ) and thoracolumbar (n ) spinal cord, and revealed a significant activation of microglia/ macrophages and upregulation of myelinophagia in dogs with sci days or longer prior to euthanasia. microglial ex vivo examination showed significantly increased expressions of b - , b - , mhc ii, cd c, icam- , cd , cd , and cd , and significantly enhanced phagocytosis and generation of reactive oxygen species (ros) in sci compared to healthy controls. microglial cells seem to be highly activated following sci with an immunophenotype indicating their active role in co-stimulation of t cells, in leukocyte adhesion and aggregation, and in lipid and glycolipid presentation. microglial phagocytosis might play a pivotal role in removal of injured or damaged cells and initialize subsequent healing processes. however, as ros can be directly neurotoxic an enhanced microglial generation might lead to bystander damage of the traumatized spinal cord and might therefore add to the deleterious effects of the secondary wave. modulating the microglial response in sci might be a valuable novel therapeutic strategy alleviating further damage to the spinal cord. thymidine kinase (tk) is a soluble biomarker present in s-phase of a salvage pathway for dna synthesis, and can be measured in serum. tk activity correlates with stage, prognosis, and relapse in canine and human lymphoma. we previously reported the results of a pilot study evaluating tk activity in archived canine osteosarcoma, transitional cell carcinoma, and hemangiosarcoma (hsa) sera, and found elevated tk activity in % of canine hsa sera evaluated. the purpose of this study was to prospectively evaluate serum tk activity in a large number of dogs presenting to emergency clinics with hemoabdomen and a splenic mass, to determine if tk activity could be used as a noninvasive means to distinguish hsa versus benign conditions in this population. dogs presenting with hemoabdomen and a splenic mass identified on ultrasound examination were studied. serum was collected prior to anesthesia, euthanasia or surgical intervention and frozen until batch analysis. tissue from all patients was evaluated histologically by a single pathologist. sera from age-matched normal dogs comprised a control population. an elisa using azidothymidine as a tk substrate was used. comparisons between groups were made using -tailed student t-tests, and receiver-operator characteristic (roc) curves were generated. sixty-two patients and normal controls were studied. there were dogs with hsa, dogs with other splenic neoplasia, and dogs with benign diseases. using a training set of normal dogs, a cutoff of . u/l was established from the roc curve. tk activity was significantly higher (p o . ) in dogs with hsa than in the validation set of normal dogs (mean /Àsd . /À . and . /À . respectively), but not between dogs with hsa and benign splenic disease (mean /Àsd . /À . , p . ). using a cutoff of . u/l, tk activity demonstrated a sensitivity of . , specificity of . , positive predictive value of . and negative predictive value of . for distinguishing hsa versus benign splenic disease. when interval thresholds of o . and . u/l were used together, diagnostic utility was markedly increased for distinguishing both hsa versus normal and hsa versus benign disease. in conclusion, serum tk evaluation may assist in detection of canine hsa, and may also discriminate between benign disease and hsa in dogs with hemoabdomen and a splenic mass. t cell chronic lymphocytic leukemia (cll) is a heterogeneous disease that affects a number of dog breeds. cll patients have variable disease outcomes. the objectives of this study were to use gene expression profiling of cd t cell leukemias with variable outcomes in order to identify markers that can be used in routine diagnostic tests to distinguish good from poor prognosis disease, and to identify potential targets for novel therapy. gene expression profiling of cd t cell leukemias ( good, poor prognosis) was conducted. samples from normal dogs were also profiled. several differentially expressed genes were found including cd , cd , and cd . these were selected for further study using flow cytometry to determine expression of protein on the cell surface. seventy nine cases of cd t cell leukemia were screened for cd expression. forty seven had associated outcome information. based on analysis to date, cd expression as assessed by flow cytometry does not appear to provide prognostic information. a monoclonal antibody to cd was recently made available. to date patients with cd t cell leukemia have been profiled. cd is variably expressed on t cell leukemias compared to normal cd t cells. cd is the receptor for interleukin . cyclosporin, a commonly used immunosuppressive drug, inhibits il- production, and has been used to treat a subset of t cell leukemias in people. thus, the finding that cd is up regulated on t cell leukemias compared with normal t cells suggests a possible new therapeutic avenue. recent molecular studies have revealed a highly complex bacterial microbiota in the intestine of dogs. there is mounting evidence that microbes play an important role in the pathogenesis of acute and chronic enteropathies of dogs, including idiopathic inflammatory bowel disease (ibd). similarly, compositional changes of the intestinal bacterial ecosystem have been associated with ibd in humans. the aim of this study was to characterize the bacterial microbiota in dogs with various gastrointestinal disorders using a next generation sequencing technique. fecal samples were obtained from healthy dogs (n ), dogs with acute uncomplicated diarrhea (n ), dogs with acute hemorrhagic diarrhea (ahd; n ), and dogs with active (n ) and therapeutically controlled ibd (n ). the bacterial composition was analyzed by massive parallel s rrna gene -pyrosequencing. differences between groups were analyzed using mann-whitney u tests and kruskal-wallis tests followed by dunn's multiple comparison tests. statistical significance was set at p o . . significant differences in the proportions of several bacterial groups were identified between healthy and diseased dogs. dogs with gastrointestinal disease had significantly higher proportions of proteobacteria (p o . ). proportions of firmicutes were lower in diseased dogs, but this difference did not reach significance (p . ). within the firmicutes the most notable findings were decreases in bacterial groups belonging to clostridium clusters iv and xiva (i.e., ruminococcus, dorea, and faecalibacterium spp.; p o . for all). dogs with ahd had the most profound changes of the microbiota, followed by dogs with acute uncomplicated diarrhea, and dogs with active ibd. faecalibacterium spp. was the bacterial group most prominently depleted in dogs with active ibd, but was not significantly different between healthy dogs and dogs with therapeutically controlled ibd (p . ). results of this study revealed bacterial dysbiosis in fecal samples of dogs with various gi disorders. bacterial changes were more profound in dogs with severe disease, but were not identified in dogs with therapeutically controlled ibd, suggesting that the microbiota is stable in non-active disease. the bacterial groups identified are considered to be important short chain fatty acid producers and may serve as candidates for the diagnosis or therapeutic monitoring of gi disease. future studies are necessary to determine if these microbial changes correlate with functional changes in the intestinal microbiota. ciprofloxacin oral tablets, available in a generic formulation for people, are widely used for treatment in dogs. oral absorption data for ciprofloxacin in dogs has been variable, and too limited to guide accurate dosing. subsequently, published doses for dogs in veterinary formularies have varied from to mg/kg. this study was undertaken to explore the factors that may affect oral absorption of generic ciprofloxacin in dogs, and to derive a pharmacokinetic-based dose for treating susceptible bacteria. six healthy adult beagle dogs were used for the study ( . kg mean weight). after placing jugular vein catheters for collecting blood samples, these dogs were administered either a single oral dose of ciprofloxacin ( mg tablet; mean dose mg/kg), or an intravenous (iv) dose ( mg/kg; mg/ml solution). a randomized crossover design was used with a washout time between treatments. blood was collected for plasma drug analysis for hours. ciprofloxacin concentration in plasma was analyzed using high pressure liquid chromatography (hplc) and pharmacokinetics analyzed using a computer program. oral absorption was also evaluated via deconvolution analysis. the oral dose was well-tolerated, but the iv dose produced transient vomiting and depression in some dogs. after the oral dose, the peak plasma concentration (c max ) was . mg/ ml (cv . %), terminal half-life (t / ) . hr (cv . %), auc . mg Á hr/ml (cv . %), and systemic absorption (f) . % (cv . %). after the iv dose, the t / was . hr (cv . %), systemic clearance . l/kg/hr (cv . %), and volume of distribution . l/kg (cv . %). after examining the pharmacokinetic results from the oral dose, it was apparent that oral ciprofloxacin was absorbed well in some dogs (approximately %), but poorly in others (approximately %). to explore the factors that may have affected oral absorption, two high absorbers and two low absorbers were administered an additional oral dose as a mg/ml solution ( mg total dose) via gastric tube. after administration of the oral solution, the plasma concentrations were more uniform and consistent among dogs. absorption of the oral solution of ciprofloxacin was . % (cv %) with a t / of . (cv . %) hr and c max of . mg/ml (cv . %). therefore, it appears that inconsistent oral absorption of ciprofloxacin in some dogs may be formulation-dependent, and affected by tablet dissolution in the canine small intestine. doses were calculated using the data for oral tablets in these dogs. the pharmacokinetic-pharmacodynamic (pk-pd) target was an auc/ mic ratio of . because of the wide range in oral absorption of tablets, a dose to reach the pk-pd target ranged from canine distemper (cd) is a highly contagious, acute or subacute systemic viral disease of dogs and other carnivores which can be controlled efficiently by the use of modified live-virus (mlv) vaccines. however, mlv strains do cross-react with molecular diagnostic tests and cause significant confusion for clinicians. the purpose of this study was to use quantitative real-time pcr viral load information to differentiate between vaccine virus used in mlv vaccines and wildtype infections in dogs. a real-time pcr test for cd virus (cdv) based on the p gene for phosphoprotein was used to determine viral loads in vaccinated and wildtype infected animals. a total of respiratory mucosal swab samples from mlv vaccinated and asymptomatic dogs were obtained within the first weeks after mlv vaccination. based on the viral load in vaccinated animals, a cutoff value was established for the differentiation of dogs with clinical signs of respiratory distress and presumably infected with a wildtype strain of cdv. two hundred clinical cases with known clinical and vaccination histories were analyzed to validate the cutoff value. the cdv real-time pcr proved to be of high analytical and diagnostic sensitivity: a standard curve was established using known numbers of cdv molecules to allow absolute quantitative cdv viral load data. the limit of detection was in the single molecule range while the limit of quantitation was established at around molecules per pcr reaction. a comparison to ifa showed real-time pcr to be % more sensitive. the cdv viral load in vaccinated animals averaged , viral particles per swab. a cutoff value of , viral particles was calculated by adding standard deviations to the average value. this cutoff value correctly detected . % of the vaccinated samples. acutely infected dogs with cdv compatible clinical signs have high viral loads normally several logs higher than the cutoff value. in dogs with clinical distress, recent cdv mlv vaccination but viral loads below the cutoff value, other infectious agents were detected by using a panel of real-time pcr tests. testing additional infectious agents in clinical settings is important in order to explain clinical signs when viral loads below cutoff values indicate that cdv is not the cause of clinical signs. in conclusion, quantitative real-time pcr is a sensitive, rapid and reliable test regardless of recent vaccination. the use of a cutoff value will be of significant help to discriminate between vaccine interference and wildtype infection in clinical settings. feline ureteral obstructions are a common urinary dilemma and traditional therapy is associated with substantial morbidity/mortality. feline nephrostomy tubes are reported as being effective when pelvic drainage is required. the biggest limitation is externalized drainage, requiring careful management to prevent infection/dislodgement. the development of an indwelling ureteral bypass using a combination locking-loop nephrostomy/cystostomy tube was modified from humans, resulting in permanent indwelling drainage, reduced complications, and improved quality of life. the objective is to describe the technical and clinical outcome using a novel device called a subcutaneous ureteral bypass (sub) in cats with ureteral obstructions. fifteen cats ( kidneys) had a sub placed for: ureterolithiasis ( ), ureteral stricture ( /À stones) ( ), and ureteral stent rejection ( ). the median pre-and post-procedure creatinine was mg/dl (range: . - ) and . mg/dl (range: . - ), respectively. the median pelvis diameter pre and post-procedure were (range: - ) and mm (range . - ), respectively. six french tubes were placed in , and fr. in . the bypass remained indwelling for a median of days (range - ). there were major complications resulting in nephrostomy tube dislodgement ( ) and port leakage ( ) days after surgery. one patient with severe coagulopathy developed a clot which resolved with tpa infusion through the port. no sub got occluded/obstructed long-term. overall, the use of a sub for cats with ureteral obstructions can be considered a functional option when other therapies have failed or are contraindicated, but shtime. oxidative stress is considered central to the pathogenesis of many systemic diseases. in humans, biomarkers of oxidative stress, antioxidant depletion and lipid peroxidation, have been correlated with disease severity and associated with poor clinical outcomes. therapeutic antioxidant supplementation with nac in glutathione (gsh)-deficient patients has shown clinical benefits, including repletion of intracellular gsh levels. we have shown that clinically ill dogs are gsh-deficient, and that gsh deficiency correlates with mortality, but it is not clear whether there are direct benefits of antioxidant intervention in these patients. the purpose of this randomized, investigator-blinded, placebo-controlled, prospective study was to evaluate the effect of nac to normalize blood antioxidants (rbc reduced gsh (rbc gsh), plasma cysteine (cys), serum vitamin e (vit e), and whole blood selenium (se)), reduce lipid peroxidation (urine isoprostane/creatinine ratio (u i/ c)), and improve illness scores (spi ) and outcome (survival to discharge) in clinically ill dogs. clinically ill client-owned dogs, admitted to the uw veterinary medical teaching hospital that did not receive blood transfusions, tpn, vitamins, or antioxidants were eligible for the study. dogs enrolled in the study were randomized to receive iv infusions q. h. of either nac (  mg/kg and  mg/kg) or equal volumes of % dextrose (placebo) over hours. at the time of enrollment, and hours following the final hour infusion, blood and urine were collected to quantify rbc gsh, cys, vit e, and se concentrations; u i/c ratios; and calculate spi scores. rbc gsh and cys concentrations were quantified by hplc. commercially available hplc, atomic absorption spectroscopy, and eia were used to quantify vit e, se, and u i/c ratios, respectively. nonparametric statistical analyses were used, with results reported as medians and p o . considered significant. sixty-one ill dogs were randomized to either nac (n ) or placebo (n ). overall this group of ill dogs had significantly decreased rbc gsh ( . vs. . mm; p . ), vit e ( vs. mg/ml; p . ), and se ( . vs. . mg/ml; p . ) levels and elevated u i/c ratios ( vs. pg/mg; p . ) in comparison to healthy control dogs. dogs in the placebo group showed a significant further decrease in rbc gsh over the next hours ( . to . ; p . ). nac supplementation significantly increased plasma cys levels ( . to . mm; p o . ), and prevented a further decline in rbc gsh ( . to . mm; p . ). however, serum vit e ( vs. mg/ml), se ( . vs. . mg/ ml), u i/c ratios ( vs. pg/mg), spi scores ( . vs. . ), and outcome ( % vs. %) were not significantly different between the nac and placebo groups after treatment. the results of this study further support that clinically ill dogs experience oxidative stress, and suggest that antioxidant supplementation with nac within the first hours of hospitalization prevents further rbc gsh depletion. further studies are necessary to investigate whether longer duration or combined antioxidant supplementation normalizes the redox state and impacts long-term outcome. diabetes mellitus in cats is very similar to type ii diabetes in humans, preceded by a period of insulin resistance. evaluating insulin resistance in a cat is a time consuming, expensive, and difficult procedure. there is a need for a simple biomarker based test predictive of insulin resistance. there is a biomarker based assay predicative of insulin resistance in humans. the purpose of this study was to evaluate the utility of this assay in overweight cats and show improvement in insulin sensitivity following weight loss and weight maintenance. the insulin resistance assay is based on the quantitative analysis of metabolites ( -hydroxybuterate, creatine, palmitate, decanoylcarnitine, and oleoyl-lpc). a proprietary algorithm (metabolon, inc, durham nc) was used to generate a predictive rd (rate of disposal) value (normal range in cats . - . ). individuals with an rd value less than will have a greater than % chance of being insulin resistant and an rd value less than will have a greater than % chance of being insulin resistant. initial studies demonstrated that the rd values indicating insulin resistance in cats correlated with age, obesity and severity of diabetes as determined by histopathology and blood glucose levels. in a feeding study of cats ( % vs. o % body fat) rd values improved from . ae . to . . (p . ). during weight maintenance, % body fat for months, further improvement was observed (rd, . . (p . e- )). these results demonstrate that long term weight maintenance following weight loss is critical for increasing insulin sensitivity in cats. the use of monoclonal antibodies and antibody fragments to directly target tumor antigens and neutralize their growth factors has shown promising results in human clinical trials. however, these targeted approaches have not been possible in dogs since specific tumor antigens have not been identified, monoclonal antibodies of canine origin are not available and the efficacy of xenogeneic antibodies in the dog is limited by neutralizing antibody responses. to overcome these obstacles, we have generated canine antibody phage display libraries from canine splenocytes. these libraries consist of single chain variable fragments (scfv) comprised of canine variable heavy (vh) and variable light (vl) immunoglobulin chains displayed on the surface of bacteriophage (fig. ) . the antigen specificity within these libraries is diverse and recapitulates the antigen-experienced immunoglobulin repertoire of the dog. we can now use simple panning techniques to isolate scfv of canine origin that bind to either known targets or unknown targets which can then be identified using standard molecular techniques. canine hsa is a highly aggressive malignancy of vascular endothelial cells that affects large breed dogs. although there are no confirmed immunological targets for hsa, serum levels of vascular endothelial growth factor (vegf) are elevated in these patients and, as in many human cancers, vegf may represent an important therapeutic target for neutralization. we used simple panning techniques to screen canine scfv libraries generated from the spleens of dogs with hsa against canine vegf and successfully isolated scfv clones that bind and neutralize canine vegf in vitro. these scfvs are now being taken into a murine model of canine hsa to determine whether they can inhibit tumor growth and metastases. in addition, we have panned the same antigen-experienced scfv phage display libraries against allogeneic primary canine hsa cells of low passage number to isolate canine-derived antibody fragments that can target malignant endothelial cell surface molecules. early results demonstrate enrichment of scfv phage libraries for malignant endothelial cell binders. these scfv can be readily linked to chemotherapeutic agents or other toxins and used to deliver high doses directly to the malignant cell. this novel approach aims to reduce side effects of systemic chemotherapy and augment therapeutic response. calcitriol, (vitamin d ), has antineoplastic activity and acts synergistically to potentiate the antitumor activity of a diverse array of chemotherapeutics. ccnu, vinblastine, corticosteroids, and tyrosine kinase inhibitors, are used to treat canine mast cell tumors (mct). vitamin d receptor is expressed in the majority of canine mcts, suggesting a role for calcitriol in the management of dogs with these tumors. the purpose of our study was to examine the in vitro effects of calcitriol in combination with ccnu, vinblastine, imatinib, or toceranib on canine mastocytoma c cells. also, we evaluated the antitumor activity of dn , a highly concentrated oral formulation of calcitriol, as single-agent treatment in dogs with naturally occurring mcts. c cells were incubated with serial dilutions of calcitriol ( . - nm). twenty-four hours later, cells were then treated with vehicle control or serial dilutions of ccnu ( . - um), vinblastine ( . - nm), imatinib ( . - . nm), or toceranib ( . - nm). cell viability was assessed with an mtt assay after hours and data was used to derive a combination index (ci: values o , , indicate synergism, additivity, antagonism, respectively). in the phase ii clinical trial, dogs were eligible if they had at least measurable, histologically confirmed, mct. calcitriol was administered orally. recist criteria were used to assess tumor response. calcitriol, ccnu, vinblastine, imatinib, and toceranib each suppressed c cell viability in a dose-dependent manner. ci values o were obtained for calcitriol ( . - . nm) combined with ccnu ( and um), vinblastine ( . and nm), imatinib ( . - . nm) and toceranib ( . - . nm). due to the occurrence of toxicity (vomiting, anorexia, hypercalcemia), the phase ii trial was terminated early; only of planned patients were treated. one dog with a metastatic muzzle mct had a complete response that lasted days. three dogs achieved partial response lasting from - days. in summary, our in vitro data demonstrate that calcitriol combined with ccnu, vinblastine, imatinib or toceranib has synergistic effects on c mastocytoma cells. antitumor responses were observed in dogs with spontaneously occurring mcts treated orally with single-agent calcitriol, but the frequency of adverse effects was high. together these results suggest calcitriol combination therapies might have significant clinical utility in the treatment of canine mcts but refinement of the calcitriol-dosing regimen must be done. cyclosporine is a potent immunosuppressive agent used to treat many canine inflammatory and immune-mediated diseases. cyclosporine has gained popularity as an immunosuppressive agent because of a favorable toxicity profile compared to many other immunosuppressive agents. optimal dosing regimens for cyclosporine in the dog remain unclear, primarily because standard methods that monitor effectiveness of immunosuppression have not been established. pharmacokinetic testing is currently used during treatment with oral cyclosporine to adjust doses based on measurement of blood drug levels. individual patients, however, often demonstrate marked variations in blood drug levels while on similar oral doses of cyclosporine, and can also demonstrate different clinical responses even at comparable drug levels, making correlation of blood cyclosporine levels and degree of disease control extremely difficult. pharmacodynamic testing offers an alternative method for regulating cyclosporine dosing by objectively measuring the effects of cyclosporine on t-cells, the drug's main cellular target in the body. our acvim foundation-funded research has focused on developing and evaluating a comprehensive panel of biomarkers of immunosuppression that can be utilized for pharmacodynamic monitoring during treatment with cyclosporine and other immunosuppressive agents that affect t-cell function. we have completed several studies using flow cytometry to evaluate activated t-cell expression of surface molecules (cd & cd ) and cytokines (il- , ifn-g & il- ) as potential biomarkers. our first study was an in vitro study evaluating expression of surface molecules and cytokines in canine t-cells exposed to varying concentrations of cyclosporine. this study established consistent drug-associated suppression of the cytokines il- , ifn-g and il- . our second study was an in vivo study in normal dogs evaluating the effects of two doses of oral cyclosporine, a high dose considered to be reliably immunosuppressive (starting dose mg/kg bid, titrated upwards as needed to attain trough drug blood levels of at least ng/ml) and a lower dose used to treat atopy ( mg/kg sid), on t-cell expression of these three cytokines. significant suppression of il- and ifn-g expression was seen at the high cyclosporine dose, while at the lower dose only ifn-g expression was suppressed. because tcell expression of il- was not significantly suppressed at the high cyclosporine dose, il- was not evaluated at the lower drug dose. because of specialized sample handling requirements, flow cytometry is not as practitioner friendly as other assays (such as pcr) for routine use in pharmacodynamic testing. we have therefore conducted an in vitro study comparing the effects of cyclosporine on activated t-cell expression of il- and ifn-g using flow cytometry and qrt-pcr, and demonstrated dose dependent and comparable suppression of il- and ifn-g using either methodology. we are currently evaluating, using qrt-pcr, the effects of oral cyclosporine on t-cell expression of il- and ifn-g in normal dogs prior to moving on to pharmacodynamic trials in our clinic patients. effect of hypothyroidism on reproduction in bitches. dl panciera , bj purswell , ka kolster , sr werre . departments of small animal clinical sciences, large animal clinical sciences, and laboratory for study design and data analysis, virginia-maryland regional college of veterinary medicine, virginia tech, blacksburg, va. numerous reproductive abnormalities, including irregular interestrous period, anestrus, and infertility have been attributed to hypothyroidism. we previously documented reduced fertility and lower birth weight and increased periparturient mortality in pups born to bitches with experimentally-induced hypothyroidism for a median duration of weeks. the purpose of this study was to evaluate reproductive function in these same bitches after hypothyroidism was treated with a replacement dose of levothyroxine. twelve multiparous bitches were studied. hypothyroidism was induced in dogs by administration of mci/kg i. hypothyroidism was confirmed by finding serum t concentrations before and hours after iv administration of human recombinant tsh that were o nmol/l. levothyroxine ( . mg/kd q h) was administered to all hypothyroid bitches. six bitches served as euthyroid, untreated controls. dogs were evaluated daily for signs of estrus and were bred by of males when serum progesterone was ! ng/ml. interestrous interval, gestation length, strength and duration of contractions during whelping, time between pups, number of live pups and stillbirths, viability of pups at birth, weight of pups, and periparturient mortality were recorded. the student's t-test and anova were used to compare differences between control and hypothyroid bitches for continuous, normally distributed data. the wilcoxon rank sum test was used to analyze data between groups that was not normally distributed. the mean duration of hypothyroidism prior to levothyroxine administration was ae . weeks. breeding took place after levothyroxine treatment for ae . weeks in the hypothyroid group. all dogs in the hypothyroid group and / control dogs were pregnant, while / hypothyroid and all control bitches became pregnant prior to levothyroxine administration. no difference in interestrus interval or gestation length was noted between groups. during whelping, no difference in strength of contractions, contraction duration, interval between pups, or viability scores of pups was found between groups. litter size, birth weight and peirparturient mortality were similar between groups. levothyroxine administration reverses the detrimental effects of hypothyroidism on fertility and neonatal health. racing sled dogs have a high prevalence of exercise-induced gastric erosions/ulcers, with reports ranging from - % of dogs running at least miles in a day or less. omeprazole reduces the severity of, but does not completely prevent, gastritis under racing conditions, and can be difficult to administer under these conditions. famotidine can be administered in food, but has only demonstrated efficacy under less intense training conditions. the purpose of these studies was to evaluate different acid suppression strategies under racing conditions for the prevention of exercise-induced gastritis. experiment # was a randomized placebo-controlled study using sled dogs ( - years) competing in a mile race over - h. treatment groups were famotidine (approx mg/kg qd) or no treatment, beginning days prior to the start of the race and proceeding until gastroscopy was performed h after the race. experiment # was a randomized positive-control study using sled dogs ( - years) running a mock race of miles in h. dogs were divided into omeprazole (approx mg/kg qd, administered min prior to a meal) or famotidine (approx mg/kg bid) groups beginning days prior to the exercise challenge and continuing for h after completion. gastroscopy was performed immediately prior to the start of dosing and h after completion of the exercise. in all cases, mucosal appearance during gastroscopy was blindly scored using previously described scoring system. famotidine ( mg/kg qd) reduced the prevalence of clinicallyrelevant, exercise-induced gastric lesions compared to no treatment ( / vs / , p . ). compared to famotidine at mg/kg bid, omeprazole significantly decreased the severity ( . vs . , p . ) and prevalence ( / vs / , p . ) of gastric lesions. although famotidine provides some benefit in the prevention of exercise-induced gastric lesions, neither the recommended dose nor the higher dose were considered acceptable in the prevention of exerciseinduced gastritis as between - % of the dogs receiving famotidine had clinically significant lesions. a previous study examining omeprazole under racing conditions, but without careful administration on an empty stomach, resulted in a % prevalence of clinically significant gastric lesions. however, the bioavailability of omeprazole is reduced in the presence of food, and when the daily administration of the drug is carefully scheduled to coincide with an empty stomach, the resulting prevalence of clinically significant lesions induced by racing-intensity exercise is reduced to just over %. the conclusions of these studies are that omeprazole is superior to famotidine in preventing gastritis in racing sled dogs during competition. routine administration of omeprazole is recommended to prevent stress-associated gastric disease in exercising and racing alaskan sled dogs. mares may be an important source of environmental contamination with rhodococcus equi on breeding farms. attempts to reduce fecal shedding of r equi by the mare and the effects of the mare's fecal r equi concentration on airborne concentrations in the foaling stall have not been previously reported. twenty-one arabian mares were treated daily with either oral gallium nitrate or placebo in a randomized double-blind study. fecal samples were collected at day of gestation (time ), the week before foaling (time ), and the week after foaling (time ). airborne concentration of r equi were measured in the stall within hours post foaling using a microbial air sampling system into which standard ( -mm) culture plates with a media selective for r. equi have been loaded. concentration of total r equi were determined by morphological characteristics. the concentration of virulent r equi was determined using a modified colony immunoblot method. concentrations of total and virulent r equi were compared among mares to examine effects of treatment, time, and treatment by time interaction. there were significant (p o . ) effects of treatment that depended on time of sample collection. at sample times and there were no significant differences between groups in the fecal concentration of virulent r equi. at time concentrations of virulent r equi were significantly lower among mares in the treatment group (p o . ) compared to control. effects of time depended significantly on groups: for the control group, there were no significant effects of time. for the treatment group, concentrations tended to decrease over time, and concentrations at time were significantly (p o . ) lower than those at time . no other differences among times for concentrations in the treatment group were statistically significant. there were no significant effects of treatment, sample time, or their interaction on the concentration of total r equi between groups; however, the pattern for these data was similar to that observed for the virulent isolates. no significant differences were determined between treatment groups for airborne concentrations of virulent or total r equi. treatment of mares with oral gallium nitrate significantly reduced the fecal concentrations of virulent r equi over time, but had no impact on the airborne concentration of r equi shortly after foaling. the purpose of this study was to evaluate the protein profile of bronchoalveolar lavage fluid (balf) in horses affected with recurrent airway obstruction (rao) and in control horses using proteomics and western blot techniques. rao-affected (n ) and control horses (n ) were subjected to an experimental exposure trial; when the rao-affected horses showed clinical signs of disease, balf was collected from all horses. balf was also collected from client-owned rao-affected horses (n ) with naturally-occurring clinical signs of disease and client-owned control horses (n ) from the same environments. the balf from the experimental exposure trial horses was subjected to trypsin digestion and proteomics analysis with mass spectrometry (ms). peaks detected with ms were identified using tandem ms analysis and database searches. western blot was used to confirm the identity and expression levels of two proteins identified using proteomics techniques in the balf of all horses. data from ms experiments were analyzed with the student's t-test to compare peak intensity between rao-affected and control horses. western blot band density data was analyzed with the kruskal-wallis anova for comparison between groups of horses. significance level was set at p o . . with ms proteomic analysis of the balf from the experimental exposure trial horses, total peaks (peptides) were identified. of these peaks, were differentially expressed between the rao-affected ( over-expressed) and control horses ( over-expressed). identifications were made for balf proteins. transferrin and secretoglobin were chosen for validation with western blot. proteomics indicated that secretoglobin was not differentially expressed between the experimental exposure trial group; this was confirmed with western blot analysis. western blot also showed that clientowned rao-affected horses had lower secretoglobin expression than client-owned control horses and control horses before experimental exposure. according to the proteomics data, transferrin was over-expressed in control horses after experimental exposure compared to rao-affected horses. while the western blot analysis did not show a statistically significant difference in this comparison, transferrin was significantly over-expressed in control horses before experimental exposure compared to client-owned rao-affected horses. in addition, both secretoglobin and transferrin band densities on western blot were negatively correlated with airway obstruction and neutrophilic pulmonary inflammation. this study demonstrates that proteomics techniques can be used in the investigation of equine balf proteins. the proteins identified as differentially expressed between rao-affected and control horses in this study including, but not limited to, secretoglobin and transferrin should undergo further evaluation for their use as biomarkers of rao, and as potential targets of new therapeutic agents for rao. cardiotoxic effects of rattlesnake venom in the horse are not well defined. the first aim of this study was to document cardiac damage in naturally envenomated horses. twenty horses with clinical diagnosis of snake bite were included. a snake venom elisa was utilized to confirm envenomation when possible. serum and plasma were collected at selected intervals. plasma was assayed for cardiac troponin i (ctni) using a flurometric assay (stratus cs s , dade behring). holter monitors (zymed s , philips) were placed at admission, week and month post presentation. echocardiography was performed on available horses - months after envenomation. the second aim of this study was to investigate potential mechanisms of the cardiac damage. serum samples were assayed for tnfalpha using a commercial assay (endogen). antibody titers to crotalus atrox venom were measured at admission, week and month after natural envenomation and compared to titers in vaccinated horses (crotalus atrox toxoid, red rock biologics). a significant number of horses showed elevations in ctni (p o . ) at one or more time point indicating myocardial damage. holter readings revealed the presence of arrhythmias or persistent tachycardia in horses. five of twenty horses were available for echocardiography; no abnormalities were noted. horses with increased ctni tended to have greater tnfalpha concentrations compared with horses without increased ctni. peak venom titers in bitten horses were significantly higher than peak titers in vaccinated horses (p o . ). rattlesnake envenomation was associated with evidence of cardiac damage in a significant proportion of bitten horses. further studies are needed to determine the cause as well as mechanisms to treat and/or prevent its occurrence. little is known about the gastric mucosal flora in healthy horses and its role in gastric disease has not been critically examined. our laboratory previously reported that a diverse microbial flora with a predominance of streptococcus spp. and lactobacillus spp. exists in healthy horses using fluorescence in situ hybridization (fish). the present study sought to further characterize the gastric mucosal flora of healthy horses using massive parallel srrna bacterial tag encoded flx-titanium amplicon pyrosequencing (btefap). biopsies of the squamous, glandular, antral and any ulcerated mucosa were obtained from healthy horses via gastroscopy after a -hour fast and horses immediately post-mortem. dna was extracted from the mucosal biopsies and btefap and data processing was performed. hierarchical cluster analysis based on relative abundance data on the genus level were performed to look for trends in bacterial diversity among the individual horses. pyrosequencing yielded between , and , reads per horse with , , reads in the antrum, squamous and glandular regions, respectively. the microbiome segregated into two distinct clusters: cluster comprised of horses that were stabled, fed hay and sampled at post-mortem and cluster consisted of horses that were pastured on grass, fed hay and biopsied gastroscopically after a -hour fast. samples from different antomic regions clustered by horse rather than region. despite being very similar at the higher taxonomic level (phyla) differences in the distribution of bacteria were seen at the genus and species level. the dominant bacteria in cluster horses were firmicutes ( % reads/sample) consisting of mainly streptococcus spp., lactobacillus jensenii, l. fornicalis and sarcina maxima. cluster had more diversity with a predominance of proteobacteria, bacteroidetes and firmicutes and genera identified such as streptococcus spp., moraxella spp., actinobacillus spp., and others. though the relative abundance of the individual taxonomic groups was significantly different between individual horses, no significant differences in the overall diversity could be found (as assesed by shanon weaver, ace and choa i diversity indices). helicobacter spp. sequences were not identified in any sample (out of , reads). the ulcerated mucosa from horse (group ) had lower diversity and higher numbers of bacteria predominated by lactobacillus equigenerosi. this data shows that the equine gastric mucosa harbors an abundant and diverse microbiome which is unique to each individual and differs by sampling method, fasting prior to sampling and diet. seasonal pasture myopathy (spm; atypical myopathy [am] in europe), typified by nonexertional rhabdomyolysis, occurs in pastured horses during autumn or spring. clinical signs rapidly progress from muscular weakness to recumbency and frequently death. extensive myonecrosis and intramyofiber lipid storage occur in highly oxidative respiratory and postural muscles. recently, a defect of lipid metabolism called madd has been identified in european horses with am. this report documents the first cases of equine madd in the united states. six midwestern us horses suspected of having spm in the spring or fall of were evaluated for madd by urine organic acids, plasma acylcarnitines and/or muscle carnitine and histopathology. five horses had clinical signs and clinicopathologic data consistent with severe rhabdomyolysis. one horse was found dead on pasture after days of rear limb stiffness and inappetance. urinary organic acid profiles revealed markedly elevated ethylmalonic and methylsuccinic acids, butyrylglycine, isovalerylglycine, and hexanoylglycine, consistent with equine madd. plasma acylcarnitine profiles from horses had marked elevations of short chain acylcarnitines, while the third horse and only survivor had minor elevations of short chain acylcarnitines. affected muscle showed extensive degeneration with intramyofiber lipid accumulation, a marked decrease in free carnitine, and high levels of carnitine esters. spm appears to be a highly fatal emerging disease of pastured horses in the us characterized by weakness, colic-like signs and myoglobinuria. the disease is associated with a defect in muscular lipid metabolism that can be diagnosed by performing lipid staining of muscle samples and urine organic acid profiles. candidatus mycoplasma haemolamae (cmhl) is a common red blood cell parasite of new world camelids. the high degree of parasitemia that develops in an infected splenectomized animal allows for the efficient collection of parasitic dna. this dna can then be used in the development of genetically-derived tools such as pcr and in-situ hybridization. thus, one splenectomized animal can replace many immunologically intact animals within a research setting. the purpose of this study was to track the natural progression of cmhl parasitemia and associated clinical signs in a splenectomized alpaca. an intact, -month-old, . kg male alpaca was used in this study. he had tested positive via pcr for cmhl on three different occasions, although no organisms were seen on peripheral blood smears. the alpaca was placed under general anesthesia and a ventral midline incision was made. the spleen was located, the vessels ligated, and the organ removed. buprenorphine and flunixin meglumine were given for and days after surgery respectively. body weight, attitude, rectal temperature, blood glucose, and pcv were recorded daily. in addition, a peripheral blood smear was examined daily and the percent of red blood cells that were infected with mycoplasma organisms was determined. the alpaca was not parasitemic prior to surgery. one percent of the rbc's contained mycoplasma on days and after splenectomy. parasitic bloom developed on day with % of the red blood cells infected, and over % containing or more organisms. the alpaca was treated with mg/kg oxytetracycline i.v. on day . on postoperative day no parasites were seen in the peripheral blood. the peripheral blood remained free of parasites for days. on the morning of the th day, % of the peripheral red blood cells contained mycoplasma. by late that afternoon, % of the observed rbc's contained - organisms. the alpaca again received oxytetracycline. there were no more parasites observed from that time until the alpaca was euthanized days later. the alpaca lost . kg between days À and after surgery. his weight fluctuated between . and . kg for the remainder of the study period. blood glucose ranged between and mg/dl there was no major change in pcv (range - %), a finding that was expected as the spleen was not available to remove infected red blood cells. body temperature ranged between . and degrees celsius except for days and when more than % of red blood cells contained parasites. on those days rectal temperature reached . and . degrees respectively. this study confirmed that a non-parasitemic, yet pcr positive alpaca did indeed harbor cmhl. the time from splenectomy to parasitic bloom was shorter, and the length of oxytetracycline suppression longer than has been observed in other species. gastro-intestinal (gi) disease frequently results in increased wall thickness in many species. identification of changes in gi wall thickness using ultrasound has proved to be a useful diagnostic tool and is widely used in human patients, small animals and horses. although gi motility has been evaluated in cattle, normal reference ranges for wall thickness has not been reported in ruminants. the aims of this study were to report normal values for wall thickness of various gi structures and to assess the repeatability of this technique in adult dairy cows, sheep and goats. eight healthy adult holstein friesian (hf) cattle ( ae kg), eight jersey (j) cattle ( ae kg), thirteen adult sheep ( ae kg) and eleven adult goats ( . ae kg) were recruited and examined on three consecutive days. ultrasonographic images were optimised for the structure of interest. structures were identified based upon appearance and anatomical position. a minimum of three cineloops were obtained of the abdominal organs per intercostal space (ics) and three along the ventral midline in each ics; images were analysed offline. data were analysed using anova and post-hoc bonferoni, student's ttest and intra-class correlation coefficients. each structure was measured per ics per species; if no differences were noted for structures in different ics, then measurements were pooled. no differences were noted between hf and j cattle so data were pooled. data are displayed in table . good repeatability (icc . ) was obtained for all measurements and no differences were noted between animals of the same species or between days. these measurements for assessment of normal gi thickness are repeatable and may allow valuable additional information to be gained from ruminants with gi disease. ocular infections with the infectious bovine keratoconjunctivitis (ibk) agent moraxella bovis (m. bovis) are associated with significant economic loss in the cattle industry.although antibiotic therapy is the treatment of choice for ibk, treatment failures are common and current vaccines are not optimally effective mainly due to antigenic variation. as a result, our laboratory has been actively investigating the therapeutic potential of bdellovibrio bacteriovorus j (b. bacteriovorus); a predatory bacterium capable of attacking and inducing lysis of gram-negative bacteria, as a new treatment for ibk. we have previously shown that b. bacteriovorus can reduce the number of m. bovis attached to bovine epithelial cells in an in vitro model of ibk and that b. bacteriovorus can be trained to kill m. bovis as effectively as e. coli using serial passages. in this study, we hypothesized that b. bacteriovorus can remain viable in bovine tears without its prey for up to hours. this hypothesis was addressed by incubating inocula of active b. bacteriovorus in its preferred media peptone yeast extract (pye) and comparing b. bacteriovorus viability in bovine tears or phosphate buffered saline (pbs) at time , , , , and hours. using a plaque assay to quantify the mean amount of plaque forming units (pfus) of b. bacteriovorus exposed to each treatment, it was determined that viability of b. bacteriovorus over time was comparable between treatment groups. overall, the results supported that b. bacteriovorus can remain viable in tears for up to hours in the absence of prey bacteria. further studies are needed to determine the therapeutic potential of b. bacteriovorus in an in vivo model of ibk. correction of the measured ionized calcium concentration (cca ) to a ph . is routinely applied in experimental studies in order to assist in the interpretation of measured values relative to a reference range. the equation most commonly used for ph correction in bovine plasma is: cca ph . cca  (- . Â{ . -ph}) . the validity of this equation for bovine plasma is unknown. accordingly, our first objective was to characterize the in vitro relationship between cca and ph for bovine plasma. feeding rations with a low dietary cation-anion difference (dcad) during late gestation mitigates periparturient hypocalcemia in dairy cows, particularly when chloride containing acidodgenic salts are fed. the mechanism for this beneficial effect remains unclear. our second objective was to determine whether hyperchloremia displaces calcium from binding sites to albumin, thereby increasing cca . the in vitro relationship between plasma log(cca ) and ph in was investigated using lithium heparin anticoagulated blood from healthy holstein-friesian calves. plasma was harvested and tonometered with co at c over a ph range of . - . . plasma chloride concentration (ccl -) was altered by equivolume dilution of plasma with electrolyte solutions of varying ccl -( ae , ae , and ae meq/l; mean ae sd). the slope of the linear regression equation relating log(cca ) to ph for tonometered plasma samples from the calves was - . ae . at normal values for cca ( . ae . meq/l), albumin concentration ( . ae . g/l), and ccl -( . ae . meq/l). the experimentally-determined value for the slope for bovine plasma was identical to that determined previously for human plasma. the formula for correcting cca in bovine plasma for change in ph from . is therefore: cca ph . cca  (À .  { . -ph}) . this equation is only valid at normal concentrations of albumin and chloride in plasma. equivolume dilution of plasma by electrolyte solutions of varying cclindicated that cca ph . increased by . meq for every meq/l increase in ccl -. in other words, plasma cca at a given ph increases directly in response to an increase in plasma ccl -, presumably because the additional chloride displaces calcium that is electrostatically bound to albumin. furthermore, the increase in cca is independent to the change in plasma ph induced by an increase in ccland decrease in plasma strong ion difference. our finding that hyperchloremia directly increases plasma cca provides an additional mechanism by which ingestion of high chloride (acidogenic) rations prevents the clinical signs of periparturient paresis. our finding is consistent with the results of other studies that indicate acidogenic salts that contain chloride as the predominant anion (ie, nh cl, cacl ) are more effective in increasing cca than equimolar quantities of acidogenic salts such as mgso . coagulase negative staphylococci (cns) are among the most common bacteria isolated from the bovine mammary gland. historically, these bacteria were lumped together as minor mastitis pathogens. modern molecular techniques have allowed accurate speciation and fingerprinting of the cns species. these methodologies have recently been applied to the study of cns in bovine mastitis. the aim of the studies presented here was to evaluate the role of individual cns species on milk somatic cell count (scc) and duration of intramammary infection (imi). in the first study, mammary quarter foremilk samples were aseptically collected from all lactating cattle ($ head) at the university of missouri dairy research center once monthly for months for bacterial culture and milk scc. staphylococcal isolates were speciated by sequencing the rpob gene and strain-typed using pulsed-field gel electrophoresis (pfge). using species and fingerprint data along with published definitions for staphylococcal imi, cns imis were identified. overall, species of cns were identified with staphylococcus chromogenes, s. cohnii, s. epidermidis, and s. simulans being most prevalent. duration of imi and scc data were analyzed using regression models accounting for repeated measures. mean milk scc and duration of imi were found to differ between cns species (p o . ). although most imis were of short duration ( month), staphylococcus capitis and s. chromogenes imis had longer mean durations of infection than or more of the other species isolated. mean sccs were under , cells/ml in most cases. however, staphylococcus simulans and s. xylosus imis were more inflammatory (mean , cells/ml) and had a higher mean scc than s. cohnii, s. epidermidis, and s. haemolyticus. to examine the relationship between cns imi and milk scc in a larger population of cattle, cns isolates from the canadian bovine mastitis research network (cbmrn) culture collection were obtained for speciation. speciation and fingerprinting were performed as above. isolates were from subclinical imi from before and after the dry period and from subclinical imi during lactation. data associated with each isolate were obtained from the cbmrn database. nine-hundred-thirty-eight isolates from mammary quarters in herds were successfully speciated. twenty-two different species of cns were identified. staphylococcus chromogenes was the most frequent species identified accounting for % of the infections. three species, s. chromogenes, s. xylosus, and s. simulans accounted for % of all infections. data were analyzed using a linear hierarchical repeated measures mixed model. differences in mean scc were found between some cns species and culture negative control quarters and also between different species of cns (p o . ). overall, our data demonstrate potential differences in pathogenicity between strains of cns that cause bovine mastitis. passive transfer of maternally derived antibodies via ingestion of good-quality colostrum within the first hours of life is crucial for the health and future productivity of dairy calves. however, infectious diseases can be transmitted via colostrum feeding, which may require use of a colostrum replacement product or pasteurization to decrease disease transmission. while pasteurization of colostrum is effective for sterilization, heating during pasteurization can alter the viscosity of colostrum, destroys important nutritional biomolecules, and has been shown to decrease colostral igg concentrations. the purpose of this study was to investigate the effect of high pressure processing (hpp) on the viscosity, igg concentration, and bacterial contamination of bovine colostrum. first milking colostrum samples were collected from cows from different farms, and ml aliquots of each sample were pooled for analysis. pooled colostrum was processed in triplicate using an isostatic press at mpa ( , psig) for , , , , and minutes. samples were tested for the effects of hpp on the viscosity, bacterial load (cfu/ml), and igg concentration. there was a significant decrease (p o . ) in bacterial load at each time point when compared to time . no significant difference in igg concentration was found between any time points. subjectively, the colostrum viscosity appeared to increase with the processing time, though the rheologic assessment has not been completed at this time. hpp appears to be an effective method to decrease bacterial contamination of colostrum while maintaining appropriate igg concentrations. minimizing the processing time or pressure may be necessary to maintain an acceptable viscosity of the colostrum. based on these results, additional studies are justified in order to determine the optimum combination of processing time and pressure and the effect of hpp on specific bovine pathogens. the heme-associated iron-binding apoprotein lactoferrin (lf) is known for its, anti-inflammatory, anti-parasitic, antimicrobial and bactericidal effects. lactoferrin demonstrates ubiquity throughout mammalian host biological fluids: saliva; tears; mammary secretions, as well as at mucosal surfaces. it is also released from immune cells under pathogenic stimulation. the purpose of this study which has been approved by western university's institutional animal care and use committee is to further characterize the mechanisms through which lf modulates inflammation in the face of bacterial endotoxin. it was hypothesized that lf would inhibit p phosphorylation. numerous studies speak to the ability of lf to alter leukocyte function, inhibit cytokine production, and bind lipopolysaccharide (lps); mechanisms through which it is believed to achieve its anti-inflammatory effects. recently, investigators demonstrated its ability to interact with host dna while others describe regulation of granulocyte adhesion and motility; elucidating its roles in the apoptotic signaling. in earlier studies, dawes me, et al. demonstrated lactoferrin's ability to limit the expression of inducible cyclooxygenase- and the gelatinase, matrix metalloproteinase - by lps-induced macrophages. the generation of these inflammatory mediators is modulated by pro-inflammatory cytokines such as interleukin- b (il- b) and tumor necrosis factor-alpha (tnf-a), the production of both being dependent on signaling through the p mitogenactivated protein kinase (mapk) pathway. peripheral mononuclear cells (  )isolated from buffy coat cells of healthy neonatal to -month old holstein calves were cultured in the presence and absence of lf ( ng/ml), lps ( mg/ml), anisomycin ( mg/ml), a known p activator -the positive control, and mm of sb , a known p inhibitor -the negative control. sample lysates obtained post culture was subjected to immunoprecipitation and kinase reactions. reactions were terminated under reducing conditions and evaluated using western immunoblotting. phosphorylation of activated transcription factor- (atf- ) by phosphorylated p served as the marker of investigation. immunologically reactive atf- expression by lps and anisomycin-treated cells was compatible with a prominent band at kd. evidence of lf-induced inhibition of lps-induced p- activation was observed in lanes representative of co-cultures of lf lps; lf anisomycin; and anisomycin sb , which was demonstrated by decreased immunological reactivity at kd. the findings here, suggest that lf interferes with lps-induced p- activation of transcription factor atf- , in vitro. this serves as additional proof of its potential use in attenuating the systemic effects of lps. six ( ) clinically normal, purpose-bred cats of similar age and body condition were imaged with [ f] fluorodeoxyglucose ([ f]fdg) and [ f]ftha by using dynamic cardiac-gated fused pet/ct for kinetic assessment of myocardial glucose and fatty acid uptake and metabolism, respectively. kinetic tracer uptake within the myocardium was achieved by initiating image data acquisition simultaneously with tracer injection. pet data were acquired over a hour period with the heart in the center of the scanner field of view. regions of interest were drawn in the left ventricular wall and thoracic aorta for the purpose of measuring the kinetics of tracer redistribution. serial blood samples were also taken during pet imaging for comparison with image data. the equilibrium biodistribution of both tracers was documented hour post-injection in a whole body pet/ct image. standard echocardiographic examination of cardiac structures was also performed. both radiotracers remained in the plasma fraction; however, [ f]ftha was cleared from the more rapidly than [ f]fdg (t / $ and $ min, respectively). the tracers were readily visualized within the feline myocardium in dynamic pet images and analysis of the blood pool clearance from the kinetic image data agreed with blood sampling data. myocardial uptake of each tracer was best described by a double exponential analysis and was rapid but variable among animals (range - bq/cc/min), although blood glucose levels were similar in all cats during image acquisition. physiologic [ f]fdg was observed in the brain, salivary tissue, gastrointestinal tract, renal pelves and urinary bladder, with [ f]ftha seen in the myocardium, liver and renal cortex. all cats were normotensive with normal echocardiographic parameters. this study demonstrates the utility of kinetic imaging using the left ventricle (lv) shape has been suggested to change from elliptical to more globular in response to chronic volume overload. real-time three-dimensional echocardiography (rt de) offers new modalities for lv assessment. the aim of the study was to investigate left ventricular changes in shape and volume occurring in response to different severities of naturally acquired myxomatous mitral valve disease (mmvd) in dogs using rt de. privately owned dogs were classified by standard echocardiography into: healthy ( ), mild ( ), moderate ( ) and severe mmvd ( ). a lv cast was obtained using semi-automated endocardial border tracking from rt de dataset, from which global and regional (automatically acquired basal, mid, and apical segments based on lv long-axis dimension) end-diastolic (edv) and endsystolic volumes (esv), lv long-axis dimension and rt de sphericity index, were derived. global and regional edv and esv increased significantly with increasing mmvd severity, assessed by mmvd group-wise comparisons and linear regression analyses using left atrial to aortic root ratio, and lv end-diastolic and end-systolic dimensions. all three segments contributed to the overall increased global volumes, but the mid edv segment was strongest associated with increasing lv end diastolic dimension (p . ). furthermore, lv long axis distance and lv sphericity index increased with increasing mmvd severity. the basal and apical edv segments were strongest associated with sphericity index (p o . ). in conclusion, this rt de study showed that increased lv edv, primarily in the mid segment, leads to rounding of lv apical and basal segments in response to increasing mmvd severity in dogs. dogs from shelters in florida with naturally acquired di infection were euthanized and necropsied. all adult di in each dog were sexed using morphological features. total worm burdens and numbers of males and females were recorded. no other information was available for any dog. all data, raw and transformed, were examined visually and descriptively. raw numerical data were further examined by a paired t-test; log-odds transformed data were examined by logistic regression. we also conducted a binomial distribution goodness of fit analysis assuming a null hypothesis of a m:f . . worm intensities ranged from to di per dog. eight dogs had unisex infections: / had all-female infections. dogs with lowintensity dual-sex infections were more likely to have greater numbers of female di. overall, sex ratios were equal (paired t-test, p . ). however, logistic regression demonstrated that the probability of being female is strongly affected by the total worm intensity, with lower intensities increasing the probability of having a predominance of female worms. our data show that di sex ratios in naturally-infected dogs equal when examining the entire dog population, but deviate to favor female worms at low worm intensities. these data could impact adulticide treatment strategies. the reasons for sex ratio distortion in di are unknown. we evaluated cardiac reverse remodeling after mitral valve repair under cardiopulmonary bypass (cpb) for mitral regurgitation in small breed dogs. fifty dogs (body weight . - . kg, age - years) with mitral regurgitation were treated between august and november . the cardiac murmur was grade / - / . the preoperative chest x-rays showed cardiac enlargement (vertebral heart scale (vhs) . - . ). echocardiography showed severe mitral regurgitation and left atrium enlargement (la/ao . - . ). after inducing anesthesia, a thoracotomy was performed in the fifth intercostal space. cpb was started by using a cpb circuit connected to carotid artery and jugular vein catheters. after inducing cardiac arrest, the left atrium was sectioned and chordae tendineae rupture confirmed. the chordae tendineae were replaced with expanded polytetrafluoroethylene. a mitral annulus plasty was also done, and the left atrium was closed. after de-clamping for restarting the heart, the chest was closed. heart rate decreased from - bpm to - bpm. the grade of cardiac murmur was reduced to / - / three months postoperatively, and the heart shadow was reduced (vhs . - . ) in the chest x-rays. echocardiography confirmed the marked reduction in mitral regurgitation and the left atrial dimensions (la/ao . - . ). mitral valve repair reduced enlarged cardiac size by reduction of regurgitant rate. pulmonary arterial hypertension (pah) is a well recognized condition in dogs leading to considerable morbidity and mortality. the majority of therapeutics has focused on endothelial dysfunction causing reduced production of vasodilators, such as nitric oxide and prostacyclin, coupled with overproduction of vasoconstrictors, such as endothelin- . more recently, it has been shown that the mitochondria play an important role in the development of pah as oxygen sensors and regulators of cellular proliferation. in pah, pulmonary artery smooth muscle cells undergo a metabolic shift from oxidative phosphorylation in the mitochondria to glycolysis in the cytoplasm as the major energy source and this leads to suppression of apoptosis and increased proliferation. dichloroacetate (dca) inhibits pyruvate dehydrogenase kinase to activate pyruvate dehydrogenase which catalyzes the rate limiting step for entry of pyruvate into the krebs cycle, thus increasing mitochondrial respiration. in three different rat models of pah, dca has been shown prevent and reverse pah by normalizing molecular pathology, stimulating apoptosis of pulmonary artery smooth muscle cells, and reducing pulmonary artery hypertrophy. dca has known toxic effects, including reversible hepatotoxicity and peripheral neuropathy, and has not been studied in any species with naturally occurring pah. the objective of this open label pilot study is to evaluate the therapeutic and toxic effects of dca in naturally occurring canine pah. three dogs with pah diagnosed by doppler echocardiography and no correctable underlying cause are enrolled in the study. dogs are orally administered mg/kg of dca divided daily for weeks, and then . mg/kg of dca divided daily for the remainder of the study. at baseline, , , , and weeks, an echocardiogram, cbc, serum chemistry profile, urinalysis, nt-probnp, blood uric acid, blood lactate, noninvasive blood pressure, nerve conduction study, and trough dca level ( hr post-dose) are obtained. the measured echocardiographic parameters include peak and mean tricuspid regurgitant flow velocity and pressure gradient, peak and enddiastolic pulmonary regurgitant flow velocity and pressure gradient, pulmonary valve flow velocity acceleration time and ejection time, pulmonary valve flow velocity time integral, right ventricular myocardial performance index, tricuspid annular plane systolic excursion, and systolic tricuspid annular tissue velocity. variables are inspected for normalcy and equality of variances and a two-sided paired t-test is used to compare the variables before and after treatment at each evaluation time. the basis for the role of the mitochondria in pah and the results of this pilot study will be presented to determine if dca warrants further study as a therapy for dogs with pah. study produced the strongest associations between the ncl phenotype and cfa markers. all ncl-affected tibetan terriers were homozygous for the same haplotype which extended for consecutive snps spanning . mb. none of the annotated genes within this target region had previously been associated with human or rodent ncl. we used dna from ncl-affected tibetan terriers to resequence the coding regions and intron-exon borders of several genes harbored within the target region and found a single base pair deletion, c. delg, in exon of positional candidate atp a . this deletion produces a frame shift and a predicted premature termination codon. we genotyped all tibetan terrier dna samples in our collection and found all ncl-affected tibetan terriers to be homozygous for the c. delg allele. eleven additional c. delg homozygotes were either less than years old, or lost to follow up. there were no known cases of ncl in the remaining tibetan terriers which were either heterozygous (n ) or homozygous for the ancestral allele (n ). atp a is a member of group of ion transport genes and has been associated with lysosomes. mutations in human atp a cause kufor-rakeb syndrome (krs), a rare neurodegenerative disorder with clinical features that include parkinsonism plus spasticity, supranuclear upgaze paresis, and dementia. post-mortem findings in krs have not been reported. we conclude that ncl in tibetan terriers is caused by a mutation in atp a . our results suggest that krs may be a form of adult onset ncl in humans. niemann-pick type c (npc) disease is a progressive neurological disorder characterized by dementia and ataxia, hepatic and pulmonary disease, and death typically within the first or second decade. despite the identification of causative mutations, the pathogenesis is not clear and therapies to successfully treat npc disease have been ineffective to date. the recent use of intravenously administered -hydroxypropylbeta-cyclodextrin (hpbcd), an fda-designated orphan drug (may ), in a small number of children with npc disease is based on favorable treatment outcome data in subcutaneously treated mouse and cat models. to rigorously evaluate the mechanistic, pharmacologic, and toxicity issues associated with hpbcd therapy in npc disease, we have utilized the spontaneous feline npc model harboring a missense mutation in npc (pc s), orthologous to the most common mutation in juvenile-onset patients. the feline npc model has clinical, neuropathological and biochemical abnormalities similar to those present in juvenile-onset patients making this model homologous to the most common disease form seen in human patients. we identified that intrathecal administration of hpbcd ameliorated all clinical aspects of neurological disease at least up to weeks of age (an age when untreated cats die) but had no effect on hepatic disease. we identified that while subcutaneous therapy with hpbcd at all doses ameliorated liver disease, only mg/kg substantially affected neurological disease but also resulted in early death due to pulmonary toxicity. finally, we identified a dose-related toxic effect of hpbcd on hearing function that had not been described in any other species. leukodystrophies are disorders of myelin synthesis and maintenance that affect cns myelin. they are subdivided as leukodystrophies, hypomyelinating disorders and spongy degenerations. although infrequently seen, several forms have been described in various dog breeds. we present a novel form of complex leukodystrophy consisting of hypomyelination and spongy degeneration that presents primarily with hind end tremors in border terrier puppies. three border terriers from two different litters (and lineages) are described here that presented with a history of shaking movements. the youngest dog was a -week old male. it was the only dog affected in the litter. the other two dogs were -week old female littermates. there were two unaffected males in the same litter. physical examination revealed no abnormalities. on neurological examination, the affected dogs displayed severe hind end tremors, with a characteristic swinging side-to-side movement (best described as ''rumpshaker''). the tremors also involved the head and thoracic limbs but to a lesser degree, and disappeared when the dogs were asleep or at rest. severe cerebellar ataxia was observed when the dogs ambulated. proprioceptive positioning was delayed in the pelvic limbs of all dogs. spinal reflexes and nociception appeared normal. necropsy was performed in all puppies. no macroscopic changes were observed. histologic evaluation of the cns revealed spongy degeneration and hypomyelination in all funiculi of the cervical and thoracic spinal cord. white matter of the frontal, temporal and parietal cortices had mild multifocal spongy degeneration and hypomyelination, whereas white matter of the cerebellum, medulla and pons showed severe diffuse spongy degeneration and hypomyelination with gliosis. the combination of reduced myelin formation combined with spongiform white matter changes in the absence of microglial responses suggest a complex pathogenesis affecting both oligodendrocytes' capacity to synthesize myelin and the stability of the myelin that was formed. the number of oligodendrocytes and axons appeared subjectively normal indicating a primarily hypomyelinating process. the clinical and pathological features of this disease have not been described in any other canine leukodystrophy. the primary and most striking clinical feature is the presence of severe tremors in the hind end, causing the ''rumpshaker'' pheynotype. genetic studies are underway to determine if the disease is inherited and the inheritance mode. a syndrome of border collie collapse (bcc) appears to be common in dogs used for working stock. this syndrome has also been called malignant hyperthermia, heat intolerance, exerciseinduced collapse and ''wobbles''. a presumptive diagnosis of bcc can only be made by eliminating other causes of exercise intolerance and weakness. the purpose of this study was to describe the clinical features of collapse in affected dogs and determine if there were characteristic clinical or laboratory features at rest or after exercise that could aid in diagnosis. seven adult border collies with a history of collapse during sheep herding (affected) and adult border collies regularly used for sheep herding but showing no signs of exercise intolerance (normal) were evaluated before and after participating in a videotaped minute exercise protocol consisting ofa series of continuous short outruns and fetches of three sheep in an outdoor pen. exercise was halted at minutes or earlier if there were signs of gait or mentation abnormalities. pre-exercise evaluation included physical examination, orthopedic and neurological exam. pre and immediate post exercise rectal temperature, pulse and respiration, patellar reflexes, ecg, cbc, serum biochemistry profile, cortisol, arterial blood gas and plasma lactate and pyruvate concentrations were measured. clinical parameters (gait, temperature, reflexes) and lactate and pyruvate concentrations were evaluated at intervals up to minutes after exercise. additional testing in affected dogs included measurement of acetylcholine receptor antibodies (achrab) and dna testing for dynamin-associated exercise induced collapse (deic) and the ryanodine receptor mutation associated with canine malignant hyperthermia(mh). one week after exercise affected dogs had thoracic radiographs and echocardiography performed and were anesthetized for emg and muscle biopsies. there were no significant differences in temperature, pulse, respiration, or any laboratory parameter at any time point between normal and affected dogs. no arrhythmias were detected. affected dogs were negative for the dna mutations tested and for achr ab. thoracic radiographs, echocardiograms, emgs and muscle biopsies were normal. the normal dogs had no alterations in mentation or gait during or after exercise. three of the affected dogs had exercise halted early ( min- min) because of altered gait or mentation. all of the affected dogs were abnormal in the minutes following exercise. abnormalities seen in all dogs included disorientation, dull mentation, swaying, falling to the side, exaggerated lifting of limbs each step, choppy gait, delayed limb protraction, scuffing of rear and/or forelegs, and crossing legs when turning. all dogs returned to normal by minutes. bcc appears to be an episodic nervous system disorder that can be triggered by exercise. genetic testing excluded deic and the described canine mh mutation. common causes of exercise intolerance were eliminated, but the cause of collapse in bcc was not determined and no clinical or biochemical marker to aid diagnosis was established. equine cushing's disease (ecd) is common in older horses. the purpose of this study was to determine the frequency of diagnosis, identify prognostic factors and assess owner satisfaction with treatment. the study was a retrospective cohort design evaluating equine accessions reported to the veterinary medical data base (vmdb) and the ohio state university from - . proportional accessions, annual incidence and demographic characteristics of horses with ecd were compared with all accessions in the vmdb. medical records for a subset of horses were extracted and owners contacted to obtain long-term follow up information. two hundred seventeen new cases of ecd were reported to the vmdb. incidence increased from . / , in to . / , in . eighty-one percent of horses were ! years of age. average delay from onset of signs to diagnosis was days (range to , days). hirsutism ( %) and laminitis ( %) were the most common clinical signs. improvement in one or more signs months after diagnosis was reported by / ( %) of horse owners. none of the clinical or laboratory data were associated with survival and, % of horses were alive, . years after diagnosis. / ( %) of horses were euthanatized and / ( %) were euthanatized due to conditions associated with ecd. twenty-eight of ( %) of horse owners said they would treat a second horse for ecd. ecd is becoming a more frequent diagnosis. fifty percent of horses survived . years after diagnosis and owners were satisfied with the horse's quality of life. supported by centers of excellence in livestock diseases and human health, college of veterinary medicine, university of tennessee. the role of the hypothalamic-pituitary-adrenal (hpa) axis in sepsis has been a subject of a great deal of research. the role that the somatogenic axis plays in sepsis is less well understood and how these two axes interact during critical illness is not clear. the purpose of this study was to assess inter-relationships of adrenocorticotropin (acth), cortisol, and insulin-like growth factor-i (igf-i), in septic and non-septic term foals. blood samples were obtained from term septic foals less than days of age (n ) admitted to texas a&m university veterinary medical teaching hospital or mid-atlantic equine hospital. the foals were classified as septic by a sepsis score ! and/ or a positive blood culture. non-septic term foals less than days of age (n ) and having a sepsis score o and a negative blood culture, were obtained from texas a&m university veterinary medical teaching hospital and mid-atlantic equine hospital. plasma and serum were processed from whole blood collected by jugular venipuncture upon admission, at hours post admission and at days post admission or at the time of discharge. plasma concentrations of acth, and serum concentrations of cortisol and igf-i were determined by specific rias. data were analyzed using linear mixed-effects modeling with foal modeled as a random effect and day of admission modeled as an ordered categorical variable; post-hoc testing of pair-wise comparisons was made using the method of sidak. significance was set at p o . , and analyses were performed using s-plus software (tibco, inc., seattle, wa). plasma concentrations of acth were not significantly different between septic and non-septic foals whereas septic foals had greater serum cortisol ( ae ng/ml vs ae ng/ml) but lower serum igf-i ( ae ng/ml vs ae ng/ml) relative to non-septic foals pooled overall sampling times. the positive association of the peripheral blood concentrations of acth and cortisol depended on disease status of the foals. specifically, cortisol and acth were positively correlated for the septic foals (p . ) but not significantly correlated in the non-septic foals. peripheral concentrations of acth and igf-i were not significantly correlated whether data were pooled overall or stratified by sepsis status. however, peripheral concentrations of cortisol and igf-i were negatively associated (p . ); disease status did not influence this association, although it appeared to be a stronger association for the septic than the non-septic foals. the negative correlation between serum concentrations of the adrenal axis steroid cortisol and the somatogenic axis peptide igf-i may reflect interactions of these homeorhetic hormones. further studies of these and other metabolic hormones in a greater number of foals are warranted to better understand how these factors contribute to survival or non-survival of critically ill foals. botulism is a potentially fatal paralytic disorder which definitive diagnosis is difficult. the purpose of this study was to investigate if repetitive stimulation of the common peroneal nerve will aid in the diagnosis of suspected botulism in foals. four healthy foals were used for its comparison with foals with suspected botulism. controls were anesthetized and affected foals were sedated to avoid risks of anesthesia. the common peroneal nerve was chosen for its superficial location and easy access. stimulating electrodes were placed along the common peroneal nerve. for recording, the active and reference electrodes were positioned over the midpoint and distal end of the extensor digitorum longus muscle, respectively. repeated supramaximal stimulation of the nerve was performed utilizing a range of frequencies ( to hz). amplitude, area under the curve and percentages of decrement or increment for each m wave over subsequent potentials for each set of stimuli were analyzed. baseline m waves were decreased in affected foals compared to controls. a decremental response was seen at all frequencies in control foals. decremental responses were also observed in affected foals at low frequencies. however, an incremental response in amplitude and area under the curve was seen in all affected foals at hz. reduced baseline m waves with incremental responses at high rates are supportive of a presynaptic neuromuscular disorder which botulism was the most likely cause in these foals. repetitive nerve stimulation is a safe, simple, fast, and non-invasive technique that can aid in the diagnosis of suspected botulism in foals. this study examined the frequency with which dogs are exposed to e. chaffeensis and e. ewingii relative to e. canis, which is transmitted by the more ubiquitously distributed brown dog tick (rhipicephalus sanguineus). a total of , canine serum samples, ranging from to from each of the participating institutions, collected at random from clinical accessions, diagnostic laboratories and/or shelters were evaluated. all serum samples were tested by three microtiter plate elisas using species-specific peptides for antibodies to e. canis, e. chaffeensis and e. ewingii. zip code information for sample origin was provided by the collaborator and was used to assess seroprevalence by region. comparisons were evaluated using the chi-square test. seroreactivity for at least of ehrlichia spp was found in samples from every institution both mississippi and oklahoma had greater than a % samples from ohio had the lowest aggregate seroprevalence ( . %) with only dogs e. canis seropositive, one e. ewingii seropositive and no e. chaffeensis seroreactors. the geospatial pattern of e. chaffeensis and e. ewingii seropositive samples was similar to that previously reported based on modeling seroreactivity to e. chaffeensis in white-tailed deer as well as the distribution of human monocytic ehrlichiosis (hme) cases reported by the cdc. this study provides the first large scale regional documentation of canine exposure to these three ehrlichia spp., highlighting where infections most commonly occur and thus identifying areas where heightened awareness about these emerging vector urinary incontinence (ui) occurs in approximately % of spayed female dogs. the most common cause is urethral sphincter mechanism incompetency (usmi). pharmacological agents are effective, however, not all dogs respond, and dogs may become refractory to treatment over time. urethral bulking, where a compound is injected submucosally in the urethra, has been used in women and in female dogs with urinary incontinence. new synthetic compounds have been used in human medicine; the most promising is polydimethylsiloxane (pdms), which has been shown to be more effective than glutaraldehyde cross-linked collagen. the purpose of this descriptive clinical trial is to evaluate the safety and effectiveness of pdms urethral bulking agent (pdms uba) in client-owned, spayed female dogs with naturally-occurring ui due to usmi.twenty-two, spayed female dogs were included. dogs had a median age of years ( to years). eighteen dog breeds were represented, and dogs weighed a median of . kg ( . to . kg). average length of time of ui was . ae . years; / dogs had been treated medically, of which / were continent, / were improved, and / had no improvement. dogs were deemed healthy based on results of physical examination, complete blood cell counts, plasma biochemical analysis, and urinalysis; urine cultures were negative.dogs were anesthetized, positioned in dorsal recumbency, and cystoscopy performed using a . mm, -or -degree, cm rigid cystoscope. urethral bulking was performed with pdms uba. on average, . ae . ml were injected in to locations approximately to . cm distal to the trigone submucosally in the proximal urethra. good coaptation was achieved in all dogs. the procedure took on average . ae . minutes. one dog experienced urethral obstruction after the procedure; a foley catheter was inserted for approximately hours and removed at which time she urinated normally and was continent. three dogs experienced an acute allergic reaction characterized by blepharedema and urticaria treated successfully with diphenhydramine. dogs were discharged on day of procedure except for the one dog that experienced urethral obstruction. all dogs were treated with meloxicam ( . mg/kg po q h for days).owners were contacted on day after discharge and / dogs were continent; / dogs was improved. dogs were re-evaluated week after discharge and / dogs were continent and / dogs polyneuropathy in large breed dogs is a relatively common clinical problem for which the genetic basis is generally unknown. the first cases of polyneuropathy in the leonberger breed (leonberger polyneuropathy or lpn) were identified in by one of the authors (gds) and a report published in (musclenerve : - ) . in this report a spontaneous, distal and symmetrical polyneuropathy with onset between to years of age was described and characterized clinically, electrophysiologically, histologically and morphometrically. there were striking similarities between lpn and the charcot-marie-tooth group of human inherited sensory and motor polyneuropathies, which have many known genetic mutations.a genome-wide case-control association study for lpn was performed with cases and controls on high-density k canine snp arrays and revealed a significantly associated region on cfa (p raw .  À p genome .  À ). a clear association of an approximately mb cfa haplotype with cases (p .  À ) was observed, particularly with those cases that were affected more severely and at a younger age (p .  À ). a positional candidate gene, arhgef , which has previously been associated with peripheral nerve abnormalities in humans, was sequenced, revealing a deletion that results in a frame shift and premature stop codon. of all leonbergers with young onset lpn (before years), . % ( of ) have two copies of this deletion, and, of all young onset leonbergers that are nerve biopsy positive for lpn, . % ( of ) have two copies of this deletion. importantly, nearly all dogs carrying two copies of the deletion ( of or . %) are affected with lpn by the age of years.the leonberger breed was generated from crossing several breeds, including the st. bernard, and a polyneuropathy clinically and histologically similar to lpn occurs in this breed. to determine if the arhgef mutation was associated with polyneuropathy in the st. bernard, dna was extracted from archived frozen muscle biopsy specimens from clinical cases (n ). the identical arhgef startle disease or hyperekplexia is caused by defects in mammalian glycinergic neurotransmission resulting in an exaggerated startle reflex and extensor hypertonia triggered by noise or touch. in humans and animals, startle disease is typically caused by mutations in one of three genes (glra , glrb, and slc a ) encoding postsynaptic glycine receptor subunits (a and b) or a presynaptic glycine transporter (glyt ). a litter of seven irish wolfhounds was recently identified in which two puppies developed muscle stiffness and tremor beginning at - days of age post-partum. signs were dramatic when the puppies were handled and resolved when the puppies were relaxed or sleeping. both puppies were euthanized due to ongoing stiffness, tremor and breathing difficulties. necropsies were performed, but no microscopic pathological abnormalities were identified in the peripheral or central nervous system.based on the clinical signs, exons from the three candidate genes were amplified from genomic dna isolated using pcr and directly sequenced. no deleterious polymorphisms were identified in either glra or glrb. however, difficulties were experienced in amplifying slc a exons and from affected animals, although control samples were positive, suggesting that the pcr primer designs and conditions were not at fault. further pcrs revealed that the reason for this anomaly was the presence of a homozygous . kb deletion encompassing exons and of the glyt gene in both affected animals. this deletion is predicted to result in the loss of part of the large cytoplasmic n-terminus that is vital for trafficking of glyt to synaptic sites, and a loss of all subsequent transmembrane domains via a frameshift. this genetic lesion was confirmed by defining the deletion breakpoint, southern blotting and multiplex ligationdependent probe amplification (mlpa). this analysis enabled the development of a rapid genotyping test that revealed heterozygosity for the deletion in the dam and sire and three other siblings, suggesting recessive inheritance of this disorder. wider testing of related animals has identified a total of carriers of the slc a deletion and enabled the identification of non-carrier animals to guide future breeding strategies. insulin resistance (ir), obesity, and type diabetes affect glucagon-like peptide (glp- ) concentrations in humans and rodents, but this incretin hormone has not been examined in horses. we therefore hypothesized that glp- concentrations would change in horses as obesity and ir were induced or exacerbated by overfeeding. six horses previously diagnosed with equine metabolic syndrome were provided with twice the amount of digestible energy required for maintenance as sweet feed and hay for weeks. intravenous and oral glucose tolerance tests (ogtts) were performed at and weeks. effects of time and period ( and weeks) were assessed by repeated measures anova.mean body weight increased from ae kg (range, to kg) to ae kg (range, to kg) over weeks, with individual horse weight gain varying from to %. mean body condition score increased (p . ) from ae (range, to . ) to ae (range, to ). three horses developed mild laminitis. glucagon-like peptide concentrations increased over time during ogtts (p . ), but the period  time effect was not significant (p . ). area under the glp- curve remained unaffected by weight gain, whereas area under the insulin curve increased (p . ) over time, indicating a reduction in insulin sensitivity. obesity and ir were induced or exacerbated when horses previously diagnosed with ems were overfed, but glp- concentrations did not change as a result. hypertonic saline solution ( . %) (hss) is an intravenous fluid used for the emergency treatment of intravascular volume deficits. the use of this fluid in horses with severe dehydration is controversial. the purpose of this study was to compare the use of hss and isotonic saline solution ( . %) (iss) for the emergency treatment of endurance horses.endurance horses eliminated from competition and requiring intravenous fluid therapy were eligible for enrollment in the study. twenty-two horses were randomly assigned to receive ml/kg of either hss or iss along with l lactate ringer's solution (lrs). following this bolus, all horses were treated with an additional l of lrs. blood and urine samples were collected before, during and after treatment. data was compared using two-way anova with repeated measures.as compared to iss, hss horses showed a greater decrease in pcv (p . ), total protein (p . ), albumin (p . ), and globulin (p . ). hss horses showed a greater increase in sodium and chloride (p o . ) as compared to iss horses. horses receiving hss had a shorter time to urination (p . ) and lower specific gravity (p o . ) than those receiving iss.results of this study indicate that hss may provide faster restoration of intravascular volume deficits than iss in endurance horses receiving emergency medical treatment. more profound electrolyte changes should be expected with hss however. b -adrenergic receptor agonists have been shown to increase erythrocyte carbonic anhydrase activity, which may stimulate the jacobs-stewart cycle and increase pulmonary circulation transvascular fluid fluxes during exercise. increase in pulmonary transvascular fluid fluxes (j v-a ) and consequent increase in the pulmonary interstitial fluid would be detrimental for alveolar o exchange during the fast erythrocyte transition time across the pulmonary capillaries. therefore, we hypothesised that treatment with inhaled b -adrenergic receptor agonist will increase j v-a and the alveolar-arterial po difference (aado ) during exercise.six stb horses were exercised on a high-speed treadmill at % vo peak until fatigue. horses were randomly assigned to treatment with salbutamol (sal: mcg) or placebo (control: con) inhalation via aeromask à min prior to exercise, with cross over treatment used at the repeated exercise test ( days later). arterial and mixedvenous blood, as well as co elimination and o uptake, were sampled simultaneously at rest, during exercise at sec intervals until fatigue, and into recovery. blood gases were analyzed. aado was calculated using the inspired po ( mmhg), and blood partial pressure of o and co . blood volume (%) changes across the lung were calculated from changes in hemoglobin and hematocrit values in venous and arterial blood. cardiac output (q) was calculated using the fick equation. j v-a was calculated using q and blood volume changes across the lung. variables were analyzed using two-way repeated-measures anova (p o . ).the duration of exercise to fatigue was . ae . min and . ae . min in both con and sal, respectively. at rest sal had no effect on j v-a , oxygen consumption (vo ), blood oxygen saturation (so ) or aado (p . ). at the onset of exercise j v-a increased in con and sal (p o . ) and at fatigue reached . ae . l/min and . ae . l/min, respectively. treatment with sal had no effect on j v-a during exercise (p . ). at the onset of exercise so and vo increased in con and sal (p o . ). treatment with sal had no effect on so or vo during exercise (p . ). aado increased during exercise in con and sal (p o . ) and at fatigue reached . ae . mmhg and . ae . mmhg, respectively. treatment with sal had no effect on aado during exercise (p . ).inhaled b -adrenergic receptor agonist salbutamol at the dose of mcg given min before exercise did not affect the duration of exercise to fatigue, j v-a , vo , so or aado . therefore, it had no detrimental effect on alveolar-capillary diffusion distance and the ventilation/perfusion mismatch in exercising horses. inflammatory airway disease (iad) and recurrent airway obstruction (rao) represent two classes of equine lung inflammatory diseases that may share some similar immunologic mechanisms. there is evidence that th cytokines and il- play some role in rao. iad is a common condition in horses, but its pathophysiology is still not understood. the aim of the present study was therefore to determine the mrna expression of th , th and th inflammatory cytokines, to understand the immunological mechanisms of iad.the mrna expression of ten inflammatory cytokines and chemokines was measured in the bronchoalveolar fluid (balf) of seventeen horses with iad and compared with ten control horses. the horses were selected based on -their clinical signs, -the inflammatory cells count in the balf, -their physical examination and -their medical history. the mrna expression of il- , il- b, il- , il- and il- was significantly up-regulated in balf from horses with iad.furthermore, the balf samples were subdivided in two groups based on the differential cells count -balf with increased mast cells (iad-mast) and -balf with increased neutrophils (iad-neutro). il- was significantly down-regulated in the iad-neutro group compared to the iad-mast group. il- , il- and il- were significantly up-regulated in the iad-neutro group compared to the iad-mast group.the present study shows that iad in horses is characterized by a th and a th mrna inflammatory expression profile and that different immunological mechanisms are involved in mast cells or neutrophils accumulation in the balf of horses with iad. b -adrenoreceptor (b -ar) agonists are a class of medications that promote smooth muscle relaxation and bronchodilation in horses and humans with airway disease. activated human peripheral blood lymphocytes (pbls) also respond to b -ar agonist stimulation by attenuating the production of cytokines associated with the pathogenesis of asthma and recurrent airway obstruction (rao). the aim of this study was to develop an in vitro technique for measuring the response of equine pbls to stimulation with salbutamol, a b -ar agonist. this method was then used to compare the response of pbls from rao-affected and non-affected horses to b -ar agonist stimulation. pbls from rao and nonaffected horses were cultured ( x /ml) in rpmi complete media with concanavalin a (cona, ug/ cells) for , , or days then stimulated with salbutamol ( minutes). using flow cytometric techniques, response was measured by detecting protein kinase a phosphorylation of vasodilator stimulated phosphoprotein (vasp). results were verified by western blot analysis. activated pbls were then incubated with cona for one day were pre-incubated with b or b-adrenoreceptor antagonist (ici , , sigma s ; atenolol, sigma s ) for minutes, followed by minutes salbutamol ( nm) stimulation. results were analyzed by anova or ancova and differences were considered significant when p o . .response to b-antagonist was only observed in activated pbls (pre-cultured with con a) and was greater in cells from rao horses as compared to cells from non-affected horses. the addition of b-antagonist attenuated the response of pbls to salbutamol while the addition of a b -antagonist had no effect. these findings indicate that activated pbls from rao-affected horses have a greater response to salbutamol as compared to pbls from non-affected horses, and this response is mediated mainly through the b -ar.human b -ar are known be polymorphic and this polymorphism results in a variable response to b -agonist binding that affects long term outcome in human asthmatics. further studies are required to determine if the difference in response of pbls from rao affected as compared to non-affected horses is due to genetic polymorphism in the equine b -ar, and whether this difference is associated with a propensity for horses to develop equine rao. key: cord- -nvzfpntu authors: nan title: research communications of the th ecvim‐ca congress date: - - journal: j vet intern med doi: . /jvim. sha: doc_id: cord_uid: nvzfpntu nan cobalamin concentrations were previously investigated in cats, but little information is available concerning the follow up of hypocobalaminemic cats. we aimed to assess the frequency of hypocobalaminemia within a large cohort of cats with gastrointestinal signs and describe the epidemiological, clinical, biological and follow-up characteristics of hypocobalaminemic cats. cats with gastrointestinal signs and for which a cobalamin assay (simultrac-snb radioassay kit vitaminb Ò , mpbiomedical) was performed between and at the ldhvet laboratory were retrospectively included in the study. ( . %) cats presented for gastrointestinal signs had an hypocobalaminemia: the majority were domestic short hair, % were males ( % castrated) and % females ( % castrated), aged from months to years. the main clinical signs included chronic diarrhea ( %), weight loss ( %), polyuropolydypsia ( %), vomiting ( %), polyphagia ( %), fatigability ( %) and dysorexia ( %) with a median duration of months before diagnosis. cobalamin values ranged from to ng/l (median: ng/l). % of the hypocobalaminemic cats had also a hyperfolatemia (folate > ng/l) at diagnosis. ft was measured in the older cats (> years) and revealed an hyperthyroidism (ft > pmol/l) in % of the cases. / hypocobalaminemic cats had a known clinical and biological follow-up (median time follow-up = days): cobalamin significantly improved month after treatment ( lg/kg im cyanocobalamin in a single dose) for % of the cats, even if % remained hypocobalaminemic. % of the followed cats were clinically improved, of which % with an associated higher cobalamin value. clinical and biological improvement after cobalamin supplementation was significantly associated with an increase in folate concentration (p-value = . ). however, % of the cats with an improved cobalamin value did not show any clinical improvement. hypocobalaminemia is frequently observed in cats as a consequence of gastrointestinal signs. cobalamin concentrations could be used as an indicator of the severity of various gut diseases more than a primary cause, because one third of the cats did not show any clinical improvement despite an improved cobalamin value. a hyperfolatemia appearing after treatment of hypocobalaminemia seems to be a good indicator of a clinical improvement associated with a return to a normal intestinal integrity. disclosures: no disclosures to report. cobalamin malabsorption is common in old cats with weight loss, macronutrient malabsorption and enteric protein loss due to idiopathic chronic enteropathy, ice (patil ap and cupp cj. proc. nestle-purina compan anim nutr summit, - , , williams and czarnecki-maulden, proc rd ecvim-ca congress, ) . high dose oral cobalamin supplementation reverses subnormal serum concentration within week but serum cobalamin can become undetectable within as little as month following cessation of supplementation (williams and czarnecki-maulden, proc rd ecvim-ca congress, ). the objectives of this study were to determine if serum cobalamin concentrations in cats with ice and the response to oral supplementation and withdrawal are associated with differences in the intestinal microbiome. the study evaluated cats older than years of age that were being fed nutritionally complete and balanced diets that included a fortification of vitamin b . thirty-one of these cats had ice demonstrated by increased fecal fat (> %), subnormal fat digestibility (< %), subnormal serum cobalamin or increased serum methylmalonic acid, but without exocrine pancreatic insufficiency as assessed by assay of serum trypsin-like immunoreactivity. serum cobalamin was determined by competitive binding assay and the fecal microbiome by roche sequencing and analysis by qiime (quantitative insights into microbial ecology), pca (principal component analysis) and opls (orthogonal projections to latent structures). of these ice cats were supplemented with oral cobalamin for months. serum cobalamin concentrations were determined monthly during supplementation and for months after cessation of supplementation. in the cats there was a significant (p ≤ . ) association between serum cobalamin and the fecal microbiome, with species being positively correlated with serum cobalamin concentration and species being negatively correlated. serum cobalamin was subnormal (< ng/l) in of the ice cats at the start of the supplementation study and subsequently became normal or supranormal. within to months after cessation of supplementation serum cobalamin was subnormal in the original cats and additional cat. it is concluded that serum cobalamin concentration and the responses to oral supplementation and subsequent cessation of supplementation are significantly associated with the composition of the intestinal microflora as reflected in the fecal microbiome. disclosures: the study described in the abstract was performed at nestle-purina facilities and funded entirely by nestle-purina. david williams is a consultant and adviser for nestle-purina, idexx laboratories, and the gastrointestinal laboratory at texas a&m university. he receives royalties from idexx laboratories and has given continuing education lectures sponsored by nestle-purina. inflammatory bowel disease (ibd) is a common cause of chronic gastrointestinal signs in cats. typically, lymphoplasmacytic inflammation is found in biopsies, but a subset of cats with ibd has neutrophilic inflammation. the clinical significance of neutrophilic infiltration is unclear. the aim of this retrospective study was to use fluorescence in situ hybridisation (fish) to look for the presence of any microorganisms within the intestinal epithelium of cats diagnosed with ibd and then to identity those micro-organisms. our hypothesis was that neutrophilic enteritis in cats would be associated with intestinal mucosal invasion by microorganisms, and specifically by campylobacter spp. the study included cats presented to the small animal hospital, langford veterinary services for investigation of gastrointestinal disease which had duodenal biopsies collected endoscopically. thirteen cats were diagnosed with neutrophilic inflammation (study group) and cats with lymphoplasmacytic inflammation (control group). fluorescence in situ hybridisation (fish) targeting either all eubacteria or campylobacter jejuni, coli and upsaliensis was used to identify and count intra-mural bacteria in the intestinal biopsy samples. neutrophils were detected simultaneously using a fish probe to neutrophil elastase. the pixel distance between different bacterial species and neutrophils was measured. all animals in both groups showed the presence of intra-epithelial bacteria and the number of bacteria present did not differ between the control and study groups. similarly, campylobacter jejuni and upsaliensis were present in some animals in each group but numbers did not differ between the groups. in contrast, campylobacter coli was present in significantly more study cats than control cats (p = . ; chi-squared test) and the study group showed significantly higher numbers of c. coli in the tissue than the control group (p = . ; mann-whitney u test). co-localisation of neutrophils and c. coli was demonstrated with c. coli closer than any of the other bacteria to the neutrophils. this association was statistically significant (p < . ; mann-whitney u test). the role of the intestinal virome in health and disease is gaining increased attention in human medicine. the use of next generation sequencing (ngs) technologies has allowed identification of diversity and distribution of the virome. these approaches can equally be applied to dogs.this study aimed to identify and characterise the virome present in faeces of dogs with chronic enteropathy (ce) compared to the virome of healthy dogs (hd). faecal samples were evaluated from hd and dogs with ce ( food, antibiotic and steroid responsive) using a ngs approach. a viral enrichment protocol, using a series of centrifugation, endonuclease treatments and bacterial filtration were performed. the enriched viral dna and rna were extracted and amplified using sequence-independent single-primer amplification (sispa) protocol, and subsequently sequenced by ngs using the illumina miseq platform at the agrf. two bioinformatic pipelines were used to analyse the viral population. after selecting high quality reads and removing dog and bacterial sequences, sequence information was compared against reference databases. we identified a total of , viral contigs, with , dna viral sequences and , rna sequences across all dog samples. the majority of viral hits from both groups of faecal samples were bacteriophage ( . % hd and . % ce), from several families mainly from the caudovirales order. after all analyses, only viral eukaryotic families were identified across all samples. two groups of sequences similar to known virus families, reoviridae and papillomaviridae, were identified in both groups (hd / and / and ce / and / , respectively). sequences similar to picornaviridae were identified only in one dog with ce and sequences similar to adenoviridae, parvoviridae and coronaviridae were identified only in healthy dogs ( / each).further genomic characterisation and phylogenetic analysis was undertaken on viruses. the genome segments of a rotavirus (reoviridae) isolate were determined. similarly, the sequence of the entire coding region of a kobuvirus (picornaviridae) isolate was determined. preliminary analyses indicated that all rotavirus gene segments exhibited between % - % nt homology to previously reported canine rotaviruses. the kobuvirus sequence exhibited moderate nt homology ( %) to previously described genomes and clustered with other canine kobuvirus sequences available in genbank. in conclusion, viral sequences from a range of different virus families, including both rna and dna families, and known pathogens were identified and characterised, and the largest proportion of viral contigs identified belonged to bacteriophages. disclosures: no disclosures to report. endoscopy is widely used to perform targeted and minimally invasive biopsies for histopathology of the gastrointestinal tract of dogs and cats. only a few studies have focused on the diagnostic contribution of cytological samples of the alimentary tract. the aims of this study were to compare 'imprint' and 'squash' techniques to obtain valuable cytological samples from endoscopic biopsies, and to evaluate the potential interest of cytology compared to histology in reaching the definitive diagnosis. eighteen dogs and cats presenting gastrointestinal symptoms that underwent an endoscopy of their alimentary tract were prospectively included. five biopsies of each area of interest were collected for regular histopathological analysis. an additional biopsy from each area was used to obtain cytological specimens by imprint and squash techniques. cytology samples were all reviewed blindly by the same pathologist. cytology samples of insufficient quality were considered as 'non diagnostic' and were excluded from further analysis. diagnostic conclusions of both cytological and histological analyses were classified into defined categories (inflammation or neoplasia with subcategories, fibrosis, epithelial hyperplasia, and normal) to allow comparison between the techniques. agreement between cytology and histology was determined by cohen's kappa coefficient. from the cases, biopsy specimens were collected from different localizations for histology and cytology slides were obtained. final diagnosis was neoplasia in cases and inflammatory disease in . considering imprint technique, / were considered 'non diagnostic'. for squash technique, only / of cytological samples were considered as 'non diagnostic'. squash cytology and histology gave the same results in . % of the cases (n = ) and agreement between the techniques was considered 'moderate' (k = . ( % confidence interval [ci] . ; . )). agreement was 'fair' between imprint cytology and histology (n = ) (k = . [ % ci . ; . ]). gastric spiral organisms (gso) were observed in cases. in cases they were identified only on cytology. amongst these cases, mast cells were identified on cytology in cases, and not on histology in any cases. mast cells were not found in any other cases. this prospective pilot study demonstrated that cytological examination of gastrointestinal biopsy squash samples obtained during endoscopy of the alimentary tract may give relevant information, which can help the clinician to initiate treatment while histopathological analysis is pending. furthermore, it can give additional information (presence of potential pathogens, mast cells) that may not be identified on histopathology. disclosures: no disclosures to report. intramucosal escherichia coli are implicated in the pathogenesis of granulomatous colitis of boxer dogs. clinical remission hinges upon its eradication, most commonly achieved with fluoroquinolones. antimicrobial resistance is not uncommon among e. coli isolated from dogs with gc and impairs successful treatment. published data is lacking on efficacious therapies for gc dogs with fluoroquinolone-resistant e. coli. the aim of this study was to characterize the antimicrobial resistance patterns and molecular characteristics of e. coli isolated from dogs with gc. additionally, to evaluate the clinical outcome of dogs treated with antimicrobials guided by culture and susceptibility results. the study population was ( boxers and french bulldogs) client-owned dogs with gc. gc biopsies with fish-confirmed intramucosal e. coli were submitted for bacterial culture. antimicrobial susceptibility was determined by broth microdilution. most strains were further characterized by phylogroup and overall genotype using triplex and random amplified polymorphic dna polymerase chain reaction, respectively. treatment and clinical outcomes data were obtained. culture yielded e. coli strains ( - per dog, med ) from / dogs. resistance to fluoroquinolones was identified in / dogs; this was correlated with resistance to other macrophage-penetrating antimicrobials (p < . ). phylogroup a was over-represented among enrofloxacin-resistant strains. in dogs with e. coli isolated at multiple time points, phylogroup changed over time. clinical remission was achieved in / dogs with fluoroquinolone-sensitive e. coli. dogs with fluoroquinolone-resistance had a more variable response; treatment with meropenem (median mg/kg sq q hours for weeks) resolved clinical signs in / . we conclude that antimicrobial resistance is a growing concern. gc-associated e. coli appear genetically diverse. clinical remission can be achieved in the face of fluoroquinolone-resistance though in vitro antimicrobial susceptibility does not consistently predict a positive response. disclosures: no disclosures to report. canine s a has potential as a biomarker of inflammation in dogs. fecal s a concentrations were increased in dogs with chronic gastroenteropathy (ce), and correlated with the severity of clinical and endoscopic disease. a negative outcome was associated with higher fecal s a concentrations in ce dogs, but the response to different forms of treatment and fecal s a has not been reported, and this information will be important to further evaluate the utility of fecal s a as a biomarker for gastrointestinal disease. aim of this study was to evaluate the association between responses to various treatments (i.e., elimination diet, antimicrobial drugs, or corticosteroids/other immunosuppressants) and fecal s a in dogs with ce. fecal samples were collected from dogs diagnosed with ce, and fecal s a was measured in all specimens using an established in-house elisa. based on the response to treatment, dogs were classified as having antibiotic-responsive diarrhea (ard), food-responsive diarrhea (frd), or steroid-responsive/therapy-resistant idiopathic inflammatory bowel disease (ibd). statistical analysis was performed using non-parametric -or multiple-group comparisons, the likelihood ratio to evaluate the association between groups of dogs and response to treatment, and a receiver operating characteristic curve to calculate sensitivity and specificity at the optimum cut-off concentration. a total of dogs with ce (median age: . years; males/ females) were included in the study, the final diagnosis of which were ard (n = ), frd (n = ), or ibd (n = ). response to treatment was complete remission (n = ), partial response (n = ), or no response (n = ). fecal s a concentrations ranged from to , ng/g, and higher s a levels were seen in dogs with ibd than in dogs with frd (p = . ) or ard (p = . ). dogs that did not respond to treatment had significantly higher s a levels than dogs with partial (p = . ) or complete (p = . ) remission, but response to treatment was associated with disease classification (p = . ). despite a small number of patients, fecal s a levels of > , ng/g at the time of diagnosis distinguished dogs that failed responding to treatment from those with at least partial remission with a sensitivity of % and specificity of %. we conclude that, in line with our previous finding that fecal s a may be a useful biomarker of disease severity in dogs with ibd, fecal s a may also have utility in predicting the lack of response to treatment in dogs with ce. the utility of serial fecal s a concentrations to monitor treatment response in dogs with ce warrants further research. disclosures: dr. heilmann and dr. steiner have filed a patent application that includes the s a elisa used for this study. constipation is a common presenting complaint in dogs and cats. differential diagnosis for this clinical sign is well-known but strictures resulting from gastro-intestinal inflammation are not commonly included and have been rarely reported in the literature. acute diarrhea and bone ingestion can lead to anal or rectal stricture which is responsible for the constipation. the aim of this retrospective study was to describe the prevalence of inflammatory rectal and anal stricture in small animals and to describe a simple and effective treatment. medical records of dogs and cats presented for constipation, dyschezia or tenesmus and diagnosed with an inflammatory stricture were obtained from the database of the gastro-intestinal diseases consultation of referral centers in gastroenterology between and ; and were reviewed. signalment, presenting complaint, clinical findings, treatment protocol and outcome were recorded. five dogs and cats were included in the study. of the cats, were purebred kitten between . and months, and among them were persians. the fifth cat was a -year-old female domestic shorthair. three out of cats had history of acute diarrhea and cats had constipation since adoption with unknown history. digital rectal examination under anesthesia revealed stricture in all cats which was treated by bougienage every days and high-fiber diet. of the dogs, age ranged from . to years; dogs had history of acute diarrhea and had ingested bones in prior days. colonoscopy and biopsies were performed in all dogs and showed a lymphoplasmocytic infiltration in all of them. dogs were treated with digital bougienage every other day until disappearance of the stricture, metronidazole, lubricant laxatives, corticosteroids and highfiber diet. the prevalence of inflammatory stricture in dogs was . % based on dogs presented with the same complaints between and . for all dogs and cats, clinical signs related to the stricture resolved for the duration of their follow-up. benign strictures secondary to gastro-intestinal inflammation should be systematically included in the differential diagnosis of constipation. strictures are easily palpated on digital rectal examination, which should always be performed during clinical examination. histology should be a routine part of the diagnosis workup to exclude neoplasia. endoscopy-assisted balloon dilatation with concurrent intralesional injection of triamcinolone has been used in dogs and reported in the human literature. the treatment described here is simpler and effective. in dogs, it can be done at home by the owner. disclosures: no disclosures to report. acute pancreatitis is a diagnostic challenge because of anatomic inaccessibility of the pancreas, vague clinical signs and physical examination findings, and inconsistent laboratory results. common, yet non-specific, clinical signs include abdominal pain, anorexia, vomiting, and diarrhea. ultrasonography is the imaging modality of choice to evaluate the pancreas. the purpose of this study was to compare clinical signs with ultrasonographic findings in dogs with acute pancreatitis to account for differences in clinical presentation depending on the region of the pancreas affected as determined by ultrasonography. the hypothesis was that there would be differences in clinical presentation depending on the pancreatic region involved. records of client-owned dogs diagnosed with acute pancreatitis based on history, clinical signs, laboratory testing, and abdominal ultrasonography were retrospectively evaluated. based on ultrasonography, dogs were divided into groups: group - dogs with changes within the left limb of the pancreas exclusively; group - dogs with changes within the right limb of the pancreas exclusively; and group - dogs with diffuse pancreatic involvement. presence of abdominal pain, anorexia, vomiting, and diarrhea was correlated between groups using chi-square and fischer's exact test. no significant differences regarding age, breed and sex were noted between groups. in group pain was noted in %, anorexia in %, vomiting in %, and diarrhea in % of dogs. in group pain was present in %, anorexia in %, vomiting in %, and diarrhea in % of dogs. in group pain was noted in %, anorexia in %, vomiting in %, and diarrhea in % of dogs. pain was noted with a significantly higher frequency in diffuse pancreatic disease as compared to disease restricted to the left or right limb of the pancreas. anorexia was significantly more common with right limb involvement. both vomiting and diarrhea were significantly more common with disease restricted to the left limb as compared to diffuse parenchymal or right limb involvement. despite overlap between groups, these findings indicate that pain response is expected to occur with a higher frequency in diffuse pancreatitis but overall is not a very common clinical sign. anorexia is more prevalent in dogs with pancreatitis of the right limb whereas vomiting and diarrhea both are more evident in dogs left limb pancreatitis. differences between the groups can possibly be ascribed to gastric involvement when the left side of the pancreas is affected. disclosures: no disclosures to report. increased physical exercise has been reported to improve the clinical symptoms of chronic enteropathies, such as inflammatory bowel disease, in human patients. the aim of this investigation was to evaluate the impact of an intervention to increase physical exercise in dogs with chronic enteropathies. twenty-two dogs ( each in the exercise and control groups) with chronic enteropathies and no response to an elimination diet were included. routine diagnostic work-up (haematology, plasma biochemistry profile, urinalysis, faecal parasitology, abdominal radiographs, and ultrasound) was conducted in all dogs to eliminate underlying causes. all dogs were given oral prednisolone ( mg/kg/day) for days, followed by a tapering dosage over weeks. after weeks of prednisolone treatment, a certified canine rehabilitation therapist instructed the owners of dogs in the exercise group on how to increase their dogs' physical exercise. the exercise protocol combined aerobic and resistance exercises in low-to moderate-intensity interval training. owners of dogs in the control group were asked to maintain the dogs' routine lifestyles. modified canine inflammatory bowel disease activity scores (cib-dais), based on the parameters of activity level, appetite, vomiting, stool consistency, stool frequency, bloating, and weight loss, were calculated pre-treatment and and weeks post-treatment for all dogs. cibdai scores were compared among timepoints (pre-treatment and post-treatment assessments) and between groups (exercise and control) using multivariate repeated-measures models for multiple comparisons. all dogs showed improvement after weeks of prednisolone treatment. modified cibdais decreased in the exercise (from . ae . to . ae . ) and control (from . ae . to . ae . ) groups. after weeks of the increased physical exercise intervention, the modified cibdai in the exercise group decreased significantly ( . ae . ) relative to the first post-treatment assessment (p = . ), whereas this index remained similar ( . ae . ) in the control group. modified cibdais differed significantly between groups after weeks of treatment (p = . ). all parameters of the modified cibdai were significantly affected by the intervention of increased physical exercise; the largest difference was found for body weight (p < . , adjusted r = . ) and faecal frequency (p < . , adjusted r = . ) and activity level (p < . , adjusted r = . ). an increased physical activity intervention had positive effects on clinical symptoms in dogs with chronic enteropathies. disclosures: no disclosures to report. corticosteroid therapy is commonly required in veterinary patients for treatment of inflammatory, immune-mediated, neurological and neoplastic diseases. some of these patients also require assisted enteral nutrition via percutaneous endoscopic gastrostomy (peg) tubes. this retrospective case-control study evaluated the complications associated with peg tube use in veterinary patients receiving corticosteroids in a referral teaching hospital. medical records of dogs and cats in which a peg tube was placed in the qmha between january and march were reviewed. patients were included if the peg tube was in use for at least hours and if complete medical records, including clinical notes from referring veterinarians, kennel sheets, communication records with patients' owners and notes from tube removal, were available. to be included in the steroid group, patients must have received corticosteroid therapy (> mg/kg/day) for at least % of the length of time the peg tube was in use. control patients were not treated with corticosteroids. forty-two cases were included ( dogs and cats). fourteen patients ( dogs and cats) were included in the steroid group and patients ( dogs and cats) were included in the control group. complications were scored in terms of severity as minor ( ), moderate ( ) and major ( ) and compared between groups using the mann-whitney u-test. values of p < . were considered significant. complications included: serous discharge (n = ), sanguineous discharge ( ), purulent discharge ( ), stoma site inflammation ( ), peg tube dislodgement ( ) , pain around the stoma ( ), peg tube blockage ( ) and peg tube chewed by the patient on its tip ( ) or at the stoma ( ). median (interquartile range) of maximum complication scores for control and steroid groups were respectively ( ) and ( ). the maximum complication scores were not significantly different between groups (u = . , p = . ), though patients receiving corticosteroids showed a trend towards higher maximum complications scores than those in the control group. in conclusion, owners of dogs and cats receiving corticosteroids in which a peg tube is planned should be appraised of the possibility of complications beyond those normally associated with tube placement alone. disclosures: no disclosures to report. canine obesity is usually treated with dietary energy restriction, but data are limited regarding nutritional adequacy. the aim of the current study was to compare intake of essential nutrients with national research council recommendations in obese dogs during weight management with a purpose-formulated diet. twenty-seven dogs were included in this non-randomized retrospective observational cohort study. all were determined to be systemically well, and without significant abnormalities based upon physical examination and clinicopathological assessments. the dogs underwent a controlled weight loss protocol of at least weeks, to achieve ideal condition and using a high protein high fiber weight loss diet. median, maximum, and minimum daily intakes of all essential nutrients were compared against nrc recommended allowances (ra) for adult dogs. median weight loss was % ( - %), median daily energy intake was kcal/kg . ( - kcal/kg . ), and no signs of nutrient deficiency were observed in any dog. based upon the average nutrient content of the diet, daily intake of the majority of essential nutrients was greater than their nrc ra (per kg body weight . ), except for selenium, choline, choline ( / dogs) and methionine+cysteine ( / dogs), all essential nutrients remained above nrc minimum requirements (mr) throughout the trial. daily intakes of most essential nutrients meet both their nrc ra and mr in obese dogs throughout a period of weight loss. in light of absence of signs of nutrient deficiency, the significance of the borderline intakes for some nutrients (especially selenium and choline) is not known, and further studies are recommended. disclosures: the following conflicts of interest apply: ajg's readership is funded by royal canin; ajg has also received financial remuneration and gifts for providing educational mate-rial, speaking at conferences, and consultancy work; slh's post at the university of liverpool is also funded by royal canin; the diet used in this study is manufactured by royal canin; ss and vb are employed by royal canin. esvcn-o- endocrine profile of obese dogs. d. j. rochel, c. amato, p. nguyen, l. jaillardon, b. siliart. oniris, nantes atlantic college of veterinary medicine, food science, engineering, nantes cedex , france obesity is a frequent condition of the dog, associated with many endocrine and metabolic disturbances leading to major organ dysfunctions. we therefore aimed to assess biochemical and hormonal profiles of a large cohort of obese dogs. obese dogs were retrospectively included in the study, based on an overweight over % the ideal body weight (ibw). endocrine profiles consisted in assessing prolactin, leptin, insulin like growth factor type (igf ), cortisol after an acth stimulation test, insulin, free thyroxine (ft ) and ctsh serum concentrations. obese dogs ( % females of which % spayed and % males of which % castrated) were from different breeds and ranged from to years [median years, . % between and years]. % of the dogs suffered from generalized (versus abdominal) obesity and long-term obesity (> year) was described in % of the cases. the main observed clinical signs were abdominal distension ( %), fatigability ( %), polyphagia ( %), decline of interest for usual activities, ( %) and polyuropolydipsia ( %). biochemical profile was unremarkable except that % of the dogs had hypercholesterolemia (cholesterol > mmol/l). a high prolactin value (> ng/ml) was observed in % of the dogs, a high leptin value (> lg/l) in %, a high igf value [igf > lg/l (ibw < kg), > ( < ibw < kg) and > (ibw> kg)] in % and a high insulin value in % (> lui/ ml), without significant correlation with glucose concentration in % of the cases. % of the dogs had a high cortisol value (> nmole/l after acth stimulation) and % had alow ft (ft < pmol/l) with % having a high tsh value (> , ng/ml). canine obesity is associated with many endocrine disorders including hyperprolactinemia, hyperleptinemia, high igf value, hypercortisolemia and/or a hypothyroxinemia associated with a high ctsh value. the endocrine profile could be very interesting for the diagnosis and prognosis of canine obesity and could allow the veterinarian to choose a better treatment, particularly when the diet is unsuccessful. further investigations could be done to assess the prognostic value of the endocrine profile at the diagnosis of canine obesity to control the treatment efficiency. disclosures: no disclosures to report. a better understanding of how dogs undergo healthy ageing would benefit owners and veterinarians alike. in july a longitudinal study began to evaluate health and longevity in labrador retrievers ( males and females, all neutered, mean age . years), continuously fed a fixed plane of nutrition with identical housing, standardised husbandry and veterinary care. body condition score was maintained between and on a -point scale. standard veterinary protocols were used for any medical conditions; cancer and severe or life threatening conditions were managed individually based on quality of life assessments. the 'average' lifespan of labrador retrievers was estimated to be years. dogs were classified according to lifespan as 'typical' if they died between and ≤ . years of age, 'long' ≥ to . years and 'exceptional' ≥ . years (corresponding to % longer than the average lifespan). data were analysed using linear mixed models with random effects for slopes and intercepts and a fixed effect for lifespan grouping variable. on st july , dogs ( %) were classified as exceptional with still alive, typical (n = ) and long (n = ). gender and age at neutering were not associated with survival time or risk of death (p≥ . ). body weight change showed a quadratic trend: up to age , body weights increased for all lifespan groups but the changes were not significantly different. there was a significant change in body weight from to years as exceptional dogs increased body weight while the long-lifespan dogs lost weight (+ . versus À . kg/dog/year, p = . ). after age the exceptional and long groups both had similar losses. dual-energy x-ray absorptiometry scans revealed that wholebody fat (g) increased in all lifespan groups to age but the change was significantly slower for the long lifespan dogs when compared with typical dogs which accumulated fat at > times the rate. all groups lost a similar amount of whole-body lean tissue (g) through age (p > . ). up to age the mean % gain in whole body fat, and % loss of whole-body lean tissue, was slower and the mean change in fat to lean ratio was lower in the exceptional and long-lived dogs compared to the typical dogs (p £ . ). typically aged labradors showed a greater gain of fat tissue, and greater loss of lean tissue, up to years of age than exceptional dogs. disclosures: the eukanuba diet used in this study is manufactured by spectrum brands whilst dmm is employed by spectrum brands. vja is an independent epidemiologist who helped analyze the data and was financially supported by spectrum brands for this work. pjw has participated in veterinary seminars organised by spectrum brands and has received an honorarium for this work. introduction: in healthy animals, the phosphate (p) in combination with calcium homeostasis is regulated in comparatively narrow limits: excessively ingested p is excreted via urine. common knowledge is, however, that p is a progressive factor in chronic renal failure (crf) wherefore typically a p restricted diet is prescribed for affected patients. in , pastoor demonstrated that a p excess (significantly at~ mg p/mj me; ca/p . / for days) impairs renal function even in healthy cats, diagnosed mainly by reduced endogenous creatinine clearance. dietary p originates from meat and other protein sources, bones and cartilages, mineral supplements and technical additives (water binding, palatability a/o texture enhancer etc.). the daily amount ingested with complete diets from the european market often exceeds the recommended daily allowance (rda; nrc , fediaf considerably (up to times, anonymus ). our own studies were done with the aim ( ) to survey the reproducibility of the results of pastoor ( ) and ( ) to test effects of different ca/p ratios and p sources on parameters of renal function in healthy adult cats. animals, materials and methods: up to adult, healthy cats were appointed to groups in every trial period. firstly, a balanced diet was fed for days including a days balance trial. one group was then switched to a high p diet whereas the other remained on the balanced diet, again completed by a balance trial. after days of wash-out (balanced diet) both groups were switched in a cross-over design repeating the days trial period. this design was carried out repeatedly with high p diets differently composed concerning ca/p ratios and p sources. endogenous creatinine clearance, glucosuria, microalbuminuria, water and mineral balance were determined at each period. the study was approved by the proper authority for animal welfare. results and discussion: the studies confirmed the results of pastoor ( ): a p content of approximately mg/mj me in a diet consumed by healthy cats at maintenance may lead to a decrease of the creatinine clearance. markers of acute tubular damage, i.e. glucose and microproteins in the urine, showed positive results in several trials. the p concentration of a diet alone is no sufficient marker of its tolerance since ca/p ratio and p origin influence the effects. therefore, high p diets cannot be considered safe and should be avoided also in healthy cats. literature the study aim was to investigate serum global proteomes in dogs with overt dilated cardiomyopathy (dcm) and to correlate protein expression in serum with that in ascitic fluid. eight dogs diagnosed with dcm based on echocardiographic evidence including increased left ventricular dimension at diastole and systole, increased e point to septal separation, and decreased fractional shortening were included in the study. serum and ascitic fluid samples were analyzed for proteomes using a label-free lc-ms/ms method. eight dogs from different breed, sex and age served as controls. proteome analyses revealed significantly different expressions of eight proteins in all samples. expressions in serum of apolipoprotein a , ig heavy chain v, superoxide dismutase and plasminogen were higher (p < . ), while expressions of clusterin, hemoglobin subunit ß, apolipoprotein c ii, b glycoprotein i (ß gpi) were lower (p < . ) in dogs with dcm than in control dogs. in addition, apolipoprotein a , clusterin, hemoglobin subunit ß, ig heavy chain v, plasminogen and ß gpi were down-regulated whereas apolipoprotein c ii and superoxide dismutase were upregulated in ascitic fluid compared with serum in dogs with dcm. data obtained in the present study suggest that serum and/or ascitic fluid proteomes may help explain some of the pathophysiological mechanisms involved in the progression of dcm. tick paralysis is an important disease of dogs and cats in australia, induced by toxins of the paralysis tick ixodes holocyclus, very commonly occurring from spring to autumn on the eastern seaboard. respiratory failure is one of the major clinical derangements occurring in severe cases of tick paralysis, although its pathogenesis is poorly characterised. there is some suggestion that the respiratory failure is secondary to toxin-induced myocardial dysfunction with the subsequent development of cardiogenic pulmonary oedema. the purpose of this study was to determine cardiac involvement in dogs infested with ixodes holocyclus, through measurement of cardiac biomarkers. a cross-sectional study of client-owned dogs was undertaken. dogs enrolled in the study belonged to one of groups: dogs with tick paralysis and no-mild respiratory compromise (group a), dogs with tick paralysis and moderate-severe respiratory compromise (group b) and a control group of dogs with neither tick paralysis nor respiratory compromise. respiratory compromise was scored using a commonly employed grading system. each animal had the following parameters determined: serum cardiac troponin i (ctni) concentration, plasma n-terminal pro-btype natriuretic peptide (nt-probnp) concentration and serum creatinine concentration. for most dogs, but not all, spo was also determined. mean nt-probnp concentrations were significantly lower in dogs with tick paralysis than those in the control group, with no statistical difference detected between dogs with and without respiratory compromise. there was no significant difference in mean ctni concentrations between groups, however there were some high outliers of ctni concentration. creatinine concentrations differed significantly between each group, with the control group having the highest mean creatinine and those in group b having the lowest mean creatinine. there was no significant difference in spo between groups. this study showed no compelling evidence of cardiac insult as measured through cardiac biomarkers in our cohort of dogs with tick paralysis; however there was evidence supporting reduced preload in these dogs. in addition, the results of this study suggested that a small subset of patients with systemic hypoxaemia might have some loss of cardiomyocyte integrity. disclosures: employee/salary: gp nicolson, r malik and nj beijerink are employees of sydney university; alh mcgrath is a student at sydney university; ra webster is an employee of the animal emergency service; carrara; s kaye is an employee of queensland veterinary specialists, stafford heights; j li is an employee of northside emergency veterinary service, forestville. grants/research: this study was funded by bequest grants provided by the faculty of veterinary science at the university of sydney. idexx laboratories provided some funding for the laboratory tests. no other disclosures. speaking & consultancies: none related to this presentation. investments/commercial interests: none related to this presentation. gifts, hospitality, travel support: none related to this presentation. other: none related to this presentation. the use of ntprobnp, troponin i (high-sensitivity, ctni) and pdk pre-screening for occult dilated cardiomyopathy(odcm) in the doberman pinscher(dp) has been previously reported. the aim of this prospective collaborative study was to identify robust pre-screening recommendations for dp utilizing the current generation of commercially available diagnostic tests. a cohort of asymptomatic dp were evaluated at the american doberman national specialty show in , , and (n = , median age years, range - ). evaluations consisted of auscultation, echocardiography (echo), -minute ecg (ecg), ntprobnp (cardiopet plus r ), ctni, and pdk . dp were classified as affected (odcm) if their lvids was > the protect entry criteria with or without vpcs (n = ). dp were classified as normal (nl) if their lvidd and lvids < protect entry criteria and they had no vpcs (ntprobnp:n = , ctni:n = , pdk ;n = ). roc analysis comparing odcm and nl was done for ntprobnp, ctni, and pdk . overall accuracy (percent correctly classified) was considered for individual tests as well as a variety of combinations. the goal of combining tests was to eliminate false negatives while minimizing false positives. the auc for ntprobnp, ctni and pdk was . , . and . respectively with the percentage correctly classified equal to . , . and . (including false negatives for pdk ) when a cut-off of pmol/l, . ng/ml or a positive pdk (hetero-or homozygous) were used respectively. when the cutoffs for ntprobnp and ctni are used in combination the auc was . and . % were correctly classified ( false negatives, false positives). disclosures: research and programatic support from idexx the lab that runs ntprobnp. the study was sponsored by boehringer ingelheim, idexx and the doberman pinscher society of america. with renal disease. r. langhorn , a.s. kloster , l.r. jessen , a. jensen , j. koch . university of copenhagen, frederiksberg c, denmark, copenhagen small animal hospital, valby, denmark cardiac troponins are sensitive and specific markers of myocardial injury. however, their reliability in renal disease has been questioned due to possible renal involvement in troponin elimination. the purpose of this study was to examine whether cardiac troponin i (ctni) is elevated in cats with renal disease and no concurrent cardiac disease, and whether ctni is measurable in urine of cats with normal and compromised renal function. cats presenting with renal disease or primary structural cardiac disease were enrolled in a renal and a cardiac group, respectively. a healthy control group was similarly included. clinical and echocardiographical examination was performed and blood and urine samples obtained for each cat. the mann-whitney u test was applied to evaluate differences between groups. seven cats with renal disease, cats with cardiac disease, and healthy cats were included. serum ctni concentrations were (median [range]) . [ . - . ] ng/ml for the renal group, . [ . - . ] ng/ml for the cardiac group, and . [ . - . ] ng/ml for the control group. the renal group had significantly higher serum ctni concentrations than the control group (p = . ), but was not significantly different from the cardiac group (p = . ). urine ctni was measurable in . % ( / ) of cats in the renal group ( . [ . - . ] ng/ml), % in the cardiac group, and . % ( / ) of controls ( . ng/ml). it was concluded that elevated serum ctni in cats with renal disease may occur without concurrent cardiac disease. moreover, compromised renal function was associated with presence of ctni in urine. disclosures: no disclosures to report. sudden death (sd) commonly occurs in dog breeds with a high predisposition to vpds and vt, occuring in about % of asymptomatic doberman pinschers (dp) and % of dp with chf, and reported in % of boxers with arvc. in human patients with atrial fibrillation (af) on anticoagulant therapy for stroke prevention (n = ), cardiac death (sd and progressive heart failure) has been reported to account for . % of all deaths. the objective of this study was to evaluate the incidence of sd in irish wolfhounds (iw) with dcm and/or af. iw from western europe (n = ) were examined by physical examination, standard echocardiography and electrocardiography between / - / (av). dogs were longitudinally followed, and owners instructed to report date and circumstances of death. dcm and/or af were diagnosed in %. long-term follow-up until death was possible in ( m, f) dogs with dcm and ( m, f) dogs with lone af. based on the initial diagnosis, disease groups were established. results: sd occurred in to % of all groups with dcm or af: ( ) out of dogs with dcm +af, sd occurred in % after median ( - ) days, median age . ae . years. ( ) out of dogs with dcm +sinus rhythm, . % died from sd after median ( - ) days, median age . ae . years. ( ) out of iw with dcm, af +chf, . % died from sd after median ( - ) days, median age . ae . years. ( ) out of iw with lone af, sd occurred in . % after median ( - ) days, median age . ae . years, of these, dogs had developed dcm prior to death. sudden cardiac death (sd) from cardiac arrest is the most common cause of death in people worldwide, accounting for > % of all deaths from cardiovascular disease. ventricular tachycardia (vt)/ fibrillation (vf) is the most common cause of sd, other causes include pulseless electrical activity. the fatal arrhythmia has not recorded in iw. in this study, vpds were recorded at one or more occasions in / iw with af, and in / with dcm, while in iws without heart disease vpds were seen in . % of males and in . % of females. in conclusion, sd occurs in . % of iw with lone af before or after development of dcm and chf, and in . % of dogs with dcm. disclosures: no disclosures to report. tachycardia may induce dilated cardiomyopathy (dcm). irish wolfhounds (iw) are commonly affected with dcm and atrial fibrillation (af). the objective of this study was to compare heart rates (hr) of iw with lone af with hr of an age and gender matched control iw cohort that had neither af nor dcm until death and to iw with dcm with either congestive heart failure (chf), af or sinus rhythm (sr). all disease groups had hr recorded before and after - months of medical therapy. out of iw with cardiovascular examinations including standard echocardiography and electrocardiography long-term follow-up until death was possible in ( m, f) dogs with dcm and ( m, f) dogs with lone af. based on the initial diagnosis, disease groups were established. dogs received single or combination treatment of metildigoxine, aceis, pimobendan, diltiazem, furosemide, spironolactone, atenolol and sotalol. mean hr during minutes ecg monitor recordings with print-outs were evaluated. the differences in hr in the disease groups before and after treatment versus controls were examined by analysis of variance with post hoc multiple comparisons (dunnett t ). mean hr in ( m, f) control dogs was . ae . bpm. mean hr in iw with lone af was . ae . bpm before, and . ae . bpm with therapy. mean hr of dogs with dcm +af was . ae . bpm before, and . ae . bpm with therapy. mean hr of dogs with dcm +sinus rhythm (sr), was . ae bpm before, and . ae sd bpm with therapy. mean hr of iw with dcm, af +chf, was . ae . bpm before, and median . ae . bpm with therapy. in conclusion, compared to control dogs, untreated iw with chf, with dcm+af, and iw with lone af had statistically significant (p = . ) increased hr, but not dogs with dcm and sr, while under medical therapy elevation of hr was only significant (p = . ) in iw with chf and dcm. disclosures: no disclosures to report. echocardiography, as a noninvasive method, is being increasingly used as a complementary means of diagnosis in small animal clinical practice. the need for standardization of techniques by ultrasound operators in the measurement of the different echocardiographic parameters is essential for a proper examination. the aim of this work was to check a potential correlation between the values obtained in right parasternal long-axis and short-axis views in -dimensional mode and m-mode. twenty persian cats were submitted to a complete physical examination, clinicopathologic tests (hematocrit, total solids and t hormone), systolic blood pressure measurement using doppler and echocardiography. seventeen cats fulfilled the criteria inclusion and were included in the study. two-dimensional mode and m-mode echocardiograms were recorded, in systole and diastole, from both short-axis and long-axis views for evaluation of left ventricular internal diameter (lvd), interventricular septum thickness (ivs), left ventricular free wall thickness (lvpw), aorta diameter, left atrium diameter (la), pulmonary artery diameter, shortening fraction (fs) and ejection fraction (fe). statistical analysis included paired t-test (wilcoxon test) and a linear regression analysis with graphical analysis to assess agreement between the methods of data acquisition. there was a highly significant correlation (p < . ) between the values obtained in short-axis and long-axis views for the parameter la diameter (longitudinal: . ae . cm; transversal: . ae . cm), a very significant correlation (p < . ) for the parameter lvds (longitudinal: . ae . cm; transversal: . ae . cm), and significant correlation (p < . ) for the parameters ivss (longitudinal: . ae . cm; transversal: . ae . cm), lvpws (longitudinal: . ae . cm; transversal: . ae . cm) and fs (longitudinal: . ae . %; transversal: . ae . %), with no significant correlation (p > . ) between the methods for the remaining parameters. in conclusion, the data obtained from right parasternal short-axis and long-axis recordings cannot be used interchangeably in the evaluation of diastolic parameters in normal adult cats. disclosures: no disclosures to report. real time three-dimensional transesophageal echocardiography (rt dtee) is an established imaging modality for interventional cardiac procedures in humans. it has been shown to yield comprehensive views of the cardiac valves and congenital heart defects. it potentially provides a more accurate echocardiographic means of evaluating cardiac chamber volumes and a more precise pre and postoperative tool. rt dtee was used in combination with conventional -dimensional transesophageal ( -d tee) and transthoracic echocardiography (tte) standard imaging protocols. the pulmonic valve anatomy and function was evaluated in client-owned dogs with severe valvular pulmonic stenosis prior to and post-balloon valvuloplasty. the -d images were obtained with the phillips ie and cx cardiac ultrasound systems using a -d transesophageal - mhz xmatrix probe. standard cardiac - mhz, - mhz and - mhz sector array probes were used to acquire d tte images. diagnostic images were obtained in all examined patients. rt dtee did not change the balloon size decision when compared with -d tee, but provided additional views, detailed anatomy of the pulmonic valve cusps and commissures, as well as thickness and mobility of the pulmonic valve cusps, when compared to those obtained with -d tee or tte. successful balloon valvuloplasty was achieved in of the patients. repeatable artifacts occurred with respiratory excursions and insufficient probe contact. no complications related to rt dtee were observed. rt dtee provided enhanced views of the pulmonic valve while aiding in the procedure guidance and evaluation of the results post-balloon valvuloplasty. a better understanding of the anatomy of the pulmonic valve may improve procedure success. immediate visualization of the results post-balloon valvuloplasty may reduce patient risk and fluoroscopy time. a larger sample and further research will be needed to establish guidelines and predict success based on particular valve anatomy. we can conclude that rt dtee provided additional anatomical and intraprocedural information and was well tolerated in this group of dogs. disclosures: no disclosures to report. pimobendan is an inodilator utilised extensively in the treatment of canine congestive heart failure. several retrospective studies evaluating clinical records have suggested that it is well tolerated in cats; however its efficacy in this species remains ill-defined. moreover, a recent pharmacokinetic study found peak plasma concentrations of the drug to be around ten times greater than those reported in the dog, thus highlighting inter-species differences in the pharmacokinetics and, potentially, pharmacodynamics of this drug. this study was conducted to evaluate the cardiovascular effects following oral doses of pimobendan in healthy cats. a placebo-controlled, randomised, operator-blinded crossover study was conducted in healthy cats (weight range . - . kg) to evaluate the effect of doses of pimobendan (high dose [hd]: . mg vetmedin chewable tablet po; low dose [ld]: . mg vetmedin chewable tablet po) and placebo ([pl]: water po) on cardiovascular parameters over time. standard echocardiography ( d, m-mode, and spectral doppler) and oscillometric blood pressure measurements (vethdo) were performed repeatedly for hours following dosing. each measured parameter was evaluated for between-and within-treatment effects over time using linear mixed modeling with reml estimation to account for intercat variability. heart rate was used as a proxy for the level of anxiety experienced by the cats, and adjustment for this was performed through inclusion of heart rate as a fixed effect in the final model. the effect of treatment with pimobendan was most evident in the left ventricular internal diameter in systole (lvids). maximal effects occurred hours following treatment with hd and ld. the predicted mean reduction from baseline following heart rate adjustment at this time for lvids was . mm ( % reduction) and . mm ( % reduction) for hd and ld, respectively. although there were no significant differences between hd and ld in the magnitude of effect at any given time point, lvids remained significantly reduced from baseline and the pl group for longer in the hd ( minutes to hours following dosing) than in the ld group ( to hours following dosing). significant treatment effects on aortic velocity and fractional shortening were also present, but to a lesser degree. these results demonstrate that treatment with pimobendan results in measurable changes to systolic indices in cats. a dose-dependent increase in duration of effect was also observed. further studies are required to characterise the optimal dose of pimobendan in cats and to evaluate its efficacy in clinical patients. disclosures: this study was funded by grants provided by the faculty of veterinary science at the university of sydney. m. yata received financial support from luoda pharma, the australian postgraduate awards scholarship, and the eric horatio maclean scholarship whilst undertaking this project. none of the authors involved in this study have current affiliations with the drug company that manufactured the product used in this study (boehringer ingelheim pimobendan has positive inotropic, positive lusitropic and vasodilator effects and is licensed for use in dogs with cardiac disease in many countries. numerous studies have shown benefit with the use of pimobendan in canine dilated cardiomyopathy and chronic degenerative mitral valve disease, and whilst not licensed for use in cats, recent studies have reported benefits with the use of oral pimobendan in a variety of cardiac diseases including dilated and hypertrophic cardiomyopathies. an intravenous formulation has been available in the uk since january . the use of intravenous pimobendan in cats in the clinical setting has not previously been described. the aim of this study was to describe the use of intravenous pimobendan in cats with naturally occurring heart failure and report tolerability and side effects/adverse reactions. the hospital data base was searched for the use of intravenous pimobendan in feline patients. signalment, presenting signs, investigations, diagnosis, dose and time of pimobendan administration, concurrent medications, short-term outcome and adverse reactions were recorded. a boarded-certified cardiologist retrospectively reviewed all the cases in order to confirm the diagnosis. all owners had signed consent forms to permit use of off licensed drugs. eight cats were included in the study. median age was . years (range, . - . ) . six ( %) were male and ( %) were domestic short-haired. weight ranged from . to . kg. all presented with dyspnoea. three out of cats ( %) had a heart murmur and out of ( %) had a gallop rhythm. different heart conditions were diagnosed including / cats with cardiomyopathy and / with suspected endocarditis. median dose of intravenous pimobendan was . mg/kg (range, . - . ). concurrent drugs administered included frusemide, dalteparin, terbutaline, dexamethasone, amoxicillin-clavulanate, maropitant, midazolam, butorphanol, methadone, glyceryl trinitrate, clopidogrel, aspirin and potassium gluconate. no immediate adverse reactions/side effects were observed in any of the cats. five of the cats were discharged from the hospital between and hours post pimobendan administration. one cat was euthanatized, one died during thoracocentesis and one had a thromboembolic episode between and hours post pimobendan administration. intravenous pimobendan was well tolerated by this clinical population of cats with heart failure. no immediate adverse reactions/ side effects were observed. the intravenous route may be considered as an alternative method of administration of pimobendan in cats with heart failure. disclosures: no disclosures to report. spironolactone (sp) is an aldosterone receptor antagonist, registered in europe for the treatment of congestive heart failure (chf) caused by valvular regurgitation in dogs, in combination with standard therapy. in cats, cardiomyopathy (cm) is the predominant cause of heart failure. to evaluate the safety and efficacy of sp in cats with cm, a double blind, randomized placebocontrolled study has been conducted with cats receiving either sp ( . to . mg/kg po once daily) or placebo for up to months in addition to benazepril and furosemide (dose at clinician's discretion). cats ( dsh, ragdoll, siamese and burmese) with cm of various types ( hypertrophic, dilated, unclassified and arrhythmogenic right ventricular) were enrolled. the cats were randomized to either group a or b according to the presence of hcm or not and whether the cat required hospitalization due to clinical need or not. cats were recruited to group a (sp) and cats recruited to group b (placebo). the only significant difference between the groups at baseline were aortic diameter (p = . ) larger in the sp group, and la:ao ratio (p = . ) larger in the placebo group. the survival analysis showed a survival rate at months respectively of % and % in the intention to treat (itt) and per protocol (pp) populations in the sp group and % and % in the placebo group. the difference between the groups was significant (log rank test: itt population p = . ; pp population p = . ). the hazard ratio indicates an % (itt) and % (pp) reduction in risk of an event occurrence in the sp group. the effect of covariates (age, weight, bcs, systolic blood pressure, ratio la/ao) was not significant. although this is a pilot study with small numbers of cats, this data would suggest that spironolactone is likely to be beneficial in the treatment of cats with congestive heart failure secondary to a cardiomyopathy. disclosures: the study was joint funded by ceva and the university of nottingham. the authors have the right to publish the results. of the study irrespective of outcome. the objectives of this study were to describe pulmonary transit time and myocardial perfusion normalized to heart rate (nptt and nmp, respectively), evaluated by means of contrast echocardiography, in dogs with stable stage c acvim myxomatous mitral valve disease (mmvd), and to assess short-term effects of pimobendan on these parameters. we hypothesized that nptt and nmp are increased in dogs with mmvd compared to normal dogs. additionally, we hypothesized that treatment with pimobendan will decrease both variables in dogs with mmvd. we prospectively enrolled normal dogs and dogs with stable stage c acvim mmvd. all dogs had a standard and contrast echocardiographic examination at the beginning of the study. at this time, mmvd dogs were randomly assigned to receive either pimobendan ( . - . mg/kg) or not. all dogs with mmvd were re-evaluated by means of standard and contrast echocardiography after week (t ), by operators blinded to the dog's treatment. our results show that nptt was significantly increased in dogs with mmvd (p = . ), compared to normal dogs. nptt was significantly decreased at t in dogs receiving pimobendan (p = . ). nmp was not significantly different in dogs with mmvd, compared to healthy dogs (p = . ), and it was not significantly different at t in the treatment group (p = . ). in conclusion, contrast echocardiography is a valid, complementary tool for echocardiographic analysis of dogs with mmvd. pimobendan decreases nptt in dogs affected by mmvd. myocardial perfusion is not different in dogs with mmvd and is not changed by pimobendan treatment. disclosures: michele borgarelli has received research funding by boheringher inghelheim for this study. in human beings, assessment of atrial function using -dimensional speckle tracking echocardiography (ste) is useful in several cardiovascular diseases. to date information on the use of ste for the evaluation of canine atrial function is lacking. we assessed the feasibility and reproducibility of ste in the assessment of left atrial (la) function in healthy dogs and dogs with myxomatous mitral valve disease (mmvd) and we compared ste derived indices with other parameters of left atrial and ventricular function and morphology. privately owned dogs including clinically healthy dogs (control, h) and dogs with mmvd subdivided according to heart failure class (b , b , c+d) were enrolled. standard echocardiographic examination was carried out in all dogs. furthermore, video clips were acquired from a -chamber apical view and ste analysis was done using dedicated software. for the ste analysis a region of interest was drawn including the entire left atrial wall. the software provided a strain/time curve that represents the degree of deformation of the la wall over the entire cardiac cycle. similarly, la areas are provided. the following variables were evaluated: peak atrial longitudinal strain (pals, %), as the point of maximal systolic strain; peak atrial contraction strain (pacs, %) just before atrial contracting phase; contraction strain index (csi, %) calculated from these variables. la areas were recorded during ventricular systole (la maximum area, laamax, cm ) and atrial contraction (la minimum area, laamin, cm ), and the la fractional area change (fac, %) was then calculated. the intra-and inter-observer variability was assessed using the coefficient of variation (cv, %). variability was low for all variables (cvs < left atrial measurements are commonly obtained by cardiologists to assess severity of left heart disease. traditionally, measurements were obtained from m-mode images, however several studies have examined measurements of left atrial dimensions and areas from -dimensional ( d) images. studies have demonstrated the interobserver variability of aortic valve measurements, and the effects of timing of the measurements throughout the cardiac cycle. however, few studies have examined interobserver variability of left atrial measurements from d images, factors affecting variability, or the consequences of this variability on ascribing degree of disease severity to a patient. images of the right parasternal short-axis view of the left atrium and aorta were provided to cardiologists or cardiology residents. the images depicted left atria of varying size, from both dogs and cats, ranging from normal to markedly enlarged. each participant placed arrows on each image to denote the start and finish points of their left atrial measurements without prior instructions -the first being near the interface with the aorta and the second being along the caudolateral border of the left atrium. thus, sets of images were analyzed. d distributions were mapped and analyzed to determine dispersion of the start and finish points. these were compared between images to look for association with severity (estimated as the median la:ao for each image) and image complexity. results: variability of measurements around the origin of the la measurement (interface with aorta) was small, and scaled with increasing heart size. variability at the distal measurement point was complex. in only / images was interobserver variability < . la:ao, and ranged up to la:ao ( % to % variability). a systematic observer effect was noted. variability did not appear to scale with severity of disease or image complexity, although the cases with the greatest variability had severe enlargement and indistinct margins. conclusion: this study demonstrates that highly trained individuals vary considerably in their measurement of left atria from the right parasternal short-axis view. the variability did not increase with increasing disease severity or image complexity. in some instances, the same patient could be classified differently by different observers if relying on la:ao thresholds. the study suggests that standardized methods of measurement should be developed to minimize this variability. disclosures: the study was supported by veterinary information network (salary, imaging software). there is growing evidence that fibrosis plays an important role in the development of remodeling and heart failure during cardiac diseases. at cellular level, fibrotic processes are prior to clinical manifestation of symptoms. fibrosis and extracellular matrix remodeling influences cardiac function in a negative manner. to our knowledge there is no biomarker, which is able to properly detect heart-specific fibrotic processes and remodeling in the peripheral blood. such a biomarker would be of great importance in cardiac diagnostics, risk stratification and therapy monitoring. in a preliminary study, using microarray method and pathway analysis in pooled samples, we were able to identify heart specific gene-expression profile representing fibrotic and inflammatory processes in the peripheral blood of tachypacing-induced cardiomyopathy model dogs. our results were validated by histopathology and quantitative real time rt-pcr (qrt-pcr). based on our microarray results, in this current study we aimed to select and investigate a panel of possible cardiac fibrosis and remodeling specific genes in blood. whole blood and left ventricular samples of tachypaced dogs (n = ), healthy controls (n = ) and blood samples of canine clinical patients (n = ) with different cardiomyopathies were collected in rna-stabilizing solution. rna integrity was confirmed by capillary electrophoresis (rin> ). exon-spanning primers were designed. expression of selected genes were measured by sybr-green based qrt-pcr and normalized to different housekeeping genes (hprt , rps ) by ddct method. quality controls were made by melting curve analysis and size determination of the pcr products by agarosegel electrophoresis. for data evaluation descriptive statistics, student's t-test and mann whitney u-test were used. the selected gene-expression panel consisted of different mrnas which represent main biological processes related to fibrosis, remodeling and impaired contractility. col a , mmp , timp , vcan, spp are directly related to collagen turnover and remodeling. il , ccl are inflammatory markers. stc , hsp , s a are early stress response genes. tgfb has central role in fibrosis while myh and myh ensure cardiac specificity. we found significant up regulation (p < . ) of col a , timp , vcan, spp , il , ccl , stc , hsp , s a , tgfb and down regulation of myh , myh genes in the heart tissue samples. all targets also showed similar changes in the blood samples except mmp , however not all alterations were significant. based on this selected panel clinical cases could be clearly differentiated from healthy dogs using their gene-expression pattern in blood samples. our findings suggest that the peripheral blood may have a potential to reveal cardiac specific fibrosis in dogs. disclosures: no disclosures to report. within the distal nephron, the enzyme -beta hydroxysteroid dehydrogenase ( bhsd ) protects the mineralocorticoid receptor (mr) from activation by cortisol, allowing it to interact with aldosterone. in humans, mutations of bhsd cause apparent mineralocorticoid excess, characterised by sodium and water retention with resultant hypertension. sodium and water retention is also a hallmark of canine congestive heart failure (chf). this could partly be explained by dysregulation of renal bhsd activity, exposing mr to activation by cortisol. the aim of this study was to investigate the activity of renal bhsd in canine chf by measuring the concentration of cortisol and its metabolites in the urine from affected dogs. owners collected urine in a home environment from healthy adult dogs (n = ), and from dogs prior to presentation with non-cardiac chronic disease (n = ), and dogs with cardiac disease (isachc ib, n = ; isachcii or iii, n = ). levels of cortisol (f) and cortisone (e) excreted in urine were measured by mass spectrometry. urinary cortisol was normalised to creatinine to account for variations in glomerular filtration rate. cortisol was also measured in plasma obtained from all unhealthy dogs. plasma cortisol levels (p = . )and urinary cortisol:creatinine ratio (p = . ) did not differ between groups. however, the f/e ratio, was increased in dogs with class ii-iii heart failure (p = . ). an increased f/e ratio, in the presence of unchanged plasma cortisol, implies decreased renal bhsd activity and enhanced mr activation by cortisol in canine chf. this data suggests that changes in renal cortisol metabolism in canine chf cannot be explained by chronicity of disease, that the urinary f/e ratio has potential as a biomarker for canine chf and that renal bhsd could offer a therapeutic target in its management. further studies investigating bhsd expression and bioactivity in canine chf are ongoing. disclosures: supported by the fiona and ian russell seed corn grant through the university of edinburgh. in humans endomyocardial biopsy (emb) is highly recommended in case of unexplained left ventricular dysfunction associated to ventricular arrhythmias (va) or high-grade atrioventricular block (avb). despite the frequency of these conditions in dogs, histopathology data are lacking. the aims of this study were to describe the feasibility of emb in dogs and to investigate a possible role of viral myocarditis in case of unexplained dilated cardiomyopathy (dcm) phenotypes, high-grade avbs, supraventricular arrhythmias (sva) and va. twenty-five dogs of different breeds, m/f , , mean age . + . years, mean body weight . + . kg, presented for third degree avb / , dcm / , va / , sva / , and va+sva / , underwent percutaneous right emb under general anesthesia throughout the jugular vein. for each dog clinical records were analyzed. in all dogs at least one right ventricular sample (range - ) was collected for histopathology and immunohistochemistry; in / dogs at least one sample (range - ) for viral pcr was collected. all histopathologic samples were stained with haematoxylin and eosin, masson's trichrome and red elastic picrocirius. in selected cases stains with monoclonal anti-cd and anti-cd were performed. nucleic acids were obtained after sample storage in rna later solution, disruption with tissue lyser and extraction with trizol; and tested for canine viruses (enteric and respiratory coronavirus, herpes virus, distemper virus, adenovirus and , and parvovirus) and for west nile virus and bartonella spp. seven out of dogs had aspecific signs of cardiomyopathy and / suggestive of arrhythmogenic right ventricular cardiomyopathy (arvc). emb gave normal samples in / dogs and not diagnostic in / dog. nine out of samples were suggestive of myocarditis at different stages ( third degree avb, dcm and sva). two of these dogs resulted positive for virus ( enteric coronavirus, herpes virus). none of the dogs had positive immunoistochemical stains. two dogs with cardiomyopathy were positive for herpes virus and for herpes virus and parvovirus, respectively. both of these dogs came from a kennel. no complication was noted in / dogs, one dog had self-limiting pericardial effusion. this study showed, similarly to human cardiology, that emb is a safe and useful technique that allows recognition and classification of unexplained myocardial and rhythm disorders, % of which possibly associated with viral myocarditis. further studies are needed to prove the relationship between viruses and myocarditis in a larger cohort of dogs. disclosures: no disclosures to report. echocardiographic evaluation of the right ventricle (rv) is challenging. studies lack quantitative assessment of the rv in dogs. the goal of this study therefore was to evaluate rv morphology and systolic function using different transthoracic echocardiographic (te) views and to compare the results with magnetic resonance imaging (mri) measurements in adult healthy anesthetized beagles. te variables were rv wall thickness (wt) in short axis and from a subcostal view, fractional shortening (fs), rv fractional area change (fac) from different apical views (an optimized view for the rv and a standard chambers view), tricuspid annular plane systolic excursion (tapse) from different apical views, right ventricular outflow tract diameter at proximal (rvot ) and valvular (rvot ) levels both obtained in long and short axis, tissue doppler imaging (tdi) derived tricuspid annulus systolic wave (s¢), isovolumic contraction velocity (ivcvel), and isovolumic contraction acceleration time (ivcat).mri variables were rv wt, rvot in short and long axis and rvot , ejection fraction (ef), and stroke volume (sv) based on flow quantification. there was no difference between rv wt measured with te in both short axis ( . ae . mm) and subcostal views ( . ae . mm) and mri ( . ae . mm). no difference was found between rvot or rvot when measured in long ( . ae . mm for the former, . ae . mm for the latter) and short axis ( . ae . mm for the former, . ae . mm for the latter) with te; however rvot in both short and long axis was overestimated by te compared to mri ( . ae . mm in long axis, and . ae . mm in short axis). both rvot in short and long axis obtained by te were lower than mri values ( . ae . mm). te fs was . ae %. values of tapse varied significantly when using different apical views, optimized ( . ae . mm) and standard chambers ( ae . mm); the same was true for fac (optimized view, . ae . %; standard chambers, . ae . mm). tdi s¢ was . ae . m/s, ivcvel was . ae . m/s, and ivcat was . ae . msec. the only te correlations found were tapse with fac and s¢. the only echocardiographic variables correlating with the mri based sv ( . ae . ml) were fac and s¢. mri based ef ( . ae . %) did not correlate with any echocardiographic variable. this new approach to assess rv function revealed problems similar to earlier attempts. without a reliable standard for comparison of quantitative results, the value of any te parameter is questionable. furthermore, te parameters obtained from different views produced different results, indicating that standardization maybe difficult, respectively increasing the risk of variability. disclosures: no disclosures to report. spontaneous echo contrast (sec) or 'smoke' is caused by low blood velocity and appears on ultrasound as a swirling blood flow pattern. it is associated with increased risk of thromboembolism in small animals. detection of sec is entirely subjective and there is limited consensus in veterinary medicine regarding the echocardiographic technique that is best for detection of sec. the main hypothesis of this study was that dimensional ( d) colour tissue doppler imaging (tdi) would significantly outperform d colour and grey scale as the best echocardiographic technique to view sec. a further hypothesis was that colour blindness would have no influence on results. echocardiographic data was obtained retrospectively from small animal cases that presented to the university of glasgow small animal hospital. all cases had evidence of sec. using a ge vivid echocardiography machine each of the cases had one video loop recorded with d colour tdi. colour tdi was then replaced by different d colours (gold, sepia and aqua) and different grey scale colours (machine presets and ) and video loops recorded. for each case the order of the recorded video loops was randomised. the video loops from the cases were viewed in standardised conditions by observers (veterinary cardiologists, residents, interns and students); observers had full colour spectrum vision (fcsv) and were red-green colour blind (deuteranopia). each observer ranked their ability to see sec with each colour, with being the least able to visualise sec and being the best able to visualise sec. binary logistic regression using minitab (version . ) was used to identify factors associated with whether tdi was ranked best or not. potential explanatory variables that were examined were view and colour blindness. observer and case were also fitted as fixed and random effects to account for clustering within these variables. tdi was chosen by the observers / ( %) occasions as the best technique to diagnose sec, which was significantly more frequently than all other d colour views together (p < . ). there was no significant difference between colour blind observers and those with fcsv (p = . ) and there was no influence of observer or case on the final model. in conclusion, d colour tdi may assist in easier diagnosis of spontaneous echo contrast in veterinary medicine regardless of colour visual spectrum of the observer. easier detection of sec would allow earlier implementation of preventative measures. disclosures: no disclosures to report. the foramen ovale (fo) is a slit-like passageway between septum secundum and primum that typically closes after birth by fusion of these septa. in - % of humans, a patent foramen ovale (pfo) persists into adulthood. the prevalence of pfo in small animals is unknown. this interatrial channel may serve as a bypass to the pulmonary circulation and is an important cause of paradoxical embolism (pe) and stroke in people. the primary aim of the study was to evaluate the prevalence of pfo in a large population of dogs and cats. secondary aims were to gather data on the prevalence of atrial septal defects (asd) and on the potential association between pfo and a) clinical/pathological signs of stroke or thromboembolism and b) the presence of right-sided heart disease. hearts of all dogs and cats that underwent a full diagnostic post mortem examination were prospectively evaluated for a pfo in a blinded fashion (to clinical history and cause of death). in selected cases with patent and closed fo respectively, a histological examination of the interatrial septum was undertaken. clinical information and the results of the post mortem examination were only evaluated after all hearts had been examined. a total of hearts ( cats, dogs) were examined, of which cats ( %) and dogs ( %) with a median age of [ day- years] and years [ day- years] respectively, exhibited a probe-patent pfo. in adult animals with presumed normal right atrial pressure the prevalence of pfo was % (cats) and % (dogs). none of the animals had an asd. one dog with a pfo also exhibited an aortic thrombus; otherwise there was no evidence of pe in this population. in % (dogs) and % (cats) with closed fo the left side of the interatrial septum (septum primum) was still partially probe-patent through a channel extending from the left atrial crescentic ridge (ostium secundum) to the limbus, but closed at this level by a thin, easily rupturable membrane. in conclusion, pfos are common in dogs and cats, but less prevalent than in humans. in contrast, asds appear to be rare in either. despite the high prevalence of pfo, clinical complications seem to be very rare. the majority of dogs with a closed fo have a fossa ovalis that is weakly fused to the limbus, which may facilitate blunt trans-septal atrial catheterisation and provide an easier access to the left atrium. disclosures: jose novo matos and tony glaus have performed consultancy work for boehringer ingelheim and vetoquinol. in human medicine diabetes mellitus (dm) is known to lead to cardiovascular dysfunction and heart failure, characterized by early diastolic and late systolic dysfunction. diabetic cardiomyopathy has been defined as the existence of left ventricular dysfunction in diabetics without coronary artery disease, hypertension or other potential etiological conditions. the prevalence of a diabetic cardiomyopathy in cats has not been previously studied. we sought to prospectively identify if cardiac diastolic dysfunction was present or would develop in a population of cats with newly diagnosed dm. cats were recruited based on a diagnosis of primary dm. patients received physical examination, biochemical and hematologic profiles including thyroxin and insulin-like growth factor , urinalysis, blood pressure measurement, thoracic and abdominal radiographs and abdominal ultrasound. echocardiography was performed at both diagnosis and months post diagnosis. echocardiographic assessment included conventional d, m-mode, spectral and tissue doppler measurements. patients with relevant concomitant systemic illness or secondary dm were excluded from the study. healthy age matched control cats were retrospectively enrolled. thirty-two diabetics (d ) were enrolled in the study. eighteen were females and were males. mean age was . years. on march , cats had received a month echocardiographic control (d ). ten control cats were enrolled (c). eight were males and females. mean age was . years. results ( fluoroscopically guided pacemaker implantation imparts a risk of radiation exposure. ideally exposure risk should be minimized or avoided. we previously reported using transthoracic (tte) and transesophageal echocardiography to guide pda occlusion and balloon valvuloplasty. therefore, we hypothesized that tte could be used to minimize fluoroscopy time during pacemaker implantation in dogs. we implanted single bipolar lead pacemakers (vvir) in dogs, using either active or passive fixation, as determined by tte assessment of the right ventricular apical myocardium thickness: in dogs with an apical rv thickness < . mm we implanted passive fixation leads. dogs were anesthetized, positioned in right lateral recumbency on a standard echocardiography table and a left jugular vein exteriorization and venotomy were performed. in all dogs, a permanent pacing lead was advanced through the left jugular venotomy and was directed from cranial vena cava through the right atrium into the rv with tte guidance. echocardiographic right parasternal views optimized to visualize the pacing lead were used, starting with a short axis image of the right atrium and ending with a long axis view of the rv optimized to image the ventricular apex. after placing the pacing lead in the rv apex with tte guidance, and after acceptable measures of the capture threshold and impedance had been obtained, fluoroscopy was used to confirm lead placement. the pulse generator was connected to the pacing lead and secured within a right dorsal cervical pocket. incisions were closed routinely and post-implantation thoracic radiographs were performed. the pacing lead appeared hyperechoic on tte images and tte guidance provided images of a quality sufficient to clearly monitor implantation in real-time. real-time monitoring allowed for immediate corrections to pacing lead malpositioning or excessive looping. with active-fixation leads, tte allowed visualization of the fixation helix being implanted into the myocardium in some cases. the right parasternal echocardiographic window allowed imaging of the different positions of the lead in the cardiac chambers during the entire procedure. the implantations were successful in all dogs but in the first cases we required fluoroscopic guidance to follow the intraventricular progression of the lead. in the last dogs we only used the fluoroscope as an intraoperative x-ray machine (no cine mode). we have demonstrated that tte monitoring can guide pacemaker lead implantation and that fluoroscopy is only required to confirm the correct placement of the lead just before the end of the procedure. disclosures: no disclosures to report. deciding whether a cardiac murmur is innocent or the result of a congenital cardiac anomaly could be challenging. the gold standard method to differentiate innocent murmurs from congenital cardiac anomalies is echocardiogram, performed by a skilled operator. the present study investigated whether objective auscultation-criteria can differentiate innocent from pathologic murmurs in asymptomatic puppies between . - months of age. the null-hypothesis was that a systolic murmur with an intensity of - out of with a musical character is innocent. a total of puppies were included between july and january . these puppies originated either from breeders who brought their puppies to the clinic for screening for congenital porto-systemic shunts, or were referred to the cardiology service for evaluation of a murmur. of the puppies that were brought for shunt-screening puppies (age - days) had a murmur that was audible on every beat. all these dogs were small terrier breeds. dogs with intermittently audible murmurs were excluded. the remaining puppies (age - days) were referrals to the cardiology service. based on the above described auscultation criteria the murmur was classified as innocent in dogs and pathologic in dogs. on each of the dogs an echocardiogram was performed. no abnormalities were seen in of the dogs whose murmur was classified as innocent. echocardiography revealed one or more congenital cardiac anomaly in of the dogs whose murmur was classified as pathologic. the congenital anomalies were patent ductus arteriosus, pulmonic stenosis ( severe, moderate and mild; in combination with a small ventricular septal defect), aortic stenosis ( severe and moderate), ventricular septal defects, mild mitral valve dysplasia and double-chambered right ventricle (both moderate, isolated and combined with a mild aortic stenosis, small ventricular septal defect and mild mitral valve dysplasia). the puppy with the isolated mild mitral dysplasia had a soft systolic murmur with a musical character. on every puppy with a murmur of the shunt-screening group also a phonocardiogram was performed. phonocardiograms of the innocent murmurs revealed early systolic murmurs with low amplitude. auscultation turned out to be a sensitive and specific method to differentiate innocent murmurs from pathologic ones. phonocardiogram did not have an additional value. limitation: the above described findings may not be used in breeds predisposed to aortic stenosis, such as boxer. disclosures: no disclosures to report. since the first description of feline hyperthyroidism (feht ) several epidemiological studies have suggested diet as a causal factor of feht . the aim was to critically assess the evidence presented in epidemiological studies suggesting food-associated factors in etiology of feht . scientific literature was screened for peer reviewed publications investigating food-associated factors in feht since it was first described in . study designs were checked against classical epidemiology biases. food-associated factors showing an increased risk for feht were assessed for compatibility with the bradford-hill (b-h) criteria, which are used to evaluate whether an association involves a causal component. evidence for a causal component is higher when more b-h criteria are met. a total of publications investigating food-associated factors in feht were identified, all retrospective. three publications investigated qualitative factors only (e.g. preferred food flavors) but not quantitative. feeding canned food was not found to be a significant risk factor for feht in one study, while it was found to be a significant and quantitative risk factor in publications. however there were important limitations in their design: controls included sick cats ( studies), cases outnumbered controls ( studies), cases and controls differed in age ( studies), or diet information did not reflect diet fed at time feht developed ( studies). three out of b-h criteria were met when considering canned diet feeding as an increased risk for feht development. from the available literature there is insufficient evidence to conclude that canned diet is a food-associated factor in the etiology of feht . retrospective studies only describe an association and not a cause to effect relationship, are sensitive to bias from host and environment, and are less likely to identify etiologic factors when prevalence is below % as is the case in feht . hypotheses linking food-associated factors to feht (such as bpa, pbdes, flavonoids and iodine content) have never been proven to date, and lifelong prospective studies are required to investigate food-associated factors in etiology of feht . an iodine-restricted food has been recently introduced as a potential therapeutic option for feline hyperthyroidism. no controlled studies have been published on this treatment. the aim of this non-randomised controlled trial was to evaluate the effects of the iodine-restricted food on serum tt concentrations, clinical and clinicopathological parameters in client-owned cats with hyperthyroidism. indoor cats with newly diagnosed hyperthyroidism (consistent clinical signs and serum tt concentration > nmol/l), with or without mild to moderate azotemia (iris stage≤ ), were included. cats with severe concurrent disorders (i.e. lymphoma, neoplasia, malabsorption or severe azotemia [iris> ]) were excluded. cats were allocated on owner preference into groups: group a received an iodine-restricted food as a single therapy; the control group (group b) received transdermal methimazole in pluronic Ò lecithin organogel (plo, mg/ml) at the starting dose of . mg/cat q h. in both groups clinical parameters, biochemistry and serum tt were evaluated at baseline (t ), (t ), (t ), (t ), and (t ) days after treatment began. twenty-five cats were enrolled in the study, were included in group a, and in group b. no statistical differences were present between group at t for signalment, clinical and laboratory findings, including tt concentrations. in group a, only / cats ( . %) completed the study. the causes of interruption were: food refusal in / ( . %), loss to follow-up in / ( . %), death of unrelated cause in / ( . %), and poor owner compliance in / ( . %). in group b, / cats ( . %) completed the study. the causes of interruption were: suspected methimazole induced hepatotoxicity in / ( . %) and loss to follow-up in / ( . %). median serum tt concentration at t was nmol/l (range - nmol/l) in group a, and nmol/l (range - nmol/l) in group b. no significant differences were found in serum tt concentrations between group a and group b in any of the evaluated timing. bodyweight and serum creatinine significantly increased only in group b between t and t (p = . and p = . , respectively); nevertheless, urea did not significantly change in both groups. ast, alt, and alp significantly decreased only in group b between t and t (p = . , p = . and p = . , respectively). these results suggest that iodine-restricted food is effective in reducing serum tt concentration in hyperthyroid cats. compared to transdermal methimazole, food does not produce an increase in serum creatinine, but apparently is less effective in improving bodyweight and liver parameters. disclosures: no disclosures to report. the objective of this study was to identify whether pre-treatment plasma ionized calcium (ica) concentrations are predictive of hypocalcemia following parathyroid removal or heat ablation in dogs with primary hyperparathyroidism. fifty-five dogs seen between january , and february , met the inclusion criteria of having persistent hypercalcemia (defined as ica > . mmol/l) due to primary hyperparathyroidism, absence of pre-emptive calcitriol therapy, and ica monitoring post-operatively. each dog was treated with surgery (n = ) or ultrasound-guided percutaneous heat ablation (n = ). hypercalcemia resolved for all dogs within hours of the procedure. dogs were split into pretreatment ica groups of . - . mmol/l, . - . mmol/l and > . mmol/l. there was a significant association between higher pretreatment hypercalcemia and lower post treatment hypocalcemia. in addition, there was a significant dose-response relationship between pretreatment calcium concentration and the absolute decline in calcium following treatment. sixteen dogs became notably hypocalcemic (ica < . mmol/l). four out of dogs with pretreatment ica ≤ . mmol/l, out of dogs with ica between . - . mmol/l and out of dogs with pretreatment ica ≥ . mmol/l became hypocalcemic (ica < . mmol/l). adverse effects of hypocalcemia were observed in dogs, of which had pretreatment ica > . mmol/l. given the risk of significant hypocalcemia following parathyroid removal in dogs with pretreatment ica > . mmol/l, these patients should be treated to prevent rapid decline and development of clinical hypocalcemia. disclosures: no disclosures to report. diabetes mellitus is estimated to affect . % of pet dogs in the uk. most cases are thought to result from insulin deficiency resulting from loss of pancreatic beta cells, similar to human type diabetes. juvenile-onset diabetes is rare (catchpole, et al., ) . seven labrador retriever puppies were diagnosed with diabetes mellitus at between and weeks of age on the basis of the presence of hyperglycaemia, glucosuria and compatible clinical signs including polyuria, polydipsia and polyphagia. two of the dogs were known to be related (full siblings); of the remaining cases, other family members (full or half siblings or one sire) were also reported to be affected, though clinical details were not available. pancreatic histopathology at post mortem in cases showed islet hypoplasia. in the only puppy tested, hypoinsulinaemia was identified. two puppies were euthanased soon after diagnosis, were lost to follow up and survived to > years of age with well controlled diabetes. due to the early age of onset and occurrence within a single breed, it was hypothesised that diabetes in these cases might be due to mutation(s) in a single gene. three candidate genes were selected on the basis that they are the more common genetic causes of monogenic diabetes in human neonates or young children (kcnj , hnf a, hnf a). the coding regions of the canine orthologues of those genes (q bmm _canfa, hnf a, hnf a, respectively) were amplified and sequenced from genomic dna from all affected puppies and unaffected siblings and control labradors. no variants were identified in the coding sequences of hnf a or hnf a. in q bmm _canfa, novel, synonymous coding, single nucleotide base substitution variants were identified in of the affected dogs. however, these variants were considered unlikely to be the cause of the clinical syndrome, as their presence did not co-segregate with disease within families or across the cohort and did not change the protein coding sequence. in conclusion, we report a case series of labrador puppies suspected to be affected with monogenic diabetes mellitus, and results of candidate gene screening which did not identify a causa-tive mutation. further investigation of a possible genetic cause would be required to elucidate the cause of early onset diabetes in this breed. disclosures: er's salary and laboratory costs were paid using a grant from the wellcome trust. samples were submitted to the canine diabetes register at the rvc which has received funding from masterfoods, kennel club charitable trust, msd animal health and eu grant fp 'lupa'. meh is a trustee of the kennel club charitable trust. he was not part of discussions to award funding to the canine diabetes register. some samples were submitted as part of ljd's phd which was funded by the rvc (clinical research fellowship) with additional funding from intervet pharma r&d (currently msd animal health). diabetes mellitus (dm) is a common feline endocrinopathy and pathophysiologically similar to human type diabetes (t dm). t dm occurs due to a combination of insulin resistance and b-cell dysfunction. several studies have identified environmental and genetic susceptibility factors for t dm. in cats, environmental factors such as obesity and physical inactivity have been linked with dm; however, identification of genetic factors has been challenging. to date, mc r is the only gene shown to be associated with increased susceptibility to dm in overweight domestic short hair (dsh) cats. the aim of the present study was to perform a genome-wide association study (gwas) to identify loci associated with dm in lean dsh cats. illumina infinium k iselect dna arrays were used to interrogate genomic dna samples from lean diabetic dsh cats from the royal veterinary college feline dm archive and control dsh cats. the data was analysed using plink whole genome data analysis toolset. significance was established at p < À . snps with a minor allele frequency below . and a call rate below % and individuals with a genotyping rate < % were excluded from analysis. a total of , snps were available for analysis. after excluding cats with low genotypic rate, control dsh and lean diabetic dsh cats were evaluated. diabetic cats had a mean (sd) age of . ( . ) years; ( %) were male, ( %) female. non-diabetic cats had a mean (sd) age of . ( . ) years; ( %) were female, ( %) male. control cats were significantly older than diabetic cats (p < . ; t-test). five significant snps were identified: chra . (p = . À ); chrun . (p = À ); chrun . (p = À ); chrun . (p = À ) and chrun . (p = À ). the first snp is located within chromosome a ; the others are located within a . mb region towards the end of chromosome a . the snp in chromosome a is located kb upstream of dipeptidyl-peptidase- (dpp ), a peptidase similar to dpp- , involved in incretin inactivation. within the identified region of chromosome a , genes of interest include tmem and acp ; both have been associated with t dm in humans, most likely causing insulin resistance. this is the first gwas of dm in cats. a number of significant snps have been identified; some of which are located in proximity to genes that have been associated with t dm in humans others could be involved in pathophysiology related to dm. further investigation of these candidate genes is warranted. disclosures: snp chips for the gwas were provided by the morris animal foundation. diabetes mellitus (dm) and its treatment have been documented to exert a negative psychosocial impact on cats and their owners. common owner concerns include hypoglycaemia worry, socialand work-life impact and a desire for more control over their cat's treatment. home blood glucose monitoring (hbgm) has been suggested to enable superior glycaemic control and could address some of the above-mentioned quality-of-life (qol) issues. conversely, hbgm could also exert negative psychosocial effects such as disturbance of the cat-owner bond. this prospective -month trial aimed to document the acceptance rate of hbgm among owners of diabetic cats, reasons for declining hbgm, possible impact on glycaemic control and whether acceptance altered pet and owner qol measured through the use of a validated psychometric diabetic pet qol-tool (diaqol-pet). at baseline (m ) all owners of recently diagnosed cats received a veterinary glucometer (alphatrak Ò , zoetis) and a standardised demonstration of its use on their cat. diabetic management was standardised and included a low carbohydrate diet, twice-daily insulin following a standardised dose-adjustment-protocol according to, initially, weekly blood glucose curves (bgcs) carried out through hbgm (if adopted; hbgm-group) or in-hospital (if not adopted; non-hbgm-group). mann-whitney u-tests assessed for significant differences in fructosamine, average bgc-value, insulin dose and diaqol-pet-scores at m and month (m ) between the hbgmand the non-hbgm-group. fisher's exact test was used to compare remission rates; average values are given as median (range). hbgm was introduced to owners and was successfully adopted by ( . %). reasons for failure were patient aggression (n = ), owner concerns about patient distress (n = ) and lack of available assistance (n = ). at m , there was no significant difference in fructosamine (hbgm: ( - )lmol/l, non-hbgm: ( - )lmol/l; p = . ), insulin dose (hbgm: . ( - . )u/kg/dose, non-hbgm: . ( . - . )u/ kg/dose; p = . ), average bgc-value (hbgm: . ( . - . ) mmol/l, non-hbgm: . ( . - . )mmol/l; p = . ) or overall diaqol-pet-score (hbgm: À . ( . to À . ), non-hbgm: À . (À . to À . ). on examination of individual diaqolpet-categories hbgm-cat owners reported no significant difference in the bond they felt with their cat (p = . ), degree of worry about hypoglycaemia (p = . ) or restriction to their work-(p = . ) or social-life (p = . ) compared to the non-hbgmgroup. four hbgm-cats ( . %) achieved diabetic remission; none of the non-hbgm-group did (p = . ). hbgm can be successfully adopted in a majority of cat-owner combinations without a demonstrable extra burden on cat and owner's qol. hbgm also did not appear to compromise owners' relationships with their cat. a larger sample size is likely needed to assess whether hbgm promotes superior glycaemic control and remission. disclosures: the research presented in this abstract was supported by zoetis. the clinic at which this research was conducted is also supported by boehringer ingelheim and nestle purina pet-care. ruth gostelow and christopher scudder both receive phd funding from the evetts-luff animal welfare trust. stijn neissen acts as a consultant for the veterinary information network (vin). a major difficulty in the management of diabetes mellitus (dm) is our inability to predict blood glucose values in response to an insulin dose. this is linked to the existence of numerous factors impacting on blood glucose in the diabetic patient (e.g. caloric intake, type of food, exercise, insulin type and dose, stress). artificial neural networks (anns), which are statistical learning algorithms, have shown potential to aid in this prediction process in human dm. their development has been hugely aided by the introduction of continuous glucose monitoring systems (cgms), enabling generation of sufficient data-points. the goal of the current study was to develop an ann for feline dm. algorithms were developed with matlab Ò (mathworks, uk) using data on exogenous insulin dose, serial blood glucoses (obtained through traditional blood glucose curves and cgms) and caloric intake over hours of diabetic cats. all cats were maintained in an environment that enabled normal activities, limiting stress. the neural network toolbox tm (mathworks, uk) was used to construct and test types of anns: multi-layer perceptron (mlp) and radial basis function (rbf). both anns were trained using the diabetic cat data, followed by so-called detrending, elimination of outliers and configuration of external delays. in order to increase accuracy, neural architecture was set as a single hidden layer with a maximum of neurons; inclusion of saturated neurons was forbidden. the accuracy of the resulting mlp and rbf models was assessed by calculating the mean squared normalised error (threshold: . ), generated through comparison of predicted data with actually registered data in recruited diabetic cats. in total, diabetic cats were recruited for this study ( males, females, . ae . [sd]kg, age . ae . ; burmese, dsh, other breeds). all had a -hour blood glucose curve performed (n = with cgms) and response to insulin was predicted by mlp and rbf. calculation of the mean squared normalized error revealed that in / diabetic cats ( %; mlp) and / ( %; rbf), the dynamics of the blood glucose curve were correctly predicted by the ann. in conclusion, our study is the first to describe the successful development of an ann to predict blood glucose dynamics in insulin-treated diabetic cats. further evaluation is indicated, though ann-model-based prediction of glucose concentration may allow clinicians in future to optimise insulin management protocols or may allow the development of an artificial pancreas for the diabetic cat. disclosures: no disclosures to report. glucagon-like peptide (glp) - analogues induce significant weight loss in humans; presumably by slowing gastric emptying and increasing satiety. in lean purpose-bred cats, short-term glp- analogue treatment also induced weight loss. we evaluated the effect of weeks exenatide, a glp- analogue approved for treatment of human type diabetes, or placebo treatment on body composition and adipokines in obese, client-owned cats. cats were randomized to subcutaneous saline (n = ) or exenatide (n = ) injections; . lg/kg q h during the initial weeks and . lg/ kg q h during the following weeks. body weight, body composition using dual-energy x-ray absorptiometry and adipokine levels were measured before and after treatment. all cats were obese (body condition score ≥ out of ). mean body weight was . kg (range . - . kg) and mean % body fat was . % ( . - . %). median percent loss of baseline body weight was . % (range . - . %) for exenatide and . % (range À . to . %) for placebo. only the exenatide group had a significant absolute weight loss; however the difference in median percent loss between groups was not significant. change in total amount or % body fat were not different between groups. correspondingly, plasma leptin and total adiponectin were unaltered by treatment. complications were limited to a single, mild hypoglycemic episode and cases of self-limiting gastrointestinal signs. we conclude that the appointed dose of exenatide was well tolerated and safe in obese healthy cats. a larger study population may be required to fully elucidate the effect of exenatide in obese cats. disclosures: no disclosures to report. feline diabetes mellitus (dm) is recommended to be treated through addressing underlying diseases, bid insulin injections and low carbohydrate diets. good glycaemic control is suggested to promote diabetic remission. a recent systematic review of feline dm literature identified studies on glargine and lente insulin to be proportionately overrepresented compared to other insulin types. additionally, most insulin studies suffered from lack of screening for concurrent disease, homogeneity in management and assessment of quality of life (qol). until recently, only porcine lente insulin was available as a veterinary-licensed product. this prospective trial evaluated the impact of newly veterinary-licensed human-recombinant protamine zinc insulin (prozinc tm , boehringer ingelheim) on clinical signs, glycaemic control and qol in diabetic cats. recently (< months) diagnosed diabetic cats, treated with caninsulin Ò (msd) bid for at least weeks and receiving a specific low carbohydrate diet were recruited. a full history, physical examination, diabetic clinical score (dcs; range [no diabetic signs] - [many diabetic signs]), fructosamine concentration, hour blood-glucose-curve (bgc) and qol-assessment (diaqolpet-score) were performed before and months after transition to prozinc tm (start dose: . - . unit/kg bid), following a set protocol of weekly bgcs and dose adjustments ( . - unit change/injection/week guided by nadir). cats were excluded if screening (biochemistry, urinalysis, fpli, tt , igf- , abdominal ultrasound) identified: ketoacidosis, clinical pancreatitis, glucocorticoid/ progestogen administration, hyperthyroidism, acromegaly, other conditions impairing treatment response. data were assessed for normality and reported as meanae-standard deviation; changes were assessed using paired t-tests (p < . ; without multiple comparisons correction following latest statistical guidelines). sixteen cats were recruited ( male neutered, female neutered; age ae months); all completed the trial. at time of entry cats received . ae . unit/kg caninsulin bid, had a dcs of . ae . ; diaqol-pet-score of - . ae . ; bgc-value of . ae . mmol/l; and fructosamine of ae lmol/l. three months after transitioning to prozinc tm , cats were receiving . ae . unit/kg bid; had a significantly lower mean dcs ( . ae . , p = . ) and diaqol-pet-score (À . ae . , p = . ); bgc-value ( . ae . mmol/l) and fructosamine ( ae lmol/l) were also lower, though not significantly (p = . and p = . , respectively). three cats entered diabetic remission ( %). these results show that transitioning cats from caninsulin to prozinc tm produced a significant improvement in clinical signs and qol. more cases are likely needed to document any additional significant glycaemic impact after transition. finally, in veterinary dm research, this represents the first clinical trial to include validated quantitative qol-assessment as an outcome parameter. disclosures: the study described in this abstract received financial support from boehringer ingelheim. the clinic in which this research was produced also receives support from nestle purina petcare and zoetis. ruth gostelow and christopher scudder both receive phd funding from the evetts-luff animal welfare trust. stijn niessen is a consultant for the veterinary information network (vin). a. hope, s. spence, i.k. ramsey. university of glasgow small animal hospital, bearsden, uk serial blood glucose measurements are currently used as an accepted method to assess diabetic control, however extended curves are expensive and can be technically challenging. a new day continuous glucose monitoring system (cgms) called the dexcom g platinum Ò that incorporates a glucose oxidase-based sensor to measure interstitial blood glucose was evaluated by comparing the results to the blood glucose measured contemporaneously on a glucometer (alphatrak Ò ). these measurements were made at least twice daily at the time of calibration of the cgms, which was a variable period (but not more than hours) after the previous calibration. a total of measurements from eight dogs' glucose curves (blood glucose range - . mmol/l, with a median of . mmol/l) were compared to a paired measurement of interstitial glucose by calculation of the pearson correlation coefficient (r). a minimum of measurements were obtained from each dog. the device only provides a specific measurement of interstitial glucose in the range . to . mmol/l. values above and below this range were not included in the study. subjectively, the device was easy to use with an intuitive user interface that provided wireless real time measurements and was much smaller than older cgms systems. overall, there was excellent correlation between the glucometer and the cgms readings (r = . ), which was statistically significant (p = < . ). the range of differences between the blood and interstitial glucose concentrations was - . mmol/l with a median of . mmol/l. problems encountered with the system included detachment of the system from the dog's skin, as well as variably correlated glucometer and cgms readings in individual dogs (r range . - . , median . ). in conclusion, the dexcom Ò g cgms can be used to assess interstitial glucose concentrations in dogs, and these are generally well correlated with blood glucose concentrations. more work is needed (with larger numbers of patients) to determine the relationship of the correlation to the timing of calibration, and to determine why some dogs seem to have poorer correlation than others. disclosures: no disclosures to report. several continuous glucose monitoring systems that measure interstitial glucose (ig) are currently available. however, they require multiple calibrations and therefore multiple blood sampling; moreover, the sensors are quite expensive and can be used only for a few days. a new human flash glucose monitoring system (fgm) (freestyle libre, abbott, uk) measures ig, does not require calibration, is rather inexpensive and the sensor can be used as many as days. it is composed by a small sensor applied subcutaneously that has to be "scanned"with a reader to obtain real time glucose values. the aim of this study was to assess the accuracy of this fgm in diabetic dogs. in all dogs the sensor was placed on the neck area and fixed with an adhesive patch. during the st - nd, th - th, th - th days from the application, the ig measurements were compared with the peripheral blood (edta plasma) glucose (pg) concentrations analysed by a reference hexochinase based method (olympus/beckman coulter au ). linear regression, bland altman plots and the clarke error grid analysis were used to assess the accuracy. ten client-owned diabetic dogs on insulin treatment were included. median age was . years (range - ), were female (spayed), were male ( neu-tered). median body weight was . kg (range . - . ). four hundred and sixty four simultaneous measurements were taken with fgm (ig) and with the reference method (pg): samples were in the hypoglycemic range (< mg/dl), in the euglycemic range ( - mg/dl) and in the hyperglycemic range (> mg/dl). considering all the measurements together a positive significant correlation between ig and pg concentrations (r = . ) was found. meanaestandard deviation difference from the reference method was À . ae mg/dl in the hypoglycemic range, À . ae mg/dl in the euglycemic range, À . ae mg/dl in the hyperglycemic range. ig values differed > mg/dl from the reference method in %, % and % and > mg/dl in %, % and % in the hypoglycemic, euglycemic and hyperglycemic range, respectively. underestimation-overestimation of ig compared to pg was observed in - %, - % and - % of hypoglycemic, euglycemic and hyperglycemic measurements, respectively; . % and . % of glucose values measured by fgm fall in zone a and zones a+b of the error grid analysis, respectively. the application of the sensor was easy and apparently painless; a mild local erythema after sensor removal was found in / dogs. fgm is a simple and promising glucose monitoring system that seems accurate for the clinical use in diabetic dogs. disclosures: no disclosures to report. the cornerstone of treatment in diabetic cats is insulin. among other issues, insufficient duration of insulin action may lead to poor metabolic control and persistence of clinical signs. with the aim to improve current therapeutic options, the present study evaluated pharmacodynamics parameters, such as onset of action, time to glucose nadir and duration of action, of protamine zinc insulin (prozinc Ò , boehringer ingelheim) and insulin degludec (tresiba Ò , novo nordisk) in healthy cats. additionally, the accuracy of different sensors, sof-and enlite-sensor, of the continuous glucose monitoring system (cgms) ipro Ò (medtronic) was determined with particular attention to the low glycemic range, since reliability of cgms in case of hypoglycemia is crucial. three different doses ( . , . and . iu/kg) of each insulin and both ipro Ò sensors were tested in healthy purpose bred cats in a randomized crossover trial. the sensors were placed in the neck area for days. paired glucose measurements were obtained every - hours with a validated portable blood glucose meter (alphatrak Ò , abbot) set as standard and accuracy was assessed by using iso criteria. additionally, to determine onset of insulin action, time to glucose nadir and duration of action, glucose concentrations were measured and minutes before and , , , , , , and minutes after each insulin administration, then every hours for hours. median (range) onset of action was . ( . - ) hours for pro-zinc Ò and . ( . - ) hours for tresiba Ò . median (range) time to glucose nadir and duration of action were ( . - ) hours and ( - ) hours for prozinc Ò and . ( ) ( ) ( ) ( ) ( ) ( ) hours and ( -> ) hours for tresiba Ò , respectively. with regard to ipro Ò , % of paired glucose measurements with both sensor types were in zone a and b of the consensus error grid. at glucose concentrations < . mmol/l % ( / ) of sof-sensor measurements and % ( / ) of enlite-sensor measurements were within ae . mmol/l of the standard; at glucose concentrations > . mmol/l % ( / ) of sof-sensor measurements and % ( / ) of enlite-sensor measurements were within ae % of the standard. in conclusion, healthy cats injected with prozinc Ò and tresiba Ò showed similar onset of action. later glucose nadir and longer duration of action was seen in cats treated with tresiba Ò compared to those treated with prozinc Ò . both ipro Ò sensors revealed good clinical accuracy and performed similarly in the low glycemic range. disclosures: no disclosures to report. the canine adrenal cortex consists of concentric zones: the zona glomerulosa (zg), the zona fasciculata (zf) and the zona reticularis (zr), which produce mineralocorticoids, glucocorticoids and androgens, respectively. in humans, critical step for the production of either aldosterone or cortisol are the zg-specific aldosterone synthase (cyp b ) and the zf-specific b-hydroxylase cytochrome p (cyp b ). the fact that humans and dogs have the same adrenocortical end products, i.e. aldosterone and cortisol, has led to the assumption that canine steroidogenesis is identical to that of humans. however, in dogs, the zonal expression of steroidogenic enzymes has not been studied previously. moreover, in dogs the expression of cyp b / is unclear, as only one coding gene sequence (cyp b ) has been published in the ncbi database and, adjacent to this, a large non-sequenced gap is present. we hypothesized that canine adrenals possess only one cyp b gene, similar to sheep and pigs. zg and zf tissue was isolated separately by use of laser-guided microdissection of adrenals of healthy dogs. the zone-specificity of the tissues was confirmed by specific markers, with mrna relative expression of wnt , angiotensin ii receptor and disabled- being significantly higher (p = . , p = . , p = . , respectively) in the zg compared to the zf. rt-qpcr analysis of mrna relative expression of steroidogenic enzymes demonstrated a significantly higher fold change of steroidogenic acute regulatory protein (star), cytochrome p side chain cleavage (cyp a ) and a-hydroxylase/ , -lyase (cyp ) (p = . , p = . , p = . , respectively) in the zf compared to the zg. the zfspecific presence of cyp was also demonstrated by immunohistochemistry. no significant difference (p = . ) in the mrna relative expression of cyp b mentioned in the database was found, and southern blot analysis showed that the non-sequenced gap does not contain another cyp b gene. we conclude that there is only one functional cyp b enzyme in canine adrenals. the zone-specific production of aldosterone and cortisol is probably due to zone-specific cyp expression. its presence in the zf is crucial for cortisol synthesis, while lack of cyp in the zg conducts steroidogenesis to mineralocorticoid production. this is the first report providing insights in one of the most important physiological mechanisms of the canine adrenal cortex, its zone-dependent steroidogenesis. disclosures: no disclosures to report. the pathogenesis of cortisol-secreting adrenocortical tumors (ats) has become more clear recently. mutations of the gnas gene provide an explanation for acth-independent hormonal activity of ats, but the autonomous growth remains greatly undisclosed. an approach to elucidate the proliferative capacity of ats is to learn from the current understanding of adrenal growth biology. the sonic hedgehog (shh) signaling pathway plays an essential role in the development of the adrenal gland and in regulating adrenocortical cell proliferation. the members of the shh signaling pathway are present in progenitors of the steroidogenic cells of the normal adrenal gland and dysregulation of shh signaling has been implicated in adrenal cancer in humans. we hypothesized that shh signaling is also enhanced in canine ats, predominantly in carcinomas. we examined the relative expression of shh pathway components (shh, ptch , smo, gli , gli and gli ) by rt-qpcr analysis in cortisol-secreting adenomas (n = ) and carcinomas (n = ) and normal canine adrenals (n = ). the relative expression of members of the shh pathway was detected in both ats and normal adrenal tissue. a significant (p < . ) lower mrna expression of gli was detected in carci-nomas when compared to normal tissue. amongst the other genes, no significant differences were found. since gli is mainly a repressor of genes activated by the shh pathway, a down regulation of gli in carcinomas could point to enhanced shh signaling in adrenocortical carcinomas and could theoretically be responsible for their expansive growth. in conclusion, dysregulation of shh pathway might be involved in the pathogenesis of canine cortisol-secreting carcinomas. modulating shh expression might provide a new target therapy for adrenocortical carcinomas and will need to be explored in the future. disclosures: no disclosures to report. feline hypersomatotropism (acromegaly) has been suggested to be an underdiagnosed endocrinopathy among diabetic cats. treatment options include management of the secondary diabetes mellitus alone, medical pituitary inhibition, radiotherapy (rt) and hypophysectomy (hpx). tools to diagnose the disease and monitor treatment effect are limited, with insulin-like growth factor- (igf- ) currently being the only easily accessible blood test. serum igf- has previously been shown to be insensitive when assessing rt effect. development of additional serological diagnostic tools that can be measured alongside serum igf- is therefore desirable. a pilot study previously validated an n-terminal type iii pro-collagen propeptide (piiinp) elisa for use in cats and found this peripheral biomarker of collagen turnover to be significantly elevated in a small number of hypersomatotropic diabetic (hsdm) cats. this study therefore aimed to: . further evaluate the use of serum piiinp to differentiate hsdm from dm; and . to evaluate piiinp as a marker for treatment success. piiinp concentrations were measured in cats with uncomplicated diabetes mellitus (dm) (igf- < ng/ml [radioimmunoassay], < . iu/kg/injection exogenous insulin requirement) and with confirmed hsdm (igf- > ng/ml, pituitary mass on computed tomography) using the previously validated elisa. additionally, piiinp and igf- were measured in preand post-treatment ( - months) samples of hsdm cats that responded favourably (decreased insulin requirement) to radiotherapy (rt; n = ) or hypophysectomy (hp; n = , of which had achieved diabetic remission at time of sampling). serum piiinp concentrations were significantly higher in hsdm cats (median . ng/ml; range: . - . ) compared to dm cats (median . ng/ml; . - . ; p < . , mann whitney u-test). receiver-operator-curve-analysis revealed a . ng/ml cut-off to differentiate between dm and hsdm cats with % sensitivity and % specificity (auc: . ; % confidence interval: . - . ). after rt, piiinp increased significantly (median pre-rt . ng/ml, . - . ; post-rt . ng/ml, . - . ; p = . , wilcoxon signed rank-test) despite absence of significant change in igf- concentrations (median pre-rt ng/ml, - ; post-rt ng/ml, - ; p = . , wilcoxon signed rank-test). following hpx, serum piiinp concentrations did not change significantly (median pre-hpx . ng/ml, . - . ; post-hpx . ng/ml, . - . ; p = . , wilcoxon signed rank-test) despite significant serum igf- decreases (median pre-hpx ng/ml, - ; post-hpx ng/ml, - ; p = . , wilcoxon signed rank-test). in conclusion, serum piiinp concentration was confirmed to be a useful additional parameter when differentiating hsdm from dm in cats. however, the current data do not suggest piiinp to be a reliable marker of treatment success following rt or hpx. disclosures: no disclosures to report. decreased frequency and function of peripheral regulatory t cells (tregs) have been documented in people with immune-mediated haemolytic anaemia (imha), suggesting that defects in peripheral tolerance may play a role in the pathogenesis of this disease. the aim of the current study was to test the hypothesis that the frequency of peripheral tregs is decreased in dogs with primary imha, accompanied by increases in t helper (th) cells, cytotoxic t (tc) cells and b cells. residual edta-anticoagulated blood samples were obtained from dogs with primary imha (n = ), dogs with inflammatory diseases (n = ) and healthy dogs (n = ). primary imha was diagnosed in dogs with regenerative anaemia (packed cell volume [pcv] < %) and either persistent agglutination of erythrocytes after saline dilution or detection of spherocytosis on a fresh blood smear. the study was approved by an institutional ethics and welfare committee. after erythrocyte lysis, peripheral blood mononuclear cells were stained with fluorophore-conjugated antibodies specific for extracellular (cd , cd , cd ) and intracellular (cd b, foxp ) antigens. multicolour flow cytometry was undertaken to determine the proportion of lymphocytes that were b cells (cd b hi ), th cells (cd hi cd + ), tc cells (cd hi cd + ) and tregs (cd hi cd +-foxp + ); the kruskal-wallis test was used to compare proportions between groups. correlations between the proportions of tregs and pcv and serum total bilirubin concentration (tbil) in dogs with imha at presentation were assessed using spearman's rho coefficients. the median proportion of cd + t cells that expressed foxp was . % (inter-quartile range [iqr]: . - . ) in dogs with imha, . % (iqr: . - . ) in dogs with inflammatory diseases and . % (iqr: . - . ) in healthy dogs, with no difference between groups (p = . ). there was no difference in proportions of t cells that were cd + (p = . ) or cd + (p = . ) between groups, nor in the proportion of b cells (p = . ). the proportion of cd hi cd + foxp + tregs was positively correlated with tbil in dogs with imha (spearman's rho . , p = . ), but not pcv (rho À . , p = . ). though limited by its size, the results of this pilot study suggest that the frequency of tregs is not decreased in dogs with imha; proportions of th, tc and b cells were also comparable to those in control dogs. further work is required to determine whether the function of tregs is altered, or whether other defects in peripheral tolerance contribute to development of this disease. disclosures: no disclosures to report. xenotransfusion of canine blood to cats may be a life-saving procedure when treating an acute anaemic syndrome and compatible feline blood cannot be obtained. published evidence in a limited number of cases dating from the s indicates that cats do not appear to have naturally-occurring antibodies against canine red blood cell antigens. in fact compatibility tests before the first transfusion did not demonstrate evidence of agglutination or haemolysis of canine erythrocytes in feline serum and no severe acute adverse reactions have been reported in cats receiving a single transfusion with canine blood. severe acute reactions not reported so far cannot however be excluded and we decided to perform a pilot study to evaluate the presence of naturally occurring antibodies against canine red blood cell antigens in cats and viceversa. surplus material from diagnostic samples (blood edta and blood serum samples) of cats and dogs was used to perform test-tube major and minor cross-match tests (at °c, °c and room temperature (rt)) and blood typing, after obtaining the informed consent from owners. hemolysis, macro-and micro-ag-glutination were investigated in each test tube and were considered markers of a positive matching. blood from each cat was tested with blood from to different dogs for a total of major and minor cross-matchings each one performed at the different temperatures of incubation. thirty-eight overall major cross-matchings proved positive at °c, at rt and at °c respectively. the minor cross-matching was positive in all but tests performed at °c. no cat tested totally negative ( °c, °c, rt) at both major and minor cross-matching procedures performed towards any single dog. ten cats experienced positive major and minor cross-matching at °c, rt and °c towards - different dogs. five cats were positive in the major cross-match, at least at °c, towards - different different dogs. seven cats obtained a positive major cross-match at rt and/or at °c towards - dogs. only cats tested completely negative at °c, rt and °c, in one out of the different major cross-matchings performed. in conclusion, naturally occurring antibodies against canine red blood cell antigens appear to be frequently detected in cats as well as those against feline red blood cell antigens in dogs. xenotrasfusion of canine blood to cats should only be performed after the selection of a compatible donor by means of at least a negative major cross-match test result. disclosures: no disclosures to report. anti-thymocyte serum (ats), a potent immunosuppressive agent, is commonly used perioperatively in human patients to increase graft survivaland decrease rejection of transplanted tissue. ats has been reported as part of an immunosuppressive protocol to treat immune-mediated diseases including aplastic anemiaand myelodysplastic syndromes (mds). rabbit anti-dog thymocyte serum (radts) has been used in veterinary medicine for perioperative immunosuppression in canine renal transplants. however, there are no reports regarding the use of radts in the treatment of dogs with immune-mediated disorders. the medical records of dogs diagnosed with imha, dogs with itp and dog with mds were reviewed. median age was . years ( . months to years). all dogs failed to respond to traditional immunosuppressive therapy and received radts. none of the dogs experienced any adverse reaction. lymphocyte counts were used to monitor the response to therapy. all dogs, except , had a significant decrease in their lymphocyte count; / had a decrease to < % of the initial lymphocyte count, which was the aim in previous studies on radts. all dogs were discharged, however, the same dog with no changes in his lymphocyte count experienced a relapse of his imha after week and was euthanized. all other cases achieved clinical remission with immunosuppressants being tapered or discontinued. radts appeared to be a safe immunosuppressant agent of interest in refractory immune mediated diseases. disclosures: no disclosures to report. in cats with upper respiratory tract aspergillosis (urta), invasive disease is common. in other species, invasive mycoses are associated with immunodeficiency. characterisation of the humoral immune response in feline urta serves to identify whether selective immunodeficiency underlies susceptibility and to determine the utility of class-specific antibody detection for early diagnosis. we have shown that serum anti-aspergillus igg has high sensitivity and specificity for diagnosis. the aims of the study were to ( ) determine whether serum anti-aspergillus iga can be detected in cats with urta, and ( ) evaluate the sensitivity and specificity of iga detection for diagnosis. sera were collected from groups of cats; group -confirmed urta (n = ), group -upper respiratory disease without aspergillosis (n = ), group -healthy cats (n = ) and cats with non-fungal, non-respiratory illness (n = ). an indirect elisa to detect anti-aspergillus iga was developed. inter-assay and intraassay coefficients of variation were . % and . %, respectively. serum iga was detected in . %, % and % of group , , and cats, respectively. using an optimal elisa cut-off value for diagnosis ( . elisa units/ml), determined by receiver-operating curve analysis, assay sensitivity for group cats was . %. specificity was highest ( . %) when group was used as the control, compared to group ( . %) or group and combined ( . %). we found no evidence of a role for primary iga deficiency in the pathogenesis of feline urta. serum anti-aspergillus iga detection has moderate sensitivity and moderate specificity for diagnosis of urta. disclosures: there is no conflict of interest. the study was partially funded by a australian companion animal health foundation grant . non-invasive topical infusion therapies are widely used in canine sinonasal aspergillosis (sna) but are time-consuming and associated with prolonged recovery and increased costs. therefore, the main goal of the present study was to compare the efficacy of a simplified infusion protocol (d e) with a -hour infusion protocol (d eb). d eb consisted in endoscopic debridement followed by minutes % enilconazole infusion and % bifonazole cream depot into the affected frontal sinus through endoscopically placed catheter. for d e protocol, after debridement, enilconazole infusion was shortened to minutes, with the dog remaining in dorsal recumbency, head flexed at °, during the whole procedure. adjunctive oral itraconazole therapy was prescribed in both protocols. effective debridement of fungal plaques is considered as an essential therapeutic step. unfortunately, it is not always possible to achieve perfect debridement of the sinonasal cavities, due to incomplete accessibility of the whole sinusal area with the endoscope; however, its effect has never been assessed as such. therefore, the second aim of this study was to evaluate the effectiveness of debridement on success rate after the first treatment. fisher's exact test was used to assess the difference in success rate between both protocols and in function of full debridement. twenty-eight dogs with sna were treated with d e and dogs with d eb. the median (range) duration of d e was only minutes ( - ) compared to minutes ( - ) for d eb. first treatment success rate did not differ between both protocols and were % for d e and % for d eb. both protocols had an overall success rate of % after procedures. in contrast to the majority of dogs with d eb, all dogs receiving d e recovered quickly and were discharged the same day. completeness of debridement was assessed endoscopically in dogs ( treated with d e and with d eb). debridement was judged complete in / dogs and had a significant effect on first treatment success rate (p = . ). when debridement was complete, % of the dogs (d e: / dogs; d eb: / dogs) were cured after the first procedure, compared to % (d e: / dogs; d eb: / dogs) of the dogs with incomplete debridement. we concluded that ( ) the simplified infusion protocol is quick, safe, easy and effective, and offers a favourable alternative to hour infusion protocols for treatment of canine sna; ( ) completeness of the debridement undoubtfully is an important step for treatment success of infusion protocols. disclosures: no disclosures to report. gastroesophageal (ge) symptoms are commonly reported in dogs with brachycephalic upper airway obstructive syndrome (bs). since ge symptoms frequently occur during situations of increased inspiratory effort (excitement, respiratory distress), dynamic disorders of the ge junction (gej) are probably involved, due to transient increased negative intrathoracic pressure. however, according to a previous study, only few dynamic abnormalities of the gej are observed during gastroscopy. we hypothesized that both anaesthesia and endotracheal intubation during gastroscopy lead to underestimation of gej abnormalities. the aim of this study was to improve detection of dynamic gej abnormalities during gastroscopy using obstructive manoeuvers mimicking and reproducing upper airway obstruction of variable severity. twenty-six dogs presented with bs were prospectively included. respiratory and digestive symptoms as well as endoscopic abnormalities were scored at initial diagnosis and at control month after surgery. during each endoscopy, gej was assessed and scored (based on esophagitis, gej atony, ge reflux, cranial displacement of the gej) in the consecutive situations: ( ) absence of obstruction with the dog intubated (ob- ), ( ) presence of natural obstruction with the dog extubated (ob-nat) and ( ) during complete manual obstruction of the endotracheal tube during up to spontaneous breathings (ob-compl). spearman's rank test was used to assess correlations between the different clinical and endoscopic scores. taking all endoscopic procedures together, the severity of respiratory symptoms correlated significantly with the severity of respiratory endoscopic abnormalities (p < . , r = . ) and the severity of digestive symptoms (p = . , r = . ). at diagnosis, dogs ( %) presented digestive symptoms while endoscopic gej abnormalities were observed in ( %), ( %) and ( %) dogs during ob- , ob-nat and ob-compl respectively. gej atony, ge reflux, cranial displacement of the gej and sliding hiatal hernia were present in ( . %), ( . %), ( . %) and dogs during ob- , in ( . %), ( . %), ( . %) and ( . %) dogs during ob-nat and in ( . %), ( . %), ( %) and ( . %) dogs during ob-compl, respectively. a significant correlation was found between digestive and endoscopic gej scores during ob-compl (r = . , p = . ) as well as during ob-nat (r = . , p = . ) but not during ob- . it can be concluded that in dogs with bs ( ) gej abnormalities are dynamic and related to the degree of upper airway obstruction; ( ) the use of obstructive manoeuvres during gastroscopy improves the detection of gej abnormalities. disclosures: no disclosures to report. tracheobronchial foreign bodies are common causes of respiratory disease in children but they are rare in veterinary medicine. particularly in cats, reports of tracheobronchial foreign bodies are scarce. this study aimed to describe clinical presentation, diagnostic findings and treatment modalities in confirmed cases of tracheobronchial foreign bodies in cats. we hypothesize that bronchoscopy is highly effective in their extraction in cats. cases of confirmed tracheobronchial foreign bodies in cats admitted to referral centers in france, from may to november , were included. files were retrospectively analyzed for age, sex, breed, clinical signs, delay between onset of signs and presentation, diagnostic procedure, method of extraction, location and nature of foreign bodies. twelve cats were included ( males, females). mean age at presentation was years old ( . years ae . ). cough was the main chief-complaint, being present in / ( %) cats. while / ( %) cats presented to consultation in the first week after the beginning of respiratory signs, / ( %) cats exhibited clinical signs for more than week. chest radiographs were done in / cats. bronchoscopy was performed in / cats, confirming the presence of foreign body material and allowing their extraction in / animals ( %). in / cats ( %), bronchoscopic extraction was unsuccessful and a pulmonary lobectomy was required. the foreign body was located in the trachea in / cats ( %) and in the bronchial tree in the remaining cats ( / in the right caudal bronchus, / in the left caudal bronchus and / in the main left bronchus). / ( %) were vegetal foreign bodies (grain seeds and foxtail awns), / ( %) were mineral (a bone fragment, a teeth and a small stone) and / ( %) of undetermined origin. all the mineral foreign bodies were extracted from the trachea, whilst the majority of the vegetal ones ( / - %) were removed from the bronchial tree. in this case series, bronchial foreign bodies were as frequent as tracheal foreign bodies in cats. this finding differs from previous data reporting that trachea is the preferential location for feline respiratory foreign bodies. vegetal foreign bodies are more common. due to their nature and shape, they may be more prone to lodge in the bronchial tree, while mineral foreign bodies remain in the trachea. according to our results, bronchoscopy is highly effective for identification and extraction of tracheobronchial foreign bodies in cats. disclosures: no disclosures to report. mycophenolate mofetil is the prodrug of mycophenolic acid (mpa). it is a selective non-competitive inhibitor of inosine- monophosphate dehydrogenase, which is expressed in many cell types. mpa's ability to induce lymphocyte cytotoxicity, reduce monocyte recruitment, and suppress dendritic cell maturation, are useful targets in treating immune mediated and inflammatory diseases. it has been used extensively in human medicine for transplant recipients and more recently in veterinary medicine in dogs with various immune mediated diseases. however, its use in cats is limited in part because mpa is primarily metabolized by glucuronidation and cats inherently have a decreased ability to glucuronidate many drugs. we proposed that cats may glucuronidate mpa more slowly than humans and dogs and conducted a series of in vitro studies to explore this hypothesis. we used liver microsomes from cats ( individual and pooled), dogs (pooled), and humans (pooled). these liver samples were incubated at °c in a water bath with mpa and udp-glucuronic acid or udp-glucose. udp-glucose was studied since mpa glucoside is a minor metabolite in humans but may be a major metabolite in other species. hplc-ms was used to determine concentrations of mpa-glucuronide (phenol and acyl) and mpa-glucoside (phenol) formed by incubation. cats formed much less mpa phenol glucuronide ( . nmoles/ min/mg) than dogs ( . nmoles/min/mg), or humans ( . nmoles/ min/mg). cats formed similar amounts of mpa acyl glucuronide ( . nmoles/min/mg) than humans ( . nmoles/min/mg), but less than dogs ( . nmoles/min/mg). in contrast, cats ( . nmoles/min/ mg) formed more mpa phenol glucoside than humans ( . nmoles/min/mg) but less than dogs ( . nmoles/min/mg). when the pathways of metabolism were summed for each species, cats ( nmoles/min/mg) metabolized mpa much less efficiently than dogs ( nmoles/min/mg) or humans ( nmoles/min/mg). variability in metabolite formation between the individual cats was high ranging from fold for mpa glucoside, to fold for mpa phenol glucuronide, to fold for mpa acyl glucuronide. our preliminary results confirm that cats glucuronidate mpa less rapidly than dogs and humans, however cats and dogs were found to glucosidate mpa more efficiently than humans. in addition, individual cats are variable in their ability to glucuronidate and glucosidate mpa. this preliminary in vitro data will be compared to in vivo studies of mpa pharmacokinetics to elucidate proper dosing of mpa in cats. disclosures: no disclosures to report. cystocentesis is the gold standard sampling method for urine microbiology in dogs, as voided samples are associated with a higher risk of contamination. however, the accuracy of the veterinary cut-off values currently recommended for detection of clinically significant bacteriuria in voided urine has been poorly investigated. the aim of this study was to evaluate the accuracy of veterinary and human criteria for diagnosis of urinary tract infection (uti) in dogs using voided urine samples. dogs with suspected uti were prospectively enrolled. paired urine samples collected by cystocentesis and voiding, respectively, were stored at • c and cultured within hours. bacterial counts in voided urine were interpreted using both the veterinary (≥ . colony forming units (cfu) per ml) and the human (≥ . cfu/ml plus presence of clinical signs) criteria for diagnosing uti, and compared to those obtained in urine collected by cystocentesis (gold standard). significant bacteriuria in cystocentesis samples was defined as ≥ . cfu/ml. sixty-five dogs were included in the study. when applying the veterinary criteria for diagnosing uti in voided samples, the diagnostic accuracy was % (sensitivity %, specificity %, positive predictive value (ppv) % and negative predictive value (npv) %). when applying the human criteria the accuracy fell to % (sensitivity %, specificity %, ppv % and npv %). the results indicate that, in most dogs with suspected uti, an accurate diagnosis can be obtained using voided urine, if applying the current veterinary cut-off values to samples stored at refrigeration temperature and cultured shortly after collection. disclosures: the study was supported financially by the uc-care research centre, university of copenhagen, and minor external foundations. antimicrobial resistance (amr) is a major public health concern and will likely be the first cause of mortality in human medicine in . canine bacterial urinary infection is a frequent condition and might be implicated in interspecies transmission of resistance mechanisms. this study aimed to retrospectively describe the evolution of the amr of uropathogens over a -year period in a veterinary teaching hospital. positive urinary cultures obtained by cystocentesis (> cfu/ ml) from dogs treated at the national veterinary school of toulouse (envt) between and , were reviewed. annual prevalence of amr for several enterobacteriaceae, staphylococcus spp, enterococcus spp and streptococcus spp were recorded for various veterinary and human antimicrobials. frequency of extended spectrum beta-lactamase-producing enterobacteriaceae (esbl) and multidrug resistant (mdr) bacteria were noted. logistic regression was performed to analyze the evolution of amrs. a p-value < . was considered significant. over the study period, isolates with stable annual distribution were identified. considering enterobacteriaceae, amr for several antimicrobials significantly evolved over time: despite a possible increase in for some antimicrobials, a general decrease of amr was observed. prevalence of esbl and mdr bacteria remained stable with mean prevalence of % and %, respectively. trends of amr of enterobacteriaceae over the study period in envt are not in accordance with the worrisome general tendency and could be consistent with a rationalized antimicrobials use. however, the persistently elevated prevalence of esbl and mdr bacteria, and the possible increase of amr during the last studied year warrant further investigation and surveillance. disclosures: no disclosures to report. klebsiella pneumoniae are important pathogens that cause urinary tract infections (uti). antibiotic-resistant and virulent bacterial clones with high interhost transmission may play an important role in the spread of antimicrobial resistance. this study aimed to characterize the antimicrobial resistance and virulence of clinical klebsiella isolated from animals and humans with uti and to determine the lineages of companion animal klebsiella pneumoniae resistant to third-generation cephalosporins ( gc). thirty-five companion animal clinical klebsiella spp., obtained between and january , and human clinical strains isolated in were included. antimicrobial susceptibility testing was performed using disk diffusion and clsi breakpoints were applied. extended-spectrum b-lactamases (esbl) and plasmid-mediated ampc genes were detected by pcr whenever resistance to gc was detected. furthermore, gc-resistant klebsiella were characterized by multi-locus sequence typing. regarding virulence, pcr for detection of fimh (adhesin type- fimbriae), mrkd (adhesin type- fimbriae), entb (enterobactin), ybts (yersiniabactin) and rpma (regulator of mucoid phenotype-a) genes was conducted on and companion animal and human strains, respectively. k. pneumoniae was the main species isolated in companion animals ( . % n = / ) and in humans ( . %, n = / ) but klebsiella oxytoca was also identified. resistance to gc (cefotaxime or ceftazidime) was present in . % (n = / ) and . % (n = / ) of companion animal and human strains, respectively. overall, cg-resistant k. pneumoniae were found to be ctx-m group- ( . %, n = / ), cmy ( . %, n = / ) and dha ( . %, n = / ) producers. both companion animal k. oxytoca were dha producers. companion animal cg resistant klebsiella were frequently ( . %, n = / ) co-resistant to fluoroquinoles and trimethoprim/sulphamethoxazole rendering them as multidrug resistant. moreover most companion animal cg resistant strains were also resistant/intermediate to amoxicillin/clavulanate ( . %, n = / ). overall, companion animal klebsiella resistance to amoxicillin/clavulanate ( . %, n = / ), fluoroquinolones ( . %, n = / ) and trimethoprim/sulphamethoxazole ( . %, n = / ) was high. companion animal k. pneumoniae resistant to gc belonged to st (n = ), st (n = ), st (n = ) and st (n = ) lineages. concerning virulence, all k. pneumoniae were positive for fimh, mrkd and entb. yersiniabactin was present in . % (n = / ) and . % (n = / ) of companion animal and human k. pneumoniae, respectively. k. oxytoca (n = ) were positive for entb and ybts. all tested strains were negative for rpma. the detection of k. pneumoniae lineages highly important for humans in companion animals with uti raises great concerns regarding their role as reservoirs. moreover, the fact they were also found to share gc resistance genes and common virulence factors with humans further extends the risk of transfer. disclosures: conflicts of interest: the first author currently receives a phd grant funded by the portuguese foundation for science and technology. feline lower urinary tract disease (flutd) refers to a heterogeneous group of disorders with similar clinical signs. some diets are designed to manage flutd by promoting struvite stone dissolution and addressing key risk factors (overweight, low water intake, stress. . .). the goal of this study is to compare the struvite dissolution potential of diets marketed for flutd, in standardized in vitro conditions. six adult healthy cats were fed successively dry diets (a = royal canin s/o-biopeptide; b = hill's c/d-urinary-stress) for days, with urine collection on the last days. urines collected for each diet were pooled and distributed in bottles containing the mean urine volume produced daily per cat. urine ph and struvite relative supersaturation (rss) were measured for each pool. two feline struvite uroliths homogenous in shape and weight were immersed separately in a urine bottle of each diet, and put in a stove at °c. twenty-four hours later, the urine was filtered to collect the stones, which were dried and weighed. every day, the stones were placed in new bottles of the corresponding urine until the first complete stone dissolution. diet effect on urine ph, volume and rss was analysed non parametrically (wilcoxon paired rank test). diet effect was combined with a period effect (period = days dissolution), in a complete factorial design to analyse struvite stones weight evolution at each period for each diet (mixed model with diet, period, diet x period as fixed effects). fdr method was applied to compare diets at each period. diet a induced a higher mean urine volume ( . versus . ml/kg/day), and a lower rss than diet b ( . versus . ) (p < . ). the urine ph of the diets were not significantly different ( . and . ) (p > . ). after days, the struvite stone immersed in urine from diet a was totally dissolved, versus % dissolution for the stone in urine from diet b. when considering periods of days, the struvite weight diminution was significantly higher when struvite stone was immersed in urine from diet a than from diet b (diet effect: p < . ). the interaction between diet and period effect revealed that the difference in dissolution rate between the diets was significant as soon as the first day period (p < . ), and increased over the other periods (p < . ). a diet inducing a lower struvite rss and a greater urine dilution allows faster struvite stone dissolution. disclosures: the author and co-authors work for royal canin, the company commercializing one of the diets evaluated. serum cystatin c (scysc) and urinary cystatin c (ucysc) are potential markers for detection of feline chronic kidney disease (ckd). our aims were twofold. firstly, we evaluated cysc as marker for ckd. we compared scysc and ucysc between ckd and healthy cats, correlated scysc and scr with glomerular filtration rate (gfr) and calculated sensitivity, specificity for detecting decreased gfr. secondly, we compared assay performance of the turbidimetric assay (petia) with the previously validated nephelometric assay (penia). forty-nine ckd (iris stage - ) and healthy cats were included. gfr was measured with plasma exogenous creatinine (pect), endo-(penict) and exo-iohexol (pexict) clearance test in ckd and healthy cats. based on pexict, scysc was evaluated to distinguish normal, borderline and low gfr. sensi-tivity and specificity to detect pexict< . ml/min/kg were calculated. validation of petia was performed and scysc results of penia and petia were correlated with gfr. statistical analysis was performed using general linear modelling. serum cysc and ucysc were significantly higher (p < . ) in ckd cats. however, ucysc was detected only in / ckd cats. r values between gfr and scr or scysc were . and . respectively. sensitivity and specificity were % and % for scysc and % and % for scr. serum cysc could not distinguish healthy from ckd cats, nor normal from borderline or low gfr, in contrast to scr. penia appeared superior to petia. in conclusion, scysc is not a reliable marker for gfr in cats and ucysc could not be detected in all ckd cats. disclosures: for this work support was received from the institute for the promotion of innovation by science and technology in flanders (iwt) through a bursary to l.f.e. ghys. soblechero, f.j. duque, p. ruiz, r. barrera. university of extremadura, c aceres, spain serum cystatin c (scys c) is a marker of glomerular filtration rate with advantages over serum creatinine. in human medicine, some authors observed that scys c is influenced by methylprednisolone or prednisone administration. with the aim to follow the course of this maker of renal function in acutely diseased patients with a receiving glucocorticoid medication, we followed at dog's whit steroid responsive meningitis. the study was carried out on patients that where divided in groups: control group ( clinical healthy dogs), group b, dogs treated with prednisone due to steroid responsive meningitis (treated with mg/kg prednisone during days, followed to mg/kg prednisone for another days). dogs had no known preexisting renal disease, and have no previous glucocorticoid medication. group c was established to test the effects of endogenous steroids: dogs with hyperadrenocorticism were evaluated. serum cys -c was measured by turbidimetric latex and creatinine by jaffe reaction (spinreac Ò ) and were determined at time of diagnosis for group b, and on days , and in the meningitis dogs. a statistically significant increase of scys-c was observed in group b, with doses of mg/kg of prednisone ( . ae . mg/l; p < . ), and doses of mg/kg of prednisone ( . ae . mg/l; p < . ) respect to these same dogs before treatment ( . ae . mg/l) and compared to the control group ( . ae . mg/l). however, the serum concentration observed in hyperadrenocorticism ( . ae . mg/l) was similar to the one find in the healthy animals group. the creatinine concentration was not increased either, during the prednisone treatment, or in the case of hiperadrenocorticism. in conclusion, the present study agrees with that is described in human medicine, and confirms the effects of glucocorticoids on scys c concentrations in dogs. the administration of high doses of prednisone is associated with a scys c increase. on the other hand endogenous cortisol increase (hyperadrenocorticism) in dogs is not seen to modificate the scys c. disclosures: no disclosures to report. chronic kidney disease (ckd) produces progressive reduction in the number of functional nephrons and directly affects the homeostasis of the solutes excreted in the urine, including phosphorus. hyperphosphatemia is considered a factor directly related to the increased mortality in humans, cats and dogs. in order to provide data from controlled clinical studies to examine the effects of hyperphosphatemia on the progression and survival of naturally occurring canine ckd, the following study was conducted. for the present study dogs, which were followed up by the veterinary teaching hospital of the university of extremadura (spain), were used for the study. distributed in the following groups: group i ( healthy adult dogs) and group ii ( adult dogs with ckd). this second group had a subclasification attending to different factors: phosphatemia: iia ( dogs with phosphatemia < . mg/dl) and iib ( dogs with phosphatemia > . mg/dl). leishmaniasis: iic ( dogs with ckd due to leishmaniasis) and iid ( dogs with ckd not due to leishmaniasis). iris classification: iris ( dogs), iris ( dogs), iris ( dogs) and iris ( dogs). the results of survival were as followed: statistically lower survival was found between the groups iia and iib (p < . ) also between the iris grades , and and the iris grade (p < . ), iris and with iris (p < . ) and iris with iris (p < . ). no significant differences between positive and negative leishmaniasis. in conclusion, plasma phosphate concentration in dogs increases as chronic kidney disease develops. and an inverse relationship to survival in dogs with phosphorus concentrations above mg/dl, and as it progresses the iris scale was observed. disclosures: no disclosures to report. studies analyzing to which extent upc in dogs is influenced by pyuria have yielded conflicting results. moreover, there is no data on the effect of proteinuria on plasma acute phase proteins in dogs. in dogs upc was prospectively measured. upc and if available, results of plasma biochemistry including measurement of c-reactive protein (crp) from the same day were analyzed using the mann-whitney-u-test. samples without sediment analysis (n = ) were excluded resulting in urine samples for analysis. hematuria (> erythrocytes/hpf), pyuria (> leucocytes/hpf), and bacteriuria were present in , and samples, respectively. upc in samples with hematuria was significantly (p = . ) higher (median . ; th - th percentile . - . ) compared to samples without hematuria ( . ; . - . ). in dogs with pyuria upc was significantly (p < . ) higher ( . ; . - . ) compared to samples without pyuria ( . ; . - . ). % of the samples with pyuria had an upc > . this is in contrast to data reported previously where only % of pyuric urine samples had an upc > (vet clin path ; : ). bacteriuria did not influence upc (p = . ). samples of dogs with negative protein dipstick results had an upc > . ( . , . , and . , respectively). all samples had low urine specific gravity ( . - . ) and alkaline ph. for a total of samples corresponding plasma data on albumin (reference interval ri: . - . g/dl) and crp (ri: - . mg/l) were available. crp was significantly (p = . ) higher in dogs with upc > . ( . mg/l; . - . ) compared to dogs with upc ≤ . ( . mg/l; . - . ). albumin was significantly (p < . ) lower ( . g/dl; . - . ) in dogs with upc > . compared to dogs with upc ≤ . ( . g/dl; . - . ). naturally occurring pyuria has a more profound effect on upc results than previously reported. proteinuria is associated with changes of acute phase proteins such as hypoalbuminemia and increased crp. whether this is consequence or cause of the proteinuria needs further investigation. furthermore, animals with low urine specific gravity may have clinically relevant proteinuria even in the light of a negative dipstick result. therefore measurement of upc is recommended to exclude renal protein loss in hypo-and isosthenuric dogs. disclosures: no disclosures to report. high blood pressure causes an increase in vascular endothelial growth factor (vegf) secretion. feline hypertension is commonly associated with chronic kidney disease (ckd) amlodipine is the first choice antihypertensive treatment in cats but could have a negative effect on the kidney by increasing glomerular pressure through afferent arteriolar dilatation. the aims of this study were to: ( ) validate a method for the quantification of vegf in feline serum samples; ( ) assess the association between urinary vegf, serum vegf (svegf) and biochemical and clinical variables in hypertensive cats and ( ) investigate changes in urinary vegf with amlodipine treatment. a randomised, double blinded, placebo controlled parallel group study (n = ) was conducted in phases to determine the efficacy and safety of amlodipine in cats with naturally occurring hypertension. the placebo group was crossed-over to amlodipine after day . a canine vegf elisa (previously validated for feline urine) was used to measure urine and serum vegf. urine vegf concentration was normalised to urinary creatinine (urinary vegf to creatinine ratio [uvc]). univariable linear regression models, followed by a backwards multivariable linear regression model, were performed to identify independent predictors of svegf and uvc. a linear mixed measures model was used to compare the effect of placebo and amlodipine on uvc ( days) and to investigate potential changes in uvc with long-term amlodipine treatment ( days). intra-assay and inter-assay cv of svegf measurements were . - . (n = ) and . - . (n = ) respectively. dilutional parallelism indicated a mean recovery of . % ae . % (n = ). urea and urine protein:creatinine (upc) were independent negative and positive predictors of svegf respectively. plasma creatinine was an independent negative predictor of uvc, upc and sodium were independent positive predictors. no association was found between svegf and uvc. no significant changes in uvc or differences between groups were found with days of amlodipine or placebo treatment. mean uvc at screening was . and . lg/g after days of amlodipine treatment (p = . ), both within the healthy cat reference range ( . to . lg/g). the lack of correlation between urinary and serum vegf suggests that uvc reflects renal vegf production, and is possibly a biomarker of renal stress. uvc does not significantly change with amlodipine treatment suggesting that amlodipine may not cause renal stress when used in cats with hypertension and concurrent ckd. disclosures: this study was sponsored by orion inc. and ceva animal health and used residual samples collected from animals involved in a clinical trial run by orion inc. jonathan elliott provides consultancy advice to the following companies: bayer animal health, ceva animal health, orion inc., elanco animal health, zoetis ltd, boehringer ingelheim, vetoquinol ltd., waltham centre for pet nutrition, idexx ltd. the group is in receipt of research funding from the following companies: novartis animal health, royal canin ltd, zoetis, ceva animal health / orion inc. jonathan elliott serves on the following advisory boards: international renal interest society, european emesis council. diffuse large b-cell lymphoma (dlbcl) is the most frequent subtype of non-hodgkin lymphomas in dogs. in humans, common morphological variants have been recognized by the world health organization (who) classification: centroblastic, immunoblastic and anaplastic. the who classification was recently adapted to canine lymphomas. however, no study clearly correlated prognosis to each morphological variant of canine dlbcl. the objective of this retrospective study was to correlate morphological variants of dlbcl to prognosis, in dogs treated with a standardized chemotherapy protocol. medical records from dogs with a cytological diagnosis of dlbcl between and were retrospectively reviewed by a single boarded clinical pathologist. the centroblastic (dlbcl-cp) and immunoblastic morphotypes (dlbcl-ib) were defined as previously described. anaplastic variant is very rare in dogs, and no case meeting all inclusion criteria were diagnosed during the study period. a fourth borderlines morphological variant was identified and distinguished in this study for clinical considerations (immunoblasts rich centroblastics (dlbcl-irc)). it was characterized by the presence of a higher number of immunoblasts compared to dlbcl-cp. complete initial and follow-up clinical information and application of a standardized chemotherapy protocol were part of the main inclusion criteria. statistical analysis was performed using kaplan-meier analysis. fourty-nine dogs were included. thirty-four ( . %) were dlbcl-cp, ( . %) were dlbcl-ib and ( . %) were dlbcl-irc. median first remission duration for dlbcl-cp, dlbcl-ib, dlbcl-irc were respectively and . and days (p < . ). median overall survival time for dlbcl-cp, dlbcl-ib, dlbcl-irc were respectively , and . days (p = . ). a significant shorter time to obtain complete remission (p = . ) and a significant longer duration of first remission (p < . ) in dogs with dlbcl-cp in comparison to dlbcl-ib were observed when dlbcl-irc were included in the dlbcl-ib group. interestingly for cases, dlbcl-irc variant was observed in peripheral lymph nodes whereas dlbcl-ib variant was observed in the spleen. moreover, / recurrent dlbcl-cp and / of dlbcl-irc displayed progression towards dlbcl-ib variant. in conclusion, this study showed, for the first time, significant prognostic differences between the morphological variants of canine dlbcl, suggesting the prognostic impact of immunoblastic features as it is discussed in humans. disclosures: the residency program of david sayag is supported in part by zoetis. canine lymphoma is a heterogenous group of diseases and evidence exists to describe different behaviours between b-cell and tcell phenotypes of disease. this study aims to describe the response to treatment and survival of canine b-cell multicentric lymphoma (cbcml) cases treated at the royal veterinary college. signalment, clinical findings, staging, treatment, response and survival times were recorded retrospectively. sixty-three cases of cbcml were identified. forty-nine percent presented as stage , % stage , and % stage . sixty-two percent were substage b and % were substage a. forty-four percent received "chop" induction protocols, % "cop," % "coap," and the rest received various induction protocols. ninety-five percent of dogs responded to induction treatment. median first remission duration (frd) was . days. thirty-seven dogs ( %) received rescue protocols with a response rate of %. median overall survival time (os) was days. follow-up was days. this study showed that cop protocols followed by doxorubicin rescue therapy gave no significant difference in os compared with both chop induction alone and chop followed by a rescue protocol (p = . ). os was significantly increased by increased frd (p = . ), absence of an aberrant immunophenotype (p = . ), complete response to therapy (p = . ), and use of rescue protocol (p = . ). frd was significantly increased by use of a chop induction protocol compared with a cop protocol (p = . ), and complete response to therapy (p = . ). age, bodyweight, sex/neuter status, stage, substage, and cell size had no effect on frd or os. seven ( %) of the dogs had a prolonged os in excess of years, and of these dogs remain alive. dogs in the prolonged os group were more likely to be anaemic on presentation (pcv< %, p = . ), experienced a greater frd (p = . ) and were more likely to be treated with a rescue protocol (p = . ) than other dogs. no other significant differences in signalment, clinical presentation, stage, substage, or treatments received were found between this group of dogs and others. in this group of dogs, chop induction therapy gave no survival benefit over the cheaper, less intense cop protocol, providing doxorubicin rescue therapy was later employed. the proportion of dogs receiving chop induction versus cop did not significantly differ between dogs with prolonged survival and those without. the use of rescue protocol, complete response to treatment, aberrant immunophenotype and first remission duration were shown to have prognostic relevance. disclosures: no disclosures to report. lymphoma is the most common malignant haemopoietic tumour in the dog. gene expression profiling (gep) of canine lymphoma has highlighted the important signalling pathways including b-cell activation, b-cell receptor and nf-kb signalling. next-generation sequencing offers benefits over microarray technology for gep in identification of novel transcripts and sequence variants. the aim of this study was to examine gene expression and variant calling in canine lymphoma using rna-seq. lymph node samples were collected from canine multicentric lymphoma patients as part of their clinical investigations. diagnosis was confirmed cytologically or histologically and cell lineage established by pcr for antigen receptor rearrangements (parr) and flow cytometry. cdna was prepared from extracted rna and sequencing performed on an illumina nextseq sequencer generating bp paired-end reads. samples were from b-cell tumours ( stage v, stage iv, stage iii and stage ii) and t cell tumours ( stage iii, each stage ii, iv, v). million reads (mean) per sample were obtained with % mapping to the canine genome. b-and t-cell samples clustered separately on principal component analysis indicating distinct gene expression patterns. genes were upregulated (log fc > , q value < . ) in b-cell lymphomas, many involved in bcr signalling, primary immunodeficiency and haematopoietic cell lineage pathways, innate immune and inflammatory responses. genes were upregulated (log fc > , q value < . ) in t cell lymphomas, most affecting tcr signalling, but also natural killer mediated cytotoxicity, jak-stat signalling, haemopoietic cell lineage and cancer pathways. compared to the reference genome, . million sequence variants were detected across the samples; % not previously described. using the sift (sorting intolerant from tolerant) algorithm, % were predicted to be deleterious for protein function. functional analysis of the affected genes indicated many were involved in bcr signalling and cancer-related pathways. some such as bcl and map k were affected in almost all cases, although proportionally more frequent in b cell lymphoma. others such as traf were exclusive to b cell cases. genes affecting a large number of cases such as bcl tended to have a common variant present in or more cases whereas other genes had variants unique to each single case. although it remains to be confirmed if the detected variants represent true mutations rather than polymorphisms, rna-seq of canine lymphoma samples has generated interesting pilot data that need to be expanded with more samples to validate the results. disclosures: manikhandan mudaliar works for glasgow polyomics which is a commercial company within glasgow university and carries out genomic and polyomic assays. the next gen sequencing was done at glasgow polyomics, partly using an ecvim grant. feline large granular lymphocyte (lgl) lymphoma is uncommonly described in the literature and it is caused mainly by t-cell lymphocyte. to date a standard protocol has not yet been established and long term prognosis is poor. a recent study (krick et al. ) described a median survival time of days (range: - ) in cats with lgl lymphoma receiving mainly a cop-based protocol and in few cases adjuvant surgery or orthovoltage radiation therapy. surprisingly, in that study the longest survival time was achieved from a cat in the non treated group (median survival time days; range: - ) that received only prednisone and single agent cyclophosphamide. considering these data, and the advantages in treating with more than one alchylating agents t-cell lymphoma in dogs (brodsky et al ), the aim of this study was to assess if the sequential use of different alchylating agents was of any benefit in cats with lgl lymphoma. to all owners of cats with a cytological or hystopatological diagnosis of lgl lymphoma that presented to the san marco veterinary clinic from july till december were offered a treatment with sequential alchylating agents and prednisone (saa&p) protocol or palliative care with only prednisone. the saa&p protocol consisted of prednisone at mg/kg q h and chlorambucil at mg/cat (for cats > kg) to q h (for cats < kg). when despite treatment progressive disease or stable disease plus clinical signs referable to the lgl lymphoma were present chlorambucil was substituted with cyclophosphamide at mg/cat q days. finally, when cats stop responding to cyclophosphamide, this drug was substituted with lomustine at - mg/m q - week. during the study period cats were diagnosed with a lgl lymphoma. on owner request cats were euthanased at the time of diagnosis, cats were sent home with no treatment and lost to follow-up, cats received prednisone alone, and cats received the saa&p protocol. median survival time for cats treated with prednisone alone was days (range: - days, % ci - days) and for cats treated with the saa&p protocol was days (range: - days, % ci - ). survival kaplan-meier curves of the treatment group were significantly different (log rank test = . ; p = . ). survival time in cats with lgl lymphoma treated with the saa&p protocol is significantly longer than in cats receiving only prednisone and seems to be longer than historical reported data of cats receiving cop-based protocol. disclosures: no disclosures to report. mast cell tumors are often accompanied by eosinophilic inflammation as they are known to produce eosinophil chemotactic factors. however, little is known about frequency or eventual prognostic significance of blood eosinophilia in mct bearing dogs. thus, the aim of this study was to determine frequency of absolute and relative peripheral blood eosinophilia as well as eosinopenia and to evaluate potential influence on progression free interval (pfi), overall survival time (ost) and tumor specific survival (tss). dogs with mast cell tumors diagnosed between and were included into this retrospective study. data were collected from medical records and follow up phone conversations with patient owners or referring veterinarians. medical records were reviewed to rule out underlying clinical conditions other than mct that could cause eosinophilia. tumor diagnosis was made either by fine needle aspirate and/or tumor biopsy. a patient was allocated to the eosinophilic group, when the eosinophil concentration was > . * /ll or the relative percentage was > %, respectively. when the eosinophil concentration was < . * /ll patients were categorized as eosinopenic. groups were compared by the pearson chi-square test. the pfi, ost and tss curves were generated by the kaplan-meier product limit method. a log rank test was used to compare the curves. one-hundred dogs were included into this study. absolute eosinophilia was detected in / patients and in / a relative eosinophilia was present. median concentration of eosinophils was . * /ll (range, - ) and median relative percentage was . % (range, - %). eosinopenia was found in % of all dogs. a positive association between relative eosinophilia and low grade tumors was detected with both patnaik (p = . ) and kiupel (p = . ) grading system. a positive linear correlation was further noticed between absolute eosinophilia and ost (p = . ). positive correlation was confirmed between relative eosinophilia and pfs (p = . ), ost (p = . ), and tss (p = . ). accordingly a negative linear fit was found between eosinopenia and pfi (p = . ), ost (p = . ) and tss (p = . ). data indicate that peripheral blood eosinophilia might serve as an easily available additional prognostic tool for mast cell tumor bearing dogs. disclosures: no disclosures to report. limited literature is available about prognostic factors for canine renal carcinomas. in humans, histologic differentiation and tumor type are strongly associated with outcome. in dogs only one publication so far has reported this association. cox- expression is documented in several canine neoplasias with prognostic value in canine mammary carcinomas. in renal carcinomas cox- expression has been demonstrated but its significance is not known. the aim of this study was to evaluate clinical and histopathological features of canine renal carcinomas, including cox- expression, and to correlate them with outcome. our hypothesis was that advanced disease, higher histological grade and increased cox- expression would be associated with shorter survivals. this retrospective multi-institutional study within veterinary institutions, included histologically confirmed cases of canine renal carcinoma undergoing nephrectomy between - , with available follow up. histologic features and cox- immunostaining scoring were reviewed by independent pathologists where available. signalment, clinical presentation, stage, adjuvant therapy and survival times were recorded and statistical analysis performed. sixty-two cases were included. male to female ratio was : , median age was . years. cross-breed dogs (n = ) and labrador retrievers (n = ) were over-represented. on presentation dogs had metastasis. overall median survival time (mst) was days ( - days) . dogs without metastasis lived longer (mst versus days, p = . ). twenty-seven dogs received adjuvant therapy post-nephrectomy, without impact in mst (treatment days, no treatment days, p = . ). fifty samples were available for histopathological review, and for cox- immunostaining. shorter survival times were seen in solid histological type compared to others (solid days, papillary days, tubular days, p = . ). histologic degree of differentiation was associated with mst (well days, moderate days, undifferentiated days; p = . ). vascular invasion was associated with shorter survival (mst days versus days if absent; p = . ). marked invasiveness was associated with shorter mst ( days versus mild = days, moderate days; p = . ). patients with low cox- expression had longer mst ( days) than those with high ( days, p = . ). mitotic index, clear cell type, nuclear morphology, fuhrman nuclear grading, presence of pseudocapsule, necrosis, haemorrhage and type of inflammation were not significantly associated with mst. histopathological findings (degree of differentiation, invasiveness, vascular invasion, solid histologic type), cox- expression and metastasis present at diagnosis are strongly associated with survival in canine renal carcinomas and can be used as prognostic factors. disclosures: no disclosures to report. the detection of leishmania infantum-specific antibodies has been extensively exploited for specific diagnosis and monitoring of treatment in canine leishmaniosis. high levels of l. infantum-specific antibodies are commonly observed in dogs with moderate to severe disease. controversial results have been described regarding the use of kinetics of l. infantum specific antibodies during treatment monitoring. the majority of the studies reported that antibodies often decreased slowly but remained detectable over a long period of time while older studies considered that serology was not useful for treatment monitoring. a consensus statement is that measurement of antibody levels is meaningless before months of treatment. the aim of this study was to evaluate l. infantumspecific antibodies at the time of diagnosis and during treatment follow-up visits and to correlate these with the dog's clinical status. nineteen dogs were diagnosed (day ) and followed-up during treatment (days , and ). the treatment protocol was a combination of meglumine antimoniate ( mg/kg/ h sc for month) and allopurinol ( mg/kg/ h po for year). physical examination and baseline laboratory tests (cbc, biochemistry profile, serum electrophoresis, urinalysis and urinary protein/creatinine ratio) were performed during all visits. leishmania infantumspecific antibodies were assessed by an end point sera dilution elisa method. the majority of dogs (n = ) were classified as leishvet stage ii (moderate disease) at the time of diagnosis. three dogs were classified as leishvet stage iii (severe disease, n = ) or iv (very severe disease, n = ). results showed high and variable levels of specific antibodies at the time of diagnosis (meanaesd: ae elisa units (eu)). interestingly, a rapid significant reduction (p < . ) was observed at day during treatment (mean aesd: ae eu). a continuing significant decrease of specific antibodies was also determined at days (mean ae sd: ae eu) and (mean aesd: ae eu). all dogs improved clinically with treatment with the exception of one dog that clinically relapsed at months post-treatment and its specific antibodies level slightly increased. in conclusion, this study reports, for the first time, a rapid reduction of l. infantum specific antibodies after days of treatment by an end point sera dilution elisa method associated with clinical improvement. it is important also to highlight that a marked decrease of antibody levels was also noted after months and year of treatment. disclosures: no disclosures to report. a broad range of clinical manifestations and immune responses have been described in canine leishmaniosis. canine l. infantum infection can manifest as a chronic subclinical infection, self-limiting disease, or non-self-limiting illness. a protective cd + t-cellmediated immune response characterized by production of inter-feron-gamma, il- and tnf-alpha is believed to be present in resistant subclinical dogs while this response seems to be diminished or absent in sick dogs. however, there are few and poorly standardized assays to evaluate this response in the dog. in addition, cellular mediated immunity has been mainly investigated in subclinical or vaccinated dogs but limited information is available in sick dogs with different degrees of disease severity. the aim of this study was to investigate l. infantum-specific cellular immunity in dogs with clinical leishmaniosis at the time of diagnosis. twenty-six dogs were diagnosed based on physical examination, routine laboratory tests (cbc, biochemistry profile, serum electrophoresis, urinalysis and urinary protein/creatinine ratio) and l. infantum-specific antibody levels measured by quantitative elisa. heparin whole blood was stimulated with l. infantum soluble antigen (lsa) and the mitogen concavalin a (cona) and incubated during days. unstimulated whole blood from each dog was used as control. supernatants were collected and ifngamma concentration was measured with a commercial sandwich elisa. the majority of dogs (n = ) were classified as leishvet stage ii (moderate disease). the rest of dogs were classified as stage i (n = ), stage iii (n = ) and stage iv (n = ). twelve dogs (stage i, n = ; stage iia: n = , stage iii: n = ) produced ifngamma after stimulation with lsa (mean ae sd: ae pg/ ml) and cona (meanaesd: ae pg/ml). in contrast, dogs (stage iia: n = ; stage iib: n = ; stage iii = n = ; stage iv, n = ) did not produce detectable ifn-gamma after stimulation with lsa but dogs produced ifn-gamma after stimulation with cona (mean ae sd: ae pg/ml) while one dog was unresponsiveness to cona. no differences in ifn-gamma concentration were found between ifn-gamma producer and nonproducer dogs when blood was stimulated with cona (p = . ). interestingly, ifn-gamma producer sick dogs presented with a lower antibody levels (meanaesd: ae elisa units (eu)) when compared with ifn-gamma non-producer sick dogs (mean ae sd: ae eu) but differences were not statistically significant (p = . ). the results of this study suggest that sick dogs with a more exaggerated humoral response and a more severe disease lack l. infantum specific ifn-gamma production in stimulated blood. disclosures: no disclosures to report. canine anaplasma platys infection (capi) results in thrombocytopenia, but is considered as a subclinical disease in the united states where the disease was first identified. in europe, where the disease seems to be more severe, it has been suggested that circulating strains are more pathogenic, although co-infection with other vector-borne pathogens (vbp) may also contribute to the expression of clinical signs. however, the availability of pcrbased investigation of the impact of co-infection in naturally infected dogs is limited, compromising the assessment of their clinical significance under field conditions. the aim of the present study was to describe epidemiological and clinical features of capi under field conditions in areas endemic for several canine vbp. a study was conducted in veterinary clinics across italy, spain and portugal. sick animals were included when fitting at least clinical and/or biological criteria compatible with ehrlichial disease. serological tests (snap Ò dx, snap Ò leish tests) and diagnostic pcr for ehrlichia canis, anaplasma platys, anaplasma phagocytophilum, babesia/theileria spp, hepatozoon canis and leishmania infantum detection were performed to identify the etiological agents. capi was considered on the basis of suggestive signs associated with positive pcr-based assay for anaplasma platys. among the dogs included, were pcr positive for a. platys. the annual incidence risk of capi was . % and the probability to diagnose capi when facing clinical and/or biological signs suggestive of ehrlichial disease was evaluated at . %. nine dogs were mono-infected, and dogs were co-infected with e.canis ( ), l.infantum ( ), babesia sp. ( ) and h.canis ( ). for dogs, all tests were not performed. anorexia ( %) and weight loss ( %) were common reasons for visit. lymphadenomegaly ( %), hyperthermia and cutaneous signs ( %) were frequent findings whereas musculoskeletal disorders ( %), petechiae/ecchymosis ( %), splenomegaly ( %), dehydration and ocular lesions ( %) then epistaxis ( %) were less common. haematological abnormalities included thrombocytopenia and anaemia ( %), leucopoenia ( %) and leucocytosis ( %). a risk-analysis conducted between mono-and co-infected dogs didn't highlight significant differences except for anorexia that was significantly more frequent in mono-infected dogs. this study illustrates the magnitude of capi in the mediterranean basin and supports the existence of virulent strains in this area. co-infections were common but had a weak impact on clinical expression. these results emphasize also the importance of testing dogs for multiple vbp due to the difficulty in assigning a specific symptom or haematological abnormality to a specific vector-borne infection in endemic areas. disclosures: no disclosures to report. leptospirosis is a worldwide zoonotic disease with high mortality rate in humans and dogs. clinicopathologic changes may reflect renal disease, hepatic disease, or both causing often vomiting, polyuria/polydipsia and diarrhea. therefore, severe electrolytes and anion gap (ag) abnormalities could be expected. the aim of this cohort study was to investigate serum electrolytes and ag in dogs with natural occurring leptospirosis and to assess their prognostic values. the electronic data-base of the san marco veterinary clinic p.o.a system-plus . Ò was searched between october- and april- for dogs with diagnosis of leptospirosis (group ; n = ). inclusion criteria for group were consistent clinicopathologic signs and a positive microscopic agglutination test (titer≥ : in vaccinated dogs, titer≥ : in nonvaccinated dogs or≥ -fold increase in convalescent titer) and/or a positive pcr (urine and/or blood) for leptospirosis. parameters studied were: serum electolytes (sodium, chloride, potassium), ag, and ag albumin-adjusted (ag alb-adjusted = ag + . x ( . -[alb] ).two control populations of randomly healthy dogs (group ; n = ) and sick dogs without leptospirosis (group ; n = ) dogs were created and matched to group for age (ae months), sex (including sexual status) and breed. statistical differences between groups were evaluated by kruskal-wallis test and post-test analysis were performed by wilcoxon-mann-whitney. mortality relative risk (mrr) at days post-admission between group and group was evaluated. roc curves were used to identify the best prognostic analyte. significance level for all statistical test was set at p < . . serum sodium and chloride concentrations were significantly decreased in group compared to group and (p < . for both comparisons). serum potassium concentration was significantly decrease in group compared to group (p = . ), while no difference was present between group and (p = . ). serum ag and ag alb-adjusted were significantly increased in group compared to group and (p < . for all comparisons). there was a significantly increased in mortality rate in group (n = , . %) compared to group (n = , . %) (mrr = . ; % ci = . - . ). between the variables studied serum potassium (auc = %; p = . ) and chloride (auc = %; p = . ), ag (auc = %; p = . ) and ag alb-adjusted (auc = %; p < . ) were prognostic. serum electrolytes, ag, and ag alb-adjusted were significantly different in dogs with leptospirosis compared to healthy and sick dogs without leptospirosis with the exception of serum potassium that was similar between sick dogs with and without leptospirosis. between the variables studied the ag alb-adjusted resulted the best parameter in predicting death in dogs with leptospirosis. disclosures: no disclosures to report. enterococci causes urinary tract infection (uti) in companion animals and may carry important resistance genes such as for the bifunctional enzyme. furthermore, their virulence factors are seldomly reported in veterinary medicine. thus, this study aims to characterize the uropathogenic enterococci antimicrobial resistance, virulence genes and the clonallity of high-level gentamicin resistance (hlgr) enterococcus faecalis. antimicrobial susceptibility testing of clinical uropathogenic enterococci isolated from dogs and cats with uti, isolated between - , was performed by the disc diffusion method. clsi clinical breakpoints were applied. strains showing hlgr were screened for aac( )-ieaph( ')-ia and aph( ')- d genes by pcr. e. faecalis harbouring hlgr genes were typed by multi-locus-sequencing. fifty-nine strains were further characterized by pcr for the presence of gel e (gelatinase), ace (collagen binding antigen), asa- (aggregation substance), and efa a (endocarditis) virulence genes. e. faecalis was the most frequently isolated ( . %, n = / ) followed by enterococcus faecium ( . %, n = / ). overall, antimicrobial susceptibility results were: . % (n = / ) resistance to penicillin/ampicillin; . % (n = / ) resistance to fluoroquinolones (enrofloxacin or ciprofloxacin); . % (n = / ) resistance to nitrofurantoin; . % (n = / ) resistance to chloramphenicol and . % (n = / ) resistance to tetracycline. hlgr was detected in . % (n = / ) enterococci, namely e. faecalis and two e. faecium. all hlgr e. faecalis were aac ( )-ieaph( ')-ia carriers. one e. faecium was positive for aac ( )-ieaph( ')-ia whereas the other was positive for aph( ')- d. interestingly, ampicillin-resistance was only detected in e. faecium ( out of isolates). furthermore, all hlgr e. faecium were also ampicillin-resistant. hlgr e. faecalis were found to belong to st , st , st , and st major lineages circulating in both hospital and community settings in portugal. considering all enterococci, . % (n = / ), . % (n = / ), . % (n = / ) and . % (n = / ) were positive for gel e, ace, asa- and efa a virulence genes, respectively. uropathogenic e. faecium were only positive for ace gene ( out of ), thus e. faecalis had higher virulence genes frequencies. in this study we detected important human hlgr e. faecalis belonging to the clonal complex , such as e. faecalis st , among uropathogens in companion animals. the presence of major clonal lineages in companion animals highlights their role as communityassociated hosts and possible reservoirs of putative human pathogenic enterococci. disclosures: conflicts of interest: c atia marques currently receives a phd grant funded by the portuguese foundation for science and technology. haemotropic mycoplasmas (haemoplasmas) can cause haemolytic anaemias in many species, including people. three feline haemoplasmas have been identified: mycoplasma haemofelis (mhf), 'candidatus mycoplasma haemominutum' (cmhm), 'candidatus mycoplasma turicensis' (cmt). mhf is considered the most pathogenic, whilst cmhm and cmt usually only cause anaemia in cats with concurrent disease or immunosuppression. the aim of this study was to estimate the prevalence of feline haemoplasmas in serbia and identify potential risk factors for infection. surplus edta blood samples from cats in the belgrade region were used. for each cat, the following variables were recorded: age, health status, gender, outdoor access, presence of ectoparasites and haematological results. samples were stored at À °c and transported to the university of bristol for haemoplasma quantitative pcr testing. serology (petchek, idexx) was performed for felv (n = ) and fiv (n = ) infection. statistical analysis was performed using spss; univariable associations between variables and haemoplasma status were first evaluated (v for categorical variables, t-test/mann-whitney u test for continuous variables) followed by multivariable analysis. two samples were negative for internal control s rdna and excluded from the study. of the remaining cats, ( . %) were infected with one or more haemoplasma species; were singly infected ( mhf, cmhm, cmt) , dually infected ( mhf/cmhm, mhf/cmt, cmhm/cmt) and triple infected. the overall prevalences of mhf, cmhm and cmt were . %, . % and . %, respectively. / ( . %) cats were felv infected whilst / ( . %) were fiv infected. multivariable analysis identified significant associations between haemoplasma infection and anaemia (anaemic/non-anaemic, odds ratio (or) . , ci . - . , p = . ), male gender (male/female, or . , ci . - . , p < . ), outdoor access (yes/no, or . , ci . - . , p < . ), non-pedigree breed (non-pedigree/pedigree, or . , ci . - . , p = . ) and fiv positive status (positive/ negative, or . , ci . - . , p = . ). the overall prevalence of feline haemoplasmas in serbia ( . %) is similar to that reported in other european countries ( . - . %). cmhm was the most prevalent haemoplasma species ( . %) in the current study, similar to other european studies (range: . - . %). most previous studies reported that cmt infection is the least prevalent feline haemoplasma species, but in the current study the prevalence of cmt was greater than that of mhf. similarly to previous studies, the presence of anaemia, male gender, outdoor access, non-pedigree status and fiv infection were significantly associated with haemoplasma infection. disclosures: no disclosures to report. canine parvovirus type (cpv- ) is a frequent digestive pathogen in dogs, responsible for high mortality rates in puppies. the control of the infection by disinfection and isolation of patients is of limited efficiency, raising questions about the contagion sources. the aim of our study was to evaluate the epidemiological role of dams in viral circulation during the reproductive period. a total of bitches (mean ae standard deviation: . ae . years old) from one kennel were enrolled in the study. all were annually vaccinated (nobivac dhppi-lepto vaccine; msd, beaucouz e, france). dams were followed from mating to whelping and dams were followed from whelping until weaning. all puppies from the lactating dams (n = ) were followed since until weeks of age. cpv- fecal excretion was evaluated by real time pcr on rectal swabs [ ] every days during gestation (dams) and every days during lactation (dams and puppies). data were analyzed through logistic regression and mixed linear models. a total of samples were collected. during pregnancy, % of the bitches excreted cpv at least once, but only one sample was above the quantification threshold ( . copies/g feces). dur-ing lactation, all bitches were found positive at least once (and times in mean) and % went above the quantification threshold at least once. during lactation, excreted viral loads were significantly higher at d ( . /g feces; p = . ), d ( . /g feces; p < . ) and d ( /g feces; p < . ) compared to the early lactation (< copies/g feces; d to d ). despite threshold for a clinical parvovirosis is . /g feces, none of the bitches expressed any symptom. in % of the cases, the dam excreted before her puppies. viral loads excreted by puppies were not correlated with those excreted by dams. the proportion of puppies excreting viral loads above the clinical threshold increased from d to d (from to % per litter), with overall mortality of only % ( / ). this study demonstrates that appropriately vaccinated adult female dogs may excrete cpv during gestation and lactation. due to the high quantity of cpv- excreted, females probably represent a major source of contamination for their puppies. viral excretion by bitches after lactation until the next breeding period and by males would be interesting to follow to better understand the role of adults in cpv circulation. [ feline panleukopenia virus (fpv) is responsible for one of the most severe infectious diseases in cats, but only few studies have addressed factors of prognostic importance. in an earlier investigation spanning over years, leukopenia, thrombocytopenia, hypoalbuminemia and hypokalemia were found associated with poor outcome. here, we aimed at identifying outcome predictors during shelter outbreaks of panleukopenia between and ; we limited our analysis to fresh cases treated and followed until recovery or death at the same institution. clinical records of the affected cats were reviewed and information was collected at diagnosis and during hospitalization. the data included anamnestic history, physical examination, complete blood count, biochemical profile, blood gas analysis and treatments, including types of antibiotic, antiviral, antiemetic, analgesic, crystalloid, colloid and hemoderivative administered. outcome predictors were analyzed using logistic regression and mixed-design analysis of variance. the study included cats diagnosed with panleukopenia based on clinical findings and positive fecal elisa, of which . % were < months old, and . % females. clinical signs at diagnosis included lethargy ( . %), vomiting ( . %) and diarrhea ( . %). at admission, median (range) leukocyte counts were , /ll ( - , ) and platelets , /ll ( - , ); . % had hypoalbuminemia and . % hypokalemia. treatments included administrations of amoxicillin-clavulanate ( . %), interferon-x ( . %) and intravenous glucose solution ( %). overall, . % of the cats did not survive. lethargic cats were more likely to die (or: . , ci %: . - . , p < . ). leukocyte counts at diagnosis were not associated with outcome, but were after days of hospitalization; in particular, cats alive at days, which succumbed later, had leukocyte counts of /ll ( - , ) whereas survivors had , /ll ( - , ) (p < . ). survivors were more likely to have received amoxicillin-clavulanate (or: . , ci %: . - . , p < . ) and less likely intravenous glucose solutions (or: . , ci %: . - . , p < . ). thrombocytopenia, hypoalbuminemia, hypokalemia and administration of interferon-x were not associated with the outcome. our results suggest that infected cats with lower leukocyte counts later during hospitalization are more likely to die despite treatment. in this study, and different from previous data, lower leukocyte counts at admission did not predict outcome, possibly due to inclusion of cats with early fpv diagnosis. administration of intravenous glucose was associated with poor outcome, perhaps because of an increased risk of sepsis; also, cats in critical conditions were more likely to receive intravenous glucose. the beneficial role of amoxicillin-clavulanate in sick cats might be due to its broad-spectrum bactericidal activity; interferon-x did not show any conspicuous effect. disclosures: no disclosures to report. according to prior studies up to % of cats in southern germany do not have protective antibodies against feline panleukopenia virus and thus, are likely susceptible for feline panleukopenia infection. until now, it is unknown how healthy adult cats with different antibody titers react to feline panleukopenia vaccination in the field. therefore, the aim of the study was to measure antibody titers in healthy adult cats within days after feline panleukopenia vaccination. one hundred and twelve healthy adult cats were vaccinated with a rcp vaccine. before vaccination (day ) and on days and antibodies against panleukopenia virus were determined by hemagglutination inhibition. in . % ( / ) of the cats, no antibodies prior to vaccination were detected; of these cats were vaccinated regularly. nearly one third of the cats ( . %; / ) showed no antibody increase after vaccination and in . % ( / ) of the cats, antibody titer decreased despite vaccination within the days. however, all of these cats were likely protected by their preexisting antibody titer. in cats no antibodies were detected neither prior to nor after vaccination. a large number of adult cats has no protective antibodies and is therefore at risk for feline panleukopenia virus infection. on the other hand, many other cats show high antibody titers and do not develop antibodies due to vaccination. therefore, evaluation of individual antibody status in cats and vaccination only in those cats, that have no antibodies or low titers, should be recommended. disclosures: independent study financed by merial. there was no influence on the results of the study by merial and there is no co-authorship planned with merial. the feline coronaviruses (fcov) occur as pathotypes with an enigmatic, even controversial, relationship: the low virulence or nonvirulent feline enteric coronavirus (fecv) and the highly lethal feline infectious peritonitis virus (fipv). recently, sequence differences within the spike gene region encoding the putative fusion peptide were described and proposed to correlate with the mutated form of fecv (i.e. fipv) leading to the clinical presentation of feline infectious peritonitis (fip). in this presentation, the development and validation of an allelic discrimination real-time pcr typing test which can identify each mutation separately will be described. the diagnostic sensitivity and specificity will be reported from a set of european clinical samples acquired from either fip confirmed cats or from healthy cats that previously tested fcov positive. of these archived samples, fcov positive samples were included into the validation. from these, samples did not pass quality control and had virus levels that were below the limit of detection of the pcr assay. of the remaining samples, were typed correctly with an accuracy of . %. one fip characterized sample was typed fecv (diagnostic sensitivity . %) while all of the healthy cats were typed fecv ( % diagnostic specificity). to confirm that these spike gene mutations are not unique to european cats with fip, additional validation studies from us and japanese samples were conducted. the us clinical study included cases, from fip suspicious cases and with non-fip compatible disease. the fipv realpcr biotyping assay was able to accurately differentiate between the fip or non-fip (fecv) etiologies (p < . ) and did not biotype cats with confirmed non-fip disease as fipv, confirming the high diagnostic specificity of the molecular test. disclosures the aim of this study was to compare the diagnostic accuracy for fip of conventional clinico-pathological tests (routine hematology, serum protein electrophoresis, a -acid glycoprotein -agpmeasurement and analysis of the effusions) with that of molecular tests such as routine pcr and pcr followed by the sequencing of the spike (s) gene. blood, effusion and tissues specimens were collected from cats with symptoms imputable to fip. the in vivo examination consisted of clinico-pathological tests such as complete blood count, serum protein electrophoresis, agp measurement, cytological and biochemical examination as well as the evaluation of the sysmex dtnc of effusions, when present, and of molecular tests such as a screening pcr (directed towards the utr region) and the pcr directed towards the s gene followed by sequencing of the amplification products in order to detect the aminoacidic substitution considered diagnostic for fip. the same molecular techniques were applied to the tissues samples collected during necropsy, which also allowed to divide the cats in a fip group ( cats) and in a non fip group ( cats) based on histology and immunohistochemistry. the diagnostic accuracy (sensitivity, specificity, negative and positive predictive values) of each test was calculated. the best test on tissues was immunohistochemistry (sens: . %; spec: %), while the screening pcr suffered of low sensitivity and very low specificity (sens: . %; spec: . %). the s gene sequencing, positive when revealing the mutated nucleotide, showed very low sensitivity (sens: . ; spec: %). on effusions, the best tests resulted the screening pcr and cytology (sens and spec: %) in comparison with the dtnc measurement (sens: . %; spec: %) and the s gene sequencing (sens: . %; spec: %). in blood samples, agp measurement demonstrated the best diagnostic accuracy (sens: . %; spec: %), while serum protein electrophoresis showed a surprisingly low sensitivity (sens: . %; spec: %). screening pcr (sens: . %; spec: %) and s gene sequencing (sens: . %; spec: %) proved again low accuracy, demonstrating that a negative result with these molecular tests does not allow to exclude fip. . chang hw, egberink hf, halpin r, spiro dj, rottier pjm "spike protein fusion peptide and feline coronavirus virulence"emerg infect dis ( ) diagnosis in feline infectious peritonitis (fip) is still challenging, especially in cats without body cavity effusion. uveitis in cats with fip commonly presents without effusion, which makes a definitive confirmation of fip difficult. the aim of this study was to evaluate the diagnostic utility of an immunocytochemical (icc) assay using aqueous humor in cats suspected of having fip. samples of cats with immunohistochemically confirmed fip and cats that were suspected of having fip due to similar clinical or laboratory changes, but that were definitively diagnosed with another disease were examined. aqueous humor was collected post-mortem after the cats were euthanized due to their diagnosed diseases. icc analysis was carried out using an anti-feline coronavirus mouse monoclonal igg a and an avidin-biotin complex method. sensitivity, specificity, negative and positive predictive values were determined and % confidence intervals ( % ci) calculated. of the aqueous humor samples, ( with fip, controls) revealed positive icc results. false positive icc results were obtained in cats suffering from lymphoma and pulmonary adenocarcinoma. diagnostic sensitivity of the icc assay in aqueous humor was . % ( % ci . - . ); diagnostic specificity was . % ( % ci . - . ); the negative predictive value was . % ( % ci . - . ); the positive predictive value was . % ( % ci . - . ) . unfortunately, false positive results occurred, and specificity, which is considered the most important parameter in a fatal disease like fip, was only . %. positive icc results in aqueous humor should be interpreted cautiously and cannot confirm a suspicion of fip. disclosures: no disclosures to report. alanine aminotransferase (alt) level in plasma is the most commonly used indicator for hepatocellular injury in dogs. however, dogs with advanced liver disease can present with alt levels within reference range. recent studies have shown the potential of circulating micrornas as a biomarker for liver injury. hepatocyte-derived microrna- (mir- ) was identified to be liver specific with superior sensitivity over alt levels in mice and humans. the aim of the present study was to investigate the potential for circulating mir- to serve as a diagnostic serum biomarker of hepatocellular injury in dogs. hereto, liver biopsies of labrador retrievers were collected. liver histology, including grade of hepatitis and stage of fibrosis, was reviewed by a boardcertified veterinary pathologist (tsgamvdi). concurrently, serum samples were collected and analyzed for alt levels and mir- levels. dogs were included into one of the following groups: normal liver and normal alt levels (control group), liver pathology and normal alt levels, or liver pathology and high alt levels. comparative statistics between groups were performed using the mann-whitney u test and associations between mir- and alt levels, grade, stage and hepatic copper concentrations were analyzed using the spearman's rank correlation coefficient. logistic regression models were used to assess the accuracy of mir- and alt to detect the presence of hepatocellular injury. in total, dogs had normal liver histology and normal alt levels. thirtyeight dogs had liver pathology whereof only dogs had increased alt levels. in the high alt group the median level of mir- was (range, - ) times higher compared to the control group (p < . ). even in dogs with liver pathology and normal alt levels, the median mir- level was (range, . - ) times higher compared to the control group (p < . ). univariate logistic regression showed that only mir- and not alt level was a significant predictor for abnormal liver histology (p < . ). serum levels of mir- were positively correlated with alt levels, histological grade and stage of fibrosis (r = . , r = . , r = . , respectively, p < . for all). this study highlights the potential of mir- as an early and sensitive biomarker for liver injury in dogs and is more sensitive than alt levels. early diagnosis of hepatocellular injury opens the opportunity to institute treatment in a subclinical stage of disease with a possibly more favorable outcome. disclosures: this study was financially supported by the ecvim clinical studies fund. the authors declare no further conflict of interest. the evaluation of liver fibrosis is of major importance for the management of chronic liver disease and the prediction of prognosis. although liver biopsy is the gold standard for evaluation of fibrosis, non-invasive tests enable the clinician to stage and monitor a variety of liver diseases in human medicine. as such transforming growth factor b (tgf-b) and hyaluronic acid (ha) are biomarkers of hepatic fibrogenesis, that reflect the activity of the fibrogenic and fibrinolytic process, their use has not been validated in dogs. the aim of this study was to evaluate the measurement of tgfb and ha and assess their sensibility and specificity for the monitoring of dogs with different level of hepatic fibrosis. eighty three adult dogs were prospectively enrolled based on a persistent elevation of alt, with the exclusion of those with focal hepatic lesions on ultrasound examination. all dogs underwent liver biopsy and serum blood collection. lf was staged according to histopathological wsava criteria and the amount of collagen was measured through morphometric analysis. quantitative variables were expressed as meanaesd. bean plots described the relationships between variables. bivariate analysis between ha, alt and pal with lf were performed by spearman correlations. a parametric test (student test) was carried out to assess the relationship between tgfb and lf. diagnostic cut-offs were determined according to the maximum youden index [sensitivity (se) + specifity (spe) - ]. preliminary results in dogs showed that . % of the individuals with ha below ng/ml had a density of < . % collagen. the serum concentration of ha was significantly (p-value = . ) higher in the group whose fibrosis density was ≥ . %. tgf-b was the most sensitive marker but its specificity to diagnose dogs with more than . % of collagen was quite low. the preliminary results show a potential interest of ha as a biomarker to detect liver fibrosis in dogs but the interest of the tgfb could not be demonstrated. ha combines a good sensitivity with a fair specificity. the complete results of the study will help to refine the cut-off value and to improve the diagnostic performance of ah and to evaluate the interest of tgfb. a combination of several markers would be helpful to elaborate a sensitive and specific diagnostic test for liver fibrosis. disclosures: the speaker declares a potential conflict of interest with the company echosens . echosens covers a part of the biological measurements expenses in the clinical trial associated to this abstract. primary hepatitis is a common disorder in dogs. treatments for primary hepatitis are typically symptomatic and importantly, predicting prognosis at point of diagnosis remains challenging. in contrast to human medicine, where the type of hepatitis is defined by the inciting cause, few causes of chronic hepatitis have been identified in the dog, and the majority of cases are idiopathic. systemic inflammation is well recognised in humans with liver disease. systemic inflammatory response syndrome (sirs) is the clinical expression of the action of complex intrinsic mediators of the acute phase reaction. the presence of sirs has been linked to a poor outcome in various liver diseases. the prevalence and predictive value of a sirs in dogs with primary hepatitis has not been examined in dogs with liver disease. this is surprising given the accumulating evidence that sirs is linked to the development of hepatic encephalopathy (he) in dogs with liver disease. although the pathogenesis of he is not completely understood, it has been indicated that ammonia and inflammatory cytokines play a crucial role in the development of he. he is an important cause of morbidity and mortality in patients with liver disease. the hypothesis of this study was to examine the prevalence and severity of sirs n dogs with histological confirmed primary hepatopathies. eighty dogs with primary hepatopathies (confirmed with histopathology) were included in this study. a sirs score was calculated for each (respiration rate > breaths per minute; heart rate > beats per minute; total white blood cell count < or > ^ /l and rectal temperature < . or < . °c). sirs scores presented as a value from to . patient's date of arrival in hospital and date of death were all recorded; therefore survival time (days) could be determined. sirs scoring was applied and survival time was recorded. the median survival for sirs ( - ) was days, while sirs ( - ) had a median survival of days (p value < . , log rank test). this study demonstrates that sirs is a common feature of dogs with primary hepatitis and is valuable in predicting clinical outcome. disclosures: no disclosures to report. the aim of this study was to determine the prevalence of congenital portosystemic shunts (cpss) in deerhounds, focussing on the uk and the usa, and to determine how many deerhound breeders routinely test their puppies for cpss. congenital portosystemic shunts (cpss) are over-represented in certain breeds such as irish wolfhounds, maltese and yorkshire terriers. anecdotal evidence suggests that there is also an increased prevalence in deerhounds. this has not been confirmed, however. the study was questionnaire-based, distributed online to deerhound breeders worldwide (particularly the usa and uk). in addition it was distributed by post in the uk. the questionnaire passed ethical review at the department of veterinary medicine, university of cambridge. fifty-six breeders worldwide returned questionnaires (uk , usa , other countries ). the uk response rate was . % (including postal and online responses). the prevalence of shunts was found to be . % of puppies with prevalences in the uk and the usa of . % and . %, respectively. worldwide, % of breeders were found to test routinely for cpss in their puppies, while the proportions in the uk and the usa were % and %, respectively. the prevalence of cpss in uk and usa deerhounds found in this study was higher than was found for mixed-breed dogs ( . %) in a separate study. this suggests a genetic component to the disease in deerhounds. a lower proportion of breeders rou-tinely tested for cpss in the usa compared with the uk. the prevalence of cpss was also lower in the usa. these findings may be related. it would be advisable for all breeders to routinely test their puppies for cpss before sale, and to avoid breeding from affected animals. disclosures: st catharine's college, cambridge, contributed to the cost of this study. the deerhound club (uk) and the scottish deerhound club of america gave their support, helping to distribute and advertise the study. measurement of neuroendocrine markers can offer diagnostic, prognostic, and therapeutic information that cannot be obtained by clinical examination. nt-probnp is a potential biomarker for hypertensive target organ damage (tod). circulating concentrations of this biomarker are increased in human hypertensive patients with tod and with poor response to antihypertensive treatment. cats with hypertension and tod have significantly higher nt-probnp concentrations than non-hypertensive cats. the aim of this study was to investigate the utility of nt-probnp as a biomarker of hypertension, tod and efficacy of antihypertensive treatment. plasma samples from hypertensive cats seen at first opinion practices were retrospectively identified. hypertension was diagnosed based on systolic blood pressure (sbp) ≥ mmhg with evidence of hypertensive retinopathy (tod-group; n = ) or sbp≥ mmhg on consecutive visits - weeks apart without evidence of retinal pathology (notod-group; n = ). all cats achieved sbp control (defined as < mmhg) on . - . mg amlodipine once daily and had samples available on both a hypertensive visit and the first visit target sbp was achieved. additionally, healthy cats ≥ years old (n = ) and normotensive cats diagnosed with ckd (plasma creatinine ≥ lmol/l in conjunction with usg< . ; n = ) were identified. nt-probnp concentration was measured at an external laboratory. if necessary, variables were log-transformed to meet normality of distribution. binary logistic regression was used to investigate nt-probnp as a predictor of hypertension (using the healthy and ckd cats as comparator group) and tod (using notod as comparator group). comparisons between groups and of response to treatment were performed using mann-whitney u and wilcoxon rank sum tests respectively. higher nt-probnp concentration significantly increased the probability for a cat to be hypertensive (odds ratio log(nt-probnp) = . , [ % confidence interval . , . ], p < . ), but could not reliably predict tod (p = . ). nt-probnp concentration was however significantly higher in cats with tod than in cats with no tod ( . [ . , . ] these data suggest that increased plasma nt-probnp concentration predicts hypertensive status in cats. cats without tod have significantly lower nt-probnp concentrations at diagnosis of hypertension than cats with tod. nt-probnp concentration decreases with effective antihypertensive treatment. further studies are required to determine whether nt-probnp remains elevated in cats with poorly controlled blood pressure. disclosures: esther bijsmans's phd is funded by zoetis. hypovitaminosis d has previously been shown to be prevalent amongst dogs with protein losing enteropathy (ple). outcome is generally poor in canine ple, and there is a lack of studies identifying underlying risk factors. the hypothesis of this study was that low vitamin d serum concentrations could be a risk factor for bad outcome in such patients. medical records for dogs seen at the royal veterinary college between and were reviewed to identify dogs with a diagnosis of ple confirmed by histopathology. dogs were included in the study if they had serum samples frozen within minutes after sampling, had been kept at - degrees c until analysis, and if clinical activity scoring (cce-cai) had been recorded at the time of diagnosis. forty-three dogs were included in the study. follow-up with referring veterinarians was made to determine outcome of patients. patients were divided into groups: patients deceased due to ple (poor outcome group, n = ) and patients alive or deceased due to another disease (good outcome group, n = ). treatments for patients were allocated to groups: one group consisted of patients who were prescribed diet only and the other group received diet and immunosuppressive agents. samples were sent on dry ice to michigan state university's diagnostic center for population and animal health. ionised calcium (ica) was measured using an ion specific electrode and (oh)d was measured using a commercially available radio-immunoassay that has been validated for use in veterinary medicine. comparisons of outcome groups for age, ccecai, treatment, serum (oh)d and ica were performed using a mann-whitney u test or chi . logistic regression analysis was performed to determine possible risk factors for poor outcome. results: ccecai scores, age, and ica concentrations between the groups were not significantly different. there was a significantly greater number of dogs treated with food alone in the group with good outcome ( / ) than in the poor outcome group ( / , p = . ). furthermore, median serum (oh)d concentration was significantly lower in patients with poor outcomes ( . nmol/l, range - nmol/l) compared to patients with good outcomes ( nmol/l, range - nmol/l, p = . ). using logistical regression, (oh)d serum concentration was a statistically significant factor for poor outcome (p = . ), with an increase of (oh)d serum concentration reducing the odds of having a poor outcome (odds ratio = . , % ci: . - . ). further studies are required to investigate vitamin d as a potential adjuvant therapeutic agent in ple patients. disclosures: no disclosures to report. campylobacter jejuni (cj), c. upsaliensis (cu) and c. helveticus (ch) are commonly isolated from dog and cat faeces but association with clinical signs is discordant or lacking. cj is a recognized human pathogen, cu is considered an 'emerging' pathogen and ch is not considered pathogenic despite a high level of genetic similarity. recently, the greater wax moth, galleria mellonella, was described as an animal model of disease; these invertebrates have a high degree of functional and structural homology with the mammalian innate immune system. this study aimed to evaluate the pathogenic potential of cj, cu and ch using the galleria mellonella larvae model. twelve isolates of cj, of cu and of ch from dogs and cats were used for the inoculation of larvae. inocula were prepared by suspending isolates in phosphate-buffered saline (pbs) from which -fold dilutions were made. each dilution was tested in duplicate sets of larvae. each larva was injected with - ll into the haemocoel via the last left pro-leg using g insulin syringes. controls consisted of pbs inoculated larvae and un-inoculated larvae. survival of larvae at °c in a h enriched microaerobic atmosphere was monitored for days postinjection. one subset of isolates was grown in mueller-hinton broth and used for the preparation of secretory products, and another grown on blood-agar and suspended in pbs for heat inactivation of minutes at °c for testing of whole-cell lysates and heat-stable insoluble and soluble components. the overall median survival of larvae was % with cj [iqr - ], % with cu [iqr - ], % with ch [iqr - ], % with pbs [iqr - ] and % for un-inoculated larvae [iqr - ]. a dose-dependent association was evident for each species with larval survival being similar between a low bacterial dose and pbs. larval survival presented a consistent pattern between species for medium and high bacterial loads; cj had a higher and faster larval death rate than cu and ch (p < . ), but no difference was observed between cu and ch (p = . ). there were no significant differences between species in any of the assays with secretory products, inactivated cells and soluble/insoluble cellular components. the observations within this invertebrate disease model support a varying pathogenic potential between the species studied that appears related to the (patho)biology of the species rather than their cellular components or metabolic products. the invertebrate animal model is promising in comparative pathogenicity studies. disclosures: no disclosures to report. acute stress from medium or high duration high-intensity exercise has been reported to be associated with an increase in serum c-reactive protein (crp) concentrations, an important acute-phase reactant in dogs. however, the effect of exercise on fecal s a concentration, a biomarker of intestinal inflammation has not previously been evaluated in dogs. the goal of this study was to determine if moderate intensity short duration exercise causes an increase in crp and/or s a concentrations in dogs, potentially leading to misinterpretation of their results. adult military working dogs (german and belgian shepherd dogs; males; mean age = years [ . - . ]) were included in the study. fecal quality, fecal s a , and serum crp concentrations were evaluated just before and after standardized exercise ( minutes of bikejoring at a speed of km/h). fecal quality was evaluated based on a -point scale (from : liquid to : dry and hard feces). fecal s a and crp concentrations were assayed with previously validated elisa tests. data were analyzed with an anova test for repeated measurements (sas software). results are presented as medians and ranges. serum crp concentrations increased significantly after exercise (median before and after excercise mg/l [ - ] and mg/l [ - ] (p = . ). also, fecal s a concentrations were significantly higher after exercise compared with baseline concentrations ( ng/g [ - ] versus ng/g [ - ], p = . ). no significant effect of exercise on fecal score was observed ( [ . - . ] before and after the exercise; p = . ). our study demonstrates that a moderate-intensity, short-duration effort performed by healthy army dogs causes significant increases in fecal s a and serum crp concentrations, as compared with baseline values, but within the respective reference intervals. therefore, a moderate exercise does not present a confounding variable in the interpretation of fecal s a or serum crp concentrations in healthy dogs. disclosures: this study was performed thanks the financial support of royal canin. imaging is an integral part of the work-up of canine gastrointestinal (gi) disease. radiography and ultrasonography are noninvasive modalities that can evaluate the bowel, but many findings lack desirable sensitivity or specificity. endoscopy directly visualizes gi mucosa, but is limited by the length of the endoscope and the need for general anesthesia, advanced training and expensive equipment. ambulatory light-based imaging (ali) is a new imaging modality that utilizes high-resolution cameras, a microprocessor, and led illumination to non-invasively visualize the gastrointestinal mucosa. ali is performed by oral administration of a fully automated device the size of a pill that is propelled by peristalsis. the aim of this study was to analyze image quality and gi transit times in a series of client owned dogs undergoing ali. dogs were food-restricted for hours before and hours after capsule administration. capsules were retrieved and images were downloaded and analyzed. video clips of frames duration were obtained from the stomach; proximal, middle and distal small intestine; and proximal colon for assessment of image quality. internists rated the images on a scale of - ( = poor, = excellent) based on clarity and resolution of images, and obscuration of the mucosa by fluid, bubbles or debris. scores for each region were compared using general estimating equation analysis. gastric and small intestine transit time were calculated based on visualization of passage of the capsule from the stomach to duodenum, and ileum to colon. clinical analysis of the entire video was performed by one of the authors. ali was successfully performed in / patients, with no adverse effects. average study duration was . ae . hours and mean image acquisition count was , ae , . gastric and small intestinal transit times were . ae . minutes and . ae . minutes, respectively. median (range) image quality scores were ( - ), ( - ) and ( - ), for the stomach, si and colon, respectively. image quality scores were significantly higher in the stomach and si than in the colon (p < . ). visualized lesions were consistent with gi ulcers ( dogs), inflammatory bowel disease ( dog), and bilious vomiting syndrome ( dog). one dog receiving chronic nsaids had a normal study. ambulatory light-based imaging resulted in good to excellent image quality throughout most of the gi tract. bowel preparation should be considered to enhance visualization of the colon. ali was safe and easy to perform in ambulatory dogs, and should therefore be considered in the work-up of canine gi disease. disclosures canine pancreatitis is the most common exocrine pancreatic disorder. the prognosis of canine pancreatitis is variably and no logistic regression constructed severity scoring systems are available. four hundred and thirty nine dogs diagnosed as pancreatitis with acute onset of compatible clinical signs, a positive snap Ò cpl tm test, and/or associated abdominal ultrasonographic abnormalities between january and december were presented at national taiwan university veterinary hospital (ntuvh). one hundred and three dogs hospitalized with complete medical therapy and outcomes were selected for further analysis. the dogs were divided into survival (n = ) and non-survival (n = ) groups. forty-seven parameters including signalment, clinical signs, physical examinations, clinicopathological examination, complications and concurrent diseases were analyzed and compared between the groups. logistic regression analyses were performed in this study. variables with p ≤ . were considered for further analyses. the mortality in this study was . %. age, heart rate, respiratory rate, white blood cell count, albumin, bun, creatinine, potassium, presence of systemic inflammatory response syndrome (sirs) and presence of oliguria or anuria were selected for constructing the scores. continuous variables outside the reference interval were separated into quartiles to yield quartile-specific odds ratios (ors) for survival. based on the integer value of the or, the scoring system was then developed by incorporating weighting factors assigned to each quartile. a predictive total score was calculated for each dog by summing all weighting factors. the total scores of each dog ranged from to . the severity scores in this study achieved an area under the receiver operating characteristic (auroc) of . . the optimal cut-off point for discriminating outcome was . with a sensitivity of . % and specificity of . %, respectively. the mortality was . % with a score ≥ , whereas . % with a score ≤ . there was a significant difference (p < . ) between the groups seperated by the cut-off point. the severity scoring system of this study provides a reliable and clinical applicable method to predict clinical outcome in dogs with pancreatitis. disclosures: no disclosures to report. glucocorticoids (gcs) are known for their anti-inflammatory and immunmodulatory properties and are therefore often used in the therapy of canine inflammatory bowel disease (ibd). it was recently shown that endogenous gcs are also produced in the intestinal epithelium of men and mice and influence the gastrointestinal immune system in case of inflammatory or neoplastic conditions. thus, the aim of this project was to prove that gcs can be produced or metabolized in the canine intestinal epithelium. five healthy beagle dogs were included into this prospective study. all dogs were clinically examined, given a clinical score using the canine ibd activity index (cibdai) scoring system, also gastrointestinal endoscopy was performed. mucosal biopsy specimens from duodenum were examined histologically from a board certified pathologist using the wsava grading. biopsy incubation of - endoscopical mucosal biopsies in tissue culture medium with h-labeled progesterone in the absence of any stimulation was performed. the mean age of the included dogs was . + . years, the mean weight was . + . kg. all beagle dogs had a mean clinical score of + . the mean wsava scoring was + . . after hours, supernatant was harvested and radioactive progesterone metabolites formed were detected using high performance liquid chromatography plus liquid scintillation counting. in all dogs the h-progesterone was metabolized into various steroid species, nevertheless a local production of cortisol could not be proven. in summary, it could be shown that precursors of gcs can be metabolized by healthy canine intestinal mucosal tissue. disclosures: no disclosures to report. we studied the relationship between pancreatitis and cardiac injury in dogs and cats. previously, we validated a cardiac troponin i (ctni; vet j : - , ) assay for sensitive and specific detection of cardiac injury in domestic animals. we found various non-cardiac diseases of dogs and cats were associated with cardiac injury detected by serum cardiac troponin i, including some cases of pancreatitis. also, we validated the dggr-lipase assay for cost-effective, sensitive and specific detection of pancreatitis in dogs and cats (vet clin path : e - , ; :e - , ). herein, we tested the hypothesis that pancreatitis was associated with cardiac injury. ctni was measured by advia centaur tni-ultra assay; dggr-lipase by the randox colourimetric assay. we retrospectively analysed data from dogs and cats admitted to ucd veterinary hospital in which both ctn and lipase had been measured. upper limit of reference range for lipase in dogs is u/l; we consider - indicative of mild pancreatitis, - moderate, and > as marked. upper limit of reference range for lipase in cats is . reference range for ctni is < . ug/l for dogs and cats. we consider . - . indicative of mild cardiac injury, . - as moderate, and > . as marked. dogs and cats had both lipase and ctni measured. dogs had normal troponin; had normal lipase and had normal lipase and normal ctni. dogs ( %) had pancreatitis as indicated by increased lipase. in ( %), pancreatitis was mild, in ( %) it was moderate, and in ( %) it was marked. of dogs had increased ctni: mild in ( %), moderate in ( %), and marked in ( %). cardiac injury in dogs with pancreatitis was absent in %, mild in %, moderate in %, and marked in %. of cats had normal ctn; had normal lipase. of cats had pancreatitis, severely in . lipase and ctni was correlated (r = . ) for dogs and cats. we conclude that both pancreatitis and cardiac injury, as indicated by high-sensitivity and high-specificity assays randox-dggr-lipase and centaur-ctni, respectively, are not uncommon in veterinary hospital cases. we confirm and extend our previous work. pancreatitis in dogs and cats is typically associated with cardiac injury. severities of pancreatitis and cardiac injury are correlated. for~ % of dogs and cats with pancreatitis, cardiac injury is moderate to marked. disclosures: no disclosures to report. intracellular colonization may serve as a protected niche where helicobacter spporganisms evade effective treatment, contributing to recolonization. confocal endomicroscopy (cem) is an endoscopic modality allowing in vivo gastrointestinal imaging at high resolution; and has aided real-time identification of helicobacter pylori and intracellular and mucosally associated bacterial. in dogs, non-helicobacter pylori-helicobacter (nhph) are described intracellularly. the objective of this study was to determine the utility of cem to identify nhph in dogs compared with other diagnostic modalities; and to assess its ability to identify intracellular organisms. fourteen clinically healthy dogs underwent standard gastroduodenoscopy followed by cem using topical acriflavine. images were obtained using cem at a minimum of sites within the stomach. endoscopic pinch biopsies were obtained for histopathology, polymerase chain reaction (pcr) and fluorescence in situ hybridisation (fish). methodologies were compared for their sensitivity in detecting the presence and distribution of nhph and their ability to identify intracellular organisms. cem provided high quality images allowing in vivo identification ofnhph in dogs, as did fish post-procedure analysis. standard histopathology identified nhph in only . nhph were identified within the superficial gastric mucus, and gastric pits. distribution throughout the stomach was diffuse and multi-focal. cem findings correlated with fish and pcr, however only fish enabled identification of intracellular nhph which were present in of dogs. cem provides in vivo histology images and is capable of identifying nhph during gastroscopy, but is unable to identify intracellular organisms using the current fluorophore protocol. nhph in the canine stomach are commonly identified intracellularly. disclosures: dr sharman has shares in optiscan imaging pty ltd. chronic enteropathies (ce) and exocrine pancreatic insufficiency (epi) can both cause hypocobalaminemia in cats. current supplementation protocols for cobalamin in cats call for repeated parenteral injections. in humans, several studies have reported equal efficacy of oral administration of cobalamin. there is also evidence that oral supplementation is effective in dogs with hypocobalaminemia. recently, it has also been reported that oral cobalamin substitution restores normocobalaminemia in healthy elderly cats. the purpose of this retrospective case series was to evaluate whether oral cobalamin supplementation can restore normocobalaminemia in hypocobalaminemic cats with chronic enteropathies. a computerized database search for cats treated at evidensia specialist animal hospital, helsingborg, sweden during - was performed. inclusion criteria were cats with symptoms of ce, an initial serum cobalamin concentration below pmol/l (reference interval: - pmol/l) and daily oral treatment with cyanocobalamin ( mg/tablet; ⅛-¼ tablet/cat daily). follow-up serum cobalamin concentration was measured to days after initiation of daily oral cobalamin supplementation. thirteen cats aged - years (median ) of different breeds met the inclusion criteria. presenting complaints included vomiting ( / ), anorexia ( / ), diarrhea ( / ), weight loss ( / ), and lethargy ( / ). increased pancreas specific lipase (spec fpl Ò ) serum concentrations were reported in / cats and / had increased serum alanine transaminase activity. feline serum trypsin like immunoreactivity (ftli) was determined in / cats revealing results within the reference interval. all cats had an abdominal ultrasound, / had changes related to the gastrointestinal tract such as mild-moderate thickening of the small intestinal wall, thickening of the muscularis layer, poor definition of intestinal wall layers, and/or enlargement of the mesenterial lymph nodes, histopathology was performed in / cats, revealing small intestinal inflammation in cats and small intestinal lymphoma in one. serum cobalamin increased in all cats with treatment. the concentration difference ranged from to pmol/l (mean: pmol/l). mean (ae standard deviation) serum cobalamin concentrations were (ae ) pmol/l before and (ae ) pmol/l after supplementation. this difference was statistically significant (p < . , paired t-test). our results suggest that oral cobalamin supplementation is effective in normalizing serum cobalamin concentrations in cats with various enteropathies. prospective studies are warranted comparing cellular cobalamin status in cats being treated with parenteral or oral cobalamin supplementation. disclosures: no disclosures to report. pulmonary thromboembolism (pte) is observed in dogs with idiopathic-inflammatory-bowel disease (ibd) and particularly with protein-losing enteropathy (ple). hypercoagulability has been attributed to antithrombin (at) loss although the pathogenesis is likely to be more complex. in humans, where venous thromboembolism (te) is a wellrecognised complication of crohn's disease and ulcerative colitis, the pathogenesis of te is still not completely understood. derangements in procoagulant and anticoagulant factors have been demonstrated, including increased circulating procoagulant microparticles (mps). the aim of this pilot study was to evaluate mp-procoagulant activity in the plasma of dogs with ibd and ple using a functional elisa assay (zymuphen-mp-activity, aniara). we hypothesised that all dogs with ple and a subset of dogs with ibd but without ple would have increased levels of circulating mps. the study group consisted of dogs with ibd, including with ple. diagnosis was based on compatible clinical and histopathology and exclusion of other causes of chronic gastrointestinal disease. ple was defined as ibd plus hypoproteinaemia (serum total protein < g/l) and hypoalbuminaemia (serum albumin < g/l). pte was diagnosed in one dog with ple, and suspected in a second. a control group comprised healthy dogs undergoing blood sampling for reasons unrelated to the study including blood donor screening (n = ) and health assessment (n = ). dogs were considered healthy based on owner evaluation, physical examination, haematology and serum biochemistry median mp procoagulant activity in dogs with ibd was . nm (range . - . ) compared with . nm (range . - . ) in the control group. median mp activity in ple dogs was . nm (range . - . ) compared with . nm (range . - . ) in non-ple ibd dogs. using kruskal-wallis test for nonparametric data and dunn's multiple comparisons test the groups were not statistically different. interestingly, mp-procoagulant activity value in the dog with documented pte was . nm; in the dog with high clinical suspicion for pte, mp-procoagulant activity was . nm. the highest mp-procoagulant activity was detected in a healthy control dog, raising concerns for pre-analytical or sampling error. removing this measurement had no impact on statistical analysis, which remained nonsignificant. mp-procoagulant activity > nm is considered clinically relevant in humans. employing a similar cut-off, / of controls, / of ibd and / of ple group would be defined as having increased levels of circulating mps. further studies are required to fully evaluate the clinical relevance and diagnostic potential of mp evaluation. disclosures: no disclosures to report. the intestinal microbiota is increasingly linked to the pathogenesis of chronic enteropathies (ce) in dogs. while imbalances in duodenal and fecal microbial communities have been associated with mucosal inflammation, relatively little is known about alterations in mucosal bacteria seen with ce involving the ileum and colon. the aim of the present study was to use fluorescence in situ hybridization (fish) techniques to investigate the composition and spatial organization of mucosal microbiota in endoscopic biopsies obtained from dogs with ce and controls. tissue sections from the ileum and colon from dogs with inflammatory bowel disease (ibd), dogs with granulomatous colitis (gc), dogs with intestinal neoplasia, and controls were studied by fish targeting the s rrna genes of total bacteria, group-specific organisms, and individual bacterial species shown to be relevant in human ibd. the numbers of mucosal bacteria were analyzed using generalized linear models for each of the colon and ileum tissues, with spearman's rank correlation coefficients used to test the correlation between mucosal microbiota and inflammatory (cib-dai score, histopathology) indices. the ileal and colonic mucosa of healthy dogs and dogs with ce was predominantly colonized by bacteria localized to free and adherent mucus compartments. dogs with ce harbored more (p < . ) mucosal bacteria belonging to the clostridium-coccoides/eubacterium rectale group, bacteroides, enterobacteriaceae, and escherichia coli versus controls. within the ce group, ibd dogs had increased (p < . ) enterobacteriaceae and e. coli bacteria attached onto surface epithelia or invading within the intestinal mucosa. bacterial invasion with e. coli was present in the ileal and colonic mucosa of dogs with gc (p < . ). dogs with intestinal neoplasia had increased (p < . ) adherent (total bacteria, enterobacteriaceae, e. coli) and invasive (enterobacteriaceae, e. coli, and bacteroides) bacteria in biopsy specimens versus all other groups. increased numbers of total bacteria adherent to the colonic mucosa were associated with clinical disease severity (cibdai score) in ibd dogs (p < . ). these results indicate that histopathologic lesions of canine ce are associated with different populations in ileal and colonic mucosal microbiota. these spatial, segment-specific structure and differential response of select bacterial groups to intestinal inflammation may be pivotal regarding the functional consequences of these alterations in the pathogenesis of canine ce. disclosures: no disclosures to report. abdominal girth is used as an indicator of human adiposity, with such measurements being made by tape measure. given concerns in precision and accuracy of repeat measurements, some tape measure designs have inbuilt mechanisms to improve consistency. although body condition scoring is the most common method of assessing adiposity in dogs, zoometric systems have also been developed requiring the use of a tape measure. however, the precision and accuracy of such zoometric measurements are not known. the aim of this study was to determine the precision and accuracy of different types of tape measure for a variety of dimensional measurements. a variety of length (head, forelimb, hindlimb) and circumferential (neck, thorax, and abdomen) were made using different tape measures, of which were designed to improve precision (standard tape; myotape tm and gulick ii tm ). to assess intra-operator variability, measurements were taken for consecutive days from healthy dogs; to assess inter-operator variability, operators independently took measurements from a group of dogs of various breeds and sizes. for intra-operator comparisons, precision was good overall (coefficient of variation [cv] ≤ % for all measurements). for inter-operator comparisons, precision was more variable and, although reasonable on average (mean cv - %), it varied depending upon tape measure type (p = . ; greatest for standard tape measure, least for gulich ii tm ), and could be highly variable for some measurements in individuals dogs (maximum cv % for head measurements with standard tape measure). significant differences also existed in the absolute results of circumferential measurements taken by the different tape measure types (neck p = . ; thorax p < . ; abdomen p < . ). finally, significant operator differences were also evident for some measurements (head p = . ; hindlimb p = . ), but not for others (forelimb p = . ; neck p = . ; thorax p = . ; abdomen p = . ). in summary, although precision for individual operators making zoometric measurements is good, significant inter-operator and tape type differences exist. these results have implications for systems using a range of zoometric measures to assess adiposity. in order to ensure precision and accuracy, it is recommended that the same operator take all measurements with the same type of tape. disclosures: the study conducted was not supported by a research grant. ajg's readership is funded by royal canin; ajg has also received financial remuneration and gifts for providing educational material, speaking at conferences, and consultancy work; slh's post at the university of liverpool is also funded by royal canin. body condition score (bcs) is a method that is commonly used in the diagnosis of nutritional status in small animals. however, this method is subjective due to its sensory evaluation. therefore, the improvement of the precision of the bcs diagnosis is expected. our previous study has shown that the bcs model that we created improved the precision of the bcs diagnosis ( ). however, a palpation site was not identified. a palpation site must be the site where thickness of subcutaneous fat is able to capture for measuring animal's obesity status. therefore the objective of this study was to find a remarkable body site of the changes with obesity status using ultrasonic diagnostic equipment. nine dogs which varied in the percent of body fat were used in this study. the percent of body fat was measured by a body fat analyzer for dog (kao). the image analysis of a palpation site was evaluated using echo, xario ssa- a (toshiba) which attached to a linear probe. the measurement points were , and o'clock positions on the ribs of t , t and t . the distance (d) from skin surface to the rib was measured in the echogram. the distance (l) from scapula to ilium was measured to offset the difference in physique by dog breeds. the d/l was used to compare relative value of the quantity of fat at each measurement point. bcs of dogs which used in this study were from bcs of to bcs of . there were no dogs in bcs of and bcs of . a statistically significant correlation was found between bcs and d/l value. the d/l value increased in order of t , t and t in bcs of and . this suggests that the thickness of subcutaneous fat in the chest is thicker at the head side than the tail side. also, as for the d/ l value from back to abdomen, the highest value was found at the position of : and : . this tendency was the most remarkable in bcs of but no difference in the d/l value was recognized in the dogs in bcs of . in conclusion, the position of : or : on the t is the suitable palpation point at the chest. ( body condition score (bcs) is a method that is commonly used in the diagnosis of nutritional status in small animals. bcs has been recognized as one of the screen method of nutrition diagnosis by american animal hospital association in . however, this method is subjective due to its sensory evaluation. therefore we made a bcs model to increase the precision of the bcs diagnosis and have shown the efficacy of the bcs model ( ). however, the prototype model which we have reported before tended to have higher bcs than a target bcs. therefore, we improved the bcs model in this study. sixty seven dogs which varied in the bcs were used in this study. body fat percentage was measured by using a body fat analyzer for dogs (kao healthlab bif- ). the bcs model was improved by using several rubber sheets. relative hardness of stacking rubber sheets in each bcs was measured by durometer mj-dua-c (satotec tokyo, japan). bcs diagnosis of dogs was performed by pet owner by using the bcs model. bcs of represents the most hard in the bcs model and the hardness decreased linearly and it was the lowest in of bcs. these values were as expected. high correlation was recognized between bcs and body fat percentage. these results suggested the efficacy of bcs model. however, the body fat percentage in the dogs diagnosed as bcs of was higher than body fat percentage which has been reported in the previous paper. there were no dogs with the body fat percentage < % which were diagnosed as bcs of . we need more study in future to make clear the difference of body fat percentage between our data and date of the previous research. the completion of this bcs model will help provide the precision of nutritional diagnosis in dogs. ( in humans the metabolic syndrome (ms) is a well-recognised and extensively studied entity that comprises obesity, hypertension, dyslipidaemia, and glucose intolerance. it is associated with an increased risk of cardiovascular diseases and diabetes. recently, human ms criteria were adapted for dogs to define the condition of obesity-related metabolic dysfunction (ormd). it was observed that ormd was associated with increased circulating insulin and decreased adiponectin concentrations, suggesting that in dogs, as in humans, there are links between obesity, ormd, and associated diseases, although pathogenetic mechanisms and health significance for dogs remain unknown. the main aim of the present study was to compare plasma proteomes of obese dogs with and without ormd, so as to investigate the mechanisms associated with canine ormd and their possible significance in the health status. eight obese dogs referred for weight management at the royal canin weight management clinic, university of liverpool participated in the study. clinical assessments included physical examination, body condition scoring, blood pressure measurement and routine clinicopathological analysis. surplus plasma was used in proteomic analysis. samples were first treated with proteominer for thedepletion of high-abundance proteins and subsequently analysed by using -de dige methodology. of the dogs in the study, dogs had ormd and dogs did not. image analysis and further statistical analysis allowed identification of spots with differential expression concentration between dogs with and without ormd. among the spots, were over-expressed and were down-expressed in dogs with ormd than in dogs that did not presented ormd. although the results of the present study are preliminary and still the identification of the spots is up to be performed, the observed datareveal that dogs with ormd present alterations in their plasma proteomes that could be responsible for the development of ormd-related pathologies. disclosures: the study was funded by waltham. ajg's readership is funded by royal canin; ajg has also received financial remuneration and gifts for providing educational material, speaking at conferences, and consultancy work; slh's post at the university of liverpool is also funded by royal canin. vb is an employee of royal canin and pjm is an employee of wal-tham. the aim of the study was to assess the diagnostic value and the discrimination potential between the normal heart size and microcardia or cardiomegaly of a method which calculates the cardiothoracic ratio (ctr) using area measurement, compared to the vertebral heart scale method (vhs) used as reference for the cardiac size, in dogs. one hundred-nine dog x-rays were accepted into study. the patients belonged to small and medium size breeds, were males and females with age between and years. the analogic xrays were scanned and transferred to a computer where the vhs and ctr was calculated for each patient with a commercial software and the data was collected and processed in a statystical analysis software. the patients were distributed into groups by respiratory phase and heart size. there was a low correlation between the vhs and ctr (r = . ), but statistically significant (p? . ). a good correlation was obtained between vhs and ctr in microcardia, normal heart size and cardiomegaly groups (p < . ). furthermore, between the ctr in dogs with microcardia and those with normal cardiac size, as well as between ctr in dogs with normal cardiac size and those with cardiomegaly, a significantly statistic difference (p < . ), respectively (p < . ), was obtained. among the groups distributed by respiratory phase and vhs, a statistically significant difference was obtained only between normal cardiac size and cardiomegaly during inspiratory phase groups (p > . ). for the x-rays taken in inspiratory phase, a cutoff of . had a sensitivity of % and a specificity of % for diagnosing cardiomegaly. the ctr can be considered a valid method being able to discriminate between the patients with microcardia and cardiomegaly from those with normal heart size. moreover, it was found that a ctr over the cutoff of . , mesured during inspiratory phase is a good predictor for cardiomegaly. key words: cardiac, cardio-thoracic ratio, dog, x-ray. the canine cardiac conduction system is modified by anatomical and functional adaptations of the maternal heart during gestation. however, it is not clear if these changes persist or are modified after parturition. therefore, the aim of this study was to describe canine electrocardiographic features during the course of normal puerperium. twenty healthy pure-bred, - ( . ae . ) year-old, weighing . - kg ( . ae . ) bitches were included in this study. all the animals whelped healthy puppies at term which were weaned on day after parturition (day ). all the dogs were electrochardiographically evaluated on days À , , , , , , and . mean electrical axis (mea; degrees), p wave amplitude (pa; mv) and duration (pd; ms), p-r interval (pr; ms), qrs complex amplitude (qrsa; mv) and duration (qrsd; ms), q-t interval (qt; ms), and s-t segment (st; mv) were calculated at mm/s of velocity. the rr interval immediately preceding each complex was recorded and qt interval was corrected (qtc) by van de water formula [qtc = qt- . (rr- )]. later, lead ii was recorded at mm/sec to analyze heart rate (hr; bpm) and cardiac rhythm (cr; normal sinus rhythm or sinus arrythmia). values of hr, mea, pa, pd, pr, qrsa, qrsd, qt, rr and qtc were analyzed by anova for repeated measures followed by tukey test. cardiac rhythm was analyzed by chi square test (spss . , spss inc. chicago, il, usa). p < . was considered significant. during the study period, hr (p < . ) and qtc (p < . ) progressively decreased, while rr (p < . ) and pa increased (p < . ). qrs complex amplitude diminished in the second week after parturition and then increased during the following weeks (p < . ). mean electrical axis shifted to the right during this period (p < . ). on day À , most of the bitches presented normal sinus rhythm in contrast with day , in which most of the bitches presented sinus arrhythmia (p < . ). from day onward, all the bitches showed sinus arrhythmia. p wave duration, pr, qrsd, qt and st remained unchanged during puerperium. it is concluded that most electrophysiological adaptive changes of canine gestation reverted during normal puerperium. the pre-sent study contributes to the understanding of canine cardiac physiology during this reproductive stage. disclosures: no disclosures to report. esvc-p- echocardiographic assessment of pregnant queens. p.g. blanco, r. rodr ıguez, a. carranza, a. rube, r. vercellini, p.r. batista, m. t ortora, c. gobello. national university of la plata, la plata, argentina cardiovascular adaptation during gestation guarantees an appropriate development of the fetuses and maternal cardiovascular maladaptation is highly correlated with adverse pregnancy outcome. while, the hemodynamic changes occurring during canine pregnancy have been described there is scarce information concerning maternal cardiac variations during feline gestation. thus, the aim of this study was to describe cardiac morphology and systolic function variations during normal feline pregnancy. eighteen pregnant queens were echocardiographically evaluated (toshiba nemio xg, japan, mhz transducer) every days from day (defined as day of mating) to parturition. left ventricular dimensions were measured in the short axis view, during mmode tracing. shortening fraction was calculated as (lvdd -lvds)/lvdd x to assess systolic function. stroke volume (ml) was calculated as the product of the velocity time integral (measured by pulsed-wave doppler) and the cross-sectional area of the aorta. cardiac output (l/min) was calculated as the product of stroke volume and heart rate (bpm) derived from electrocardiographic monitoring. uterine artery resistance index (ri) was obtained by doppler ultrasound. all the parameters were analyzed by repeated measures anova. all the queens delivered healthy kittens at term. throughout the study period, interventricular septum in diastole (p < . ) and systole (p < . ) and left ventricular diameter in diastole (p < . ) augmented during gestation. shortening fraction (p < . ), cardiac output (p < . ) and maternal heart rate (p < . ) also increased up to parturition. conversely, uterine artery resistance index decreased in the same period (p < . ). it is concluded that cardiac structure and function varied during normal pregnancy in these queens. cardiac eccentric hypertrophy, systolic function and cardiac output increases appear to be the consequences of the hemodynamic modifications occurring during pregnancy. the assessment of maternal cardiovascular function may prove a useful screening tool to detect pregnancy complications in feline reproduction. disclosures: no disclosures to report. tricuspid annular plane systolic excursion (tapse) is an echocardiographic measure that allows to assess right ventricular systolic function. it has been described reference values for tapse in normal adult dogs, but there is no reference to influence of age in tapse in dogs. this influence has been reported in humans. thus, the goal of this study is to determine the reproducibility of the measure tapse in normal dogs and to determine the relationship between tapse and age in healthy beagle dogs. tapse was measured from an m-mode recording of the lateral aspect of the tricuspid valve annulus obtained through a left parasternal apical -chamber view. tapse values were averaged from measurements on consecutive beats during sinus rhythm. the measurements were recorded by different persons (c-v, a.; m-m f.) with different grade of experience in canine echocardiography studies.. all patients had a complete -dimensional and doppler study using an envisor chd (philips Ò ) ultrasound system. twenty-three healthy beagles were used. the study was approved by the ethical committee of veterinary medicine service of las palmas de gran canaria university (spain) and it was carried out in accordance with the current european legislation on animal protection. these dogs were divided in different groups according to age: group included dogs under years, group included dogs between and years, group included eight dogs older than years. we analyzed differences between groups using non-parametric (kruskal wallis and wilcoxon scores (rank sums)) test. there were no differences with respect to sex. dogs in group presented higher tapse values than group or ( . ae . cm versus . ae . cm versus . ae . cm; p < . ). statistic intra-observer and inter-observer agreement using the intraclass correlation coefficient was . (p < . ). this study showed that tapse measurement is easily obtainable with a standard echocardiography system, and has adequate interobserver agreement. this study showed higher values of tapse in normal young dogs with respect to older dogs. these results are similar to the results obtained in humans, and could reflect a less effective right ventricle with age. the values presented should be taken with caution due to the relatively small number of patients included. it may also be necessary to validate results in future studies with a second independent sample of dogs of other races. disclosures: no disclosures to report. tetralogy of fallot (tof) is a congenital heart disease characterized by abnormalities, i.e., pulmonic stenosis, ventricular septal defect (vsd), aortic overriding and secondary right ventricular hypertrophy, caused by anterior deviation and abnormal septation of the conal septum during the embryonic period. few studies have reported the hemodynamic consequences and clinical outcome of tof in small animals. the objective of this retrospective study was therefore to document the epidemiological, clinical, echo-doppler findings, and survival, in a canine and feline population with tof. the case records of animals diagnosed with tof by combined use of echocardiography and doppler examination were reviewed ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . tetralogy of fallot was identified in animals ( dogs, cats). the most commonly represented breeds were terriers for dogs ( / , . %), and domestic shorthair for cats ( / , . %). most included animals ( / , . %) were clinically affected at the time of diagnosis. pulmonic stenosis was characterized by a variable systolic doppler-derived pressure gradient both in dogs (median [range] mmhg ) and cats ( mmhg ), and associated with hypoplasia of the pulmonary trunk in one third of the cases ( . %). most vsd were large, with a median vsd:aorta ratio of . [ . - . ] in dogs and . [ . - . ] in cats. median age at death from cardiac cause was . months [ . - . ] without significant difference between dogs and cats (p = . ). these results suggest that in both cats and dogs tof-related death occurs predominantly in young adult animals with major hemodynamic consequences at the time of diagnosis. disclosures: no disclosures to report. the aim of this study was to assess whether and how radiographic and echocardiographic cardiovascular variables differ across age bands of healthy cats. a cohort of clinically healthy cats were categorized into groups: adolescent-adult ( . - years; n = ), middle-aged ( - years; n = ), and geriatric ( - years; n = ). all cats underwent a full physical examination, a complete blood count, routine biochemical profile, a baseline serum total thyroxine concentration, auscultation, non-invasive blood pressure measurements, thoracic radiography, electrocardiography, and echocardiography. cats with hypertension, hyperthyroidism, cardiac, or renal disease were excluded from the study. body weight, body condition score, systolic blood pressure, heart rate, and all echocardiographic indices were similar across the groups. the mean (ae standard deviation [sd]) vertebral heart scale (vhs) value obtained for the geriatric group ( . ae . ) was significantly greater than that obtained for the adolescent-adult group ( . ae . ; p = . ). the mean ratio of the distance between the cardiac base and dorsal sternum to thoracic cavity height at the point of the cardiac base was significantly less in the middle-aged ( . ae . ) and geriatric ( . ae . ) groups than in the adolescent-adult group ( . ae . ; both p < . ). the mean angle between the cardiac long axis and the body axis was significantly smaller in middle-aged ( . ae . °) and geriatric cats ( . ae . °) than in adolescent-adult cats ( . ae . °; both p < . ). the mean angle between the cardiac long axis and the sternum of middle-aged ( . ae . °) and geriatric cats ( . ae . °) was significantly smaller than that in adolescentadult cats ( . ae . °; p = . and p = . , respectively). additionally, the degree of undulation of the thoracic aorta correlated positively with age (r = . , p = . ). these findings suggest that differences in the horizontal alignment of the heart, thoracic-aorta undulation, and vhs in healthy geriatric cats, relative to observations in younger cats, can be considered to be agerelated. disclosures: no disclosures to report. the aim of this study was to investigate the presence of pulmonary hypertension (ph) in young cats affected by single or mixed lungworm infections. twenty-three cats infected with lungworms were examined at the veterinary teaching hospital of teramo, italy, in - . animals underwent to a complete physical examination and to -or -views radiographic analysis of the thorax. a minimum database (i.e. cbc, serum biochemistry, serology for fiv antibody and felv antigen) was obtained for each patient. nine cats were excluded for concomitant diseases, while cats were included in the study. microscopic identification of parasites was confirmed by molecular tests and all cats received an anthelmintic treatment. a single infection by aelurostrongylus abstrusus was diagnosed in cats, while cats had a troglostrongylus brevior infection either alone or in combination with a. abstrusus. transthoracic echocardiography was performed using an ultrasound unit with a mhz phased array transducer. no structural abnormalities of the tricuspid valve and sign of pulmonary stenosis were detected. the -dimensional and m-mode echocardiography showed a cardiac involvement in cats. one cat, infected by a. abstrusus and t. brevior showed a mild systolic tricuspid regurgitant jet with color doppler of . m/sec, while another a. abstrusus-infected cat, had mild tr of . m/sec with a mean paps of mmhg which resolved within weeks after therapy. one cat diagnosed with troglostrongylosis, showed a marked right-sided cardiac enlargement of mm, and a large systolic tricuspid regurgitant jet with a tr peak velocity of . m/s recorded at continuous-wave doppler via a color doppler echocardiography. the minimum pressure difference between the right ventricle and the right atrium was estimated mmhg and the paps was at least mmhg. the echocardiographic and doppler evidence of mild ph persisted at further examination performed until months after diagnosis. ph is rare in cats, despite cases of reversible ph are known in cat aelurostrongylosis. in this study the first case of irreversible ph infection in a cat affected by t. brevior is presented and this finding further supports the high pathogenicity of troglostrongylosis, especially in young patients. in cats with lungworm infection, possible cardiovascular complications must be taken into account and these infections should be always considered in the differential diagnosis in cats with cardiorespiratory signs. disclosures: no disclosures to report. although uncommonly assessed in veterinary cardiology, a right ventricular (rv) function has been shown to be an important prognostic determinant of many congenital and acquired heart diseases in human patients. our group has already demonstrated that -dimensional ( d) color tissue doppler imaging provides a noninvasive evaluation of systolic and diastolic rv function in the awake dog with adequate repeatability and reproducibility. b however, other noninvasive ultrasound imaging variables reflecting rv function need to be further investigated, particularly in correlation with pulmonary arterial pressure (pap) values and left ventricular (lv) function. the aim of this prospective study was therefore to assess several indices of systolic and diastolic rv function using conventional echocardiography and speckle tracking echocardiography (ste) in healthy awake dogs of different breeds with documented systolic pap (spap) and lv function (lv ejection fraction and global lv systolic strain assessed using the simpson's derived method of disks and ste, respectively). imaging rv tested variables included tricuspid annular plane systolic excursion (tapse), right fractional area change (rfac, %), ste longitudinal systolic strain of the rv free wall (rvfw, %) and of the whole rv (i.e., global rv strain, %), ste longitudinal systolic strain rate (sr, s À ) and diastolic early:late sr ratio. additionally, d-guided m-mode ventricular measurements included the end-diastolic rv:lv diameter ratio (rvdd: lvdd) and the end-systolic rvfw:lvfw ratio. correlations between imaging variables were calculated by using spearman's correlation coefficients. means of age and body weight (ae sd; range) of the study population were . years (ae . ; . - . ) and . kg (ae . ; . - . ), respectively. no correlations were found between rv morphological variables (i.e., rvdd:lvdd and rvfw:lvfw ratios) and all indices of systolic and diastolic rv function. global rv strain (meanaesd = . ae . %) and rvfw strain ( . ae . %) were positively correlated (p < . ) with rfac ( . ae . %, r = . and r = . , respectively), and negatively correlated (p < . ) with spap ( . ae . mmhg [ . - . ], r = À . and r = À . , respectively). spap was also negatively correlated with the tapse:body weight ratio and systolic sr (r = À . and À . respectively, p < . ). there was no correlation between indices of lv function and ste indices of rv function, and no correlation either between ste rv indices of systolic function and the diastolic early:late sr ratio. in conclusion, ste provides a rapid and non-invasive evaluation of rv function that may be used for clinical investigations in canine cardiology. doppler-derived +dp/dt and -dp/dt from mitral regurgitation are considered indexes for assessment of systolic and diastolicfunction respectively, that have less load dependence than the ejection phase indexes. this study aimed to determine correlation between doppler-deriveddp/dt and other systolic and diastolic echocardiographic indexes, and if they can be used to identify dogs with and without remodeling, with or without congestive heart failure (chf) and for evaluation of chronic mitral valve disease (cmvd) severity. fifty-seven dogs with cmvd (stages b , b , c+d) were included prospectively in an observational cross-sectional clinical study and distributed in groups regarding the presence of remodeling and chf, to evaluate+dp/dt and -dp/dt, and distributed according to tdi-diastolic pattern tocompare -dp/dt. group c+d ( mmhg/s, p -p = - )had +dp/dt significantly lower compared to b ( mmhg/s, p -p = - )and b ( mmhg/s, p -p = - ) (p = . ). groupc+d also had lower -dp/dt, compared to b ( . mmhg/sae . and mmhg/sae . ; p = . ). dogs with chf compared to those without chf, presented lower +dp/dt( mmhg/s, p -p = - ; mmhg/s, p -p = - ; p = . ) and -dp/dt ( . mmhg/sae . ; mmhg/sae . ; p = . ). regardingdiastolic function, -dp/dt was lower for the restrictive pattern group ( . mmhg/sae . )compared to those without diastolic dysfunction, ( mmhgae . ), delayedrelaxation ( mmhgae . ) and pseudonormal patterns ( mmhgae . ) (p < . ).when +dp/dt< mmhg/s, the post-test chance for the dog with cmvd to havechf is twice the chance than not having it. for -dp/dt< mmhg/s theposttest chance of having chf is times higher than not having it. in conclusion, doppler-derived +dp/dt and-dp/dt may contribute respectively, for systolic and diastolic assessment ofdogs with cmvd. disclosures: no disclosures to report. pulmonic stenosis (ps) is one of the most common congenital heart defects seen in veterinarycardiology practice. pulmonary balloon valvuloplasty (pbv) is considered to be the treatment of choice for dogs withsevere stenosis. whether dogs with moderate stenosis benefit from pbv remains unclear, and variables such as degree of hypertrophy, valve morphology, amount of tricuspid insufficiency and presence or absence of clinical signs aregenerally used when recommendations are made to pet owners. in this study we report the effect of valve type on pbv outcome in dogs treated at different academic speciality cardiology practices. baseline echocardiographic images were evaluated at each institution and valve morphology was classified as either type a (n = , . mmhg, range - ) or type b (n = , . mmhg, range - ) and 'no' (n = , mmhg, range - ) or 'yes' (n = , mmhg, range - ) for presence of pulmonary annular hypoplasia when diameter was compared to aortic annulus. twenty four hours following pbv both type a ( mmhg, range - ) and type b ( mmhg, range - ) valves had significant reduction in gradient compared to baseline (p < . ). this reduction remained significant at days (a: mmhg, range - ; b: mmhg, range - ; p < . for both). dogs with annular hypoplasia ( mmhg, range - ) and without annular hypoplasia ( mmhg range - ) had a significant reduction in gradient hours post pbv. it remained significant at days (with annular hypoplasia: mmhg, range - ; without annular hypoplasia: mmhg, range - ; p < . for both). when comparing to baseline, considering valve type, there was no significant difference in percent reduction in gradient for type a versus type b valves at both the -hour (a: %, range - ; b: %, range - ; p = . ) and -day (a: %, range - ; b: %, range - ; p = . ) recheck evaluation time points. additionally, there was no significant difference in gradient reduction when looking only at whether or not there was annular hypoplasia at hours (yes: %, range - ; no: %, range - ; p = . ) and days (yes: %, range - ; no: %, range - ; p = . ). in conclusion, classification of dogs with ps according to valve type and annulus morphology did not help predict the -day response to pbv. disclosures: no disclosures to report. hypertrophic cardiomyopathy (hcm) is the most common feline inherited cardiac disease and it is a major cause of morbidity and mortality. the osservatorio italiano hcm felina was formed in by a network of clinicians, geneticists and breeders, to monitor and study hcm in italian cats. since april , adult cats, belonging to various breeds, including maine coon, siberian, norwegian forest cats, ragdoll, sphynx, british sh, birmans and others have been prospectively enrolled. recheck evaluations were performed in cats. each cat underwent a clinical examination, echocardiography, and blood collection for genetic testing (when appropriate) and storage in the italian feline bio-bank. the disease status was defined by echocardiography according to established guidelines (left ventricular diastolic wall thickness < . mm = hcm negative, = . but < mm = hcm equivocal; = mm = hcm positive). the prevalence of hcm in the population was % ( cats); equivocal diagnoses were conferred on % ( cats). these prevalences did not differ between breeds. the prevalence of hcm in the italian feline population was lower compared to those reported by other investigators. evaluation of data from the entire population demonstrated that left ventricular end-diastolic wall thicknesses and aortic diameter showed a weak positive correlation with body weight (p < . , r < . for all variables), suggesting that weight-dependent limits on wall thickness should be considered in cats as is currently practiced in dogs. the lower prevalence of hcm in italian cat breeds compared with those examined elsewhere might be explained by different criteria for determining presence or absence of disease, differences in ages at which the subjects were examined, or a selection bias by breeders in presenting cats they consider 'normal'. disclosures: no disclosures to report. chronic mitral valve disease is by far the most common cardiovascular disease in dogs. the disease is caused by myxomatous degeneration of the mitral valve leaflets and, in approximately % of cases, it's accompanied by degeneration of the tricuspid valve. it is also described in previous studies that approximately % of affected dogs also have evidence of associated pulmonary arterial hypertension. the prevalence of the disease is higher in small breed dogs (under kg), although large breeds can also be affected and it occurs more frequently in males than in females. the present study aims to characterize the disease in a population of dogs in portugal. we retrospectively reviewed the medical records of dogs presented to hospital veterin ario do porto, with an echocardiographic diagnosis of canine chronic mitral valve disease, during a period of years. from this records, cases were identified, from which ( . %) were males and ( . %) were females. most of the dogs were mixed breed ( ) and different breeds of dogs were represented. the poodle was by far the most represented breed (n = ; . %), followed by english cocker spaniel ( . %), yorkshire terrier ( . %), boxer ( . %), epagneul breton ( . %), dalmatian ( . %), pekingese ( . %), labrador retriever ( %) and portuguese podengo ( . %). all other breeds represented . % of the population. regarding weight, . % of the dogs (n = ) weighted < kg, with a mean body weight of . kg (range . - kg). the mean age at diagnosis was . years old. we also observed that . % of the dogs (n = ) had concomitant degeneration of the tricuspid valve and . % (n = ) pulmonary arterial hypertension (ph). we categorized these dogs according to the severity of ph, in mild ph if they had a doppler echocardiography derived systolic pulmonary arterial pressure (spap) of - mm/hg, moderate ph (spap - mm/hg) and severe ph (spap > mm/hg). we found that . % (n = ) of dogs had mild ph; . % (n = ) moderate ph and . % (n = ) severe ph. as described in previous studies, the disease affects mainly males and small breed dogs, with a breed distribution that reflects the canine population in the country, including very including very popular large breed dogs in portugal, as the boxer and labrador. both the presence of concomitant tricuspid valve disease and ph had a higher prevalence in our study than previously described. disclosures: no disclosures to report. in people anemia is frequent in patients with heart failure (hf) and it is associated with poor outcomes. the most likely pathogenic factors include iron deficiency, chronic kidney disease (ckd), and cytokine production, although other factors may contribute. little is known about the prevalence of anemia in dog with cardiovascular disease. the aim of this retrospective study was to define the prevalence of anemia (hct ≤ %) in dogs with mitral valve disease (mvd) and to investigate associated risk factors (age, weight, azotemia, hf, iris/acvim class). medical records of dogs presented at the cardiology service, divet, university of milan (january -march ) were retrospectively evaluated. dogs with mvd with complete physical, thoracic and echocardiographic examinations, and serum biochemical panel, including serum creatinine (scr), were included in the study. dogs with other heart or systemic diseases, except ckd, or neoplasm were excluded. statistical analysis was performed using jmp . (sas institute). a p value < . was considered significant. two hundred and ninety dogs ( males/ females), . ae . years of age, . ae . kg of body weight fulfilled the inclusion criteria. the % of males and the % of females were neutered. the most represented breeds were mongrel ( %), miniature poodle ( %), york shire terrier ( %), and cavalier king charles ( %). dogs were % b , % b , % c and % d acvim class. while the % of the dogs were normoazotemic (scr < . mg/dl), . % were staged in iris , % in iris and . % in iris . the prevalence of anemia in dogs with mvd was % ( / ): showed mild ( ≤ hct ≤ %) and moderate ( ≤ hct ≤ %) anemia. sixteen dogs were in b , in b , in c and in d acvim class; were normoazotemic ( %). anemic dogs showed a significant higher scr. normoazotemic dog showed significant higher hb, hct and rbc both in the overall population and in the anemic group. in the overall population dogs in different iris class showed statistically different hb, hct and rbc and hb was significantly lower in decompensated hf dogs. in conclusion although a relationship between anemia and azotemia/ckd was documented in our study, it is important to emphasize that most of the anemic dog were normoazotemic: anemia is not an exclusive finding of cardiorenal syndrome and should be considered as possible complication in dogs with mvd alone. disclosures: no disclosures to report. the objective of this study was to evaluate left atrial (la) function by left atrial total fractional area change (la-factotal) and left atrial ejection fraction (laef) in dogs affected with chronic mitral valve disease (cmvd) naturally acquired with and without congestive heart failure (chf). our hypothesis was that la-factotal and laef decrease with severity of cmvd. eighty dogs were included in a prospective observational cross-section clinical study, grouped according to cmvd severity based on echocardiographic evaluation and clinical signs. the dogs were equally distributed in each group: a, b , b and c, according to american college of veterinary internal medicine staging system. indicators of la function were calculated with the following equations: la-factotal = (lamaximum area -laminimum area)/lamaximum area, measured by apical view; and laef = x (lamaximum volume -laminimum volume)/lamaximum volume, by biplane area-length method from the left apical and chamber views. la-factotal showed lower values (p < . ) in group c ( . %, p - % = . - . ) compared with groups a ( . %, p - % = . - . ), b ( . %, p - % = . - . ) and b ( . %, p - % = . - ). group c had lower laef ( . %, p - % = . - . ) than groups a ( . %, p - % = . - . ), b ( . %, p - % = . - . ) and b ( . %, p - % = . - ) (p < . ). left atrial function, assessed by la-factotal and laef, was reduced in dogs with cmvd and chf compared with healthy and asymptomatic cmvd groups. disclosures: no disclosures to report. recurrent episodes of heart and/or kidney failure are considered one of the causes leading to worsening heart/renal functions in human patients. the aim of this prospective study was to assess the influence of heart/kidney worsening on elected parameters of heart/kidney function in dogs affected by mitral valve disease (mvd). between july and may , dogs affected by mvd in acvim class b and without comorbidities were included in the study group. the control group was constituted by healthy dogs, matched with the cases for age (older than years) and gender. all the dogs underwent physical examination, thorax radiography, ecg, echocardiography, systemic blood pressure assessment, a complete blood count, serum biochemical analysis, including assessment of serum creatinine (scr), serum urea nitrogen (urea) and glycaemia (gly) and urine analysis with urine protein/creatinine ratio (upc). dogs were re-evaluated every -month until october . statistical analysis was performed using ibm spss statistics (p value significant if < . ). twenty-one dogs affected by mvd (cases) were included and healthy dogs (controls) were randomly selected among the eligible population. the % of cases experienced at least one episode of congestive heart failure (chf), but none of these patients developed chronic kidney disease (ckd). the % of cases developed ckd while remaining in acvim class b . no dogs in the control group developed ckd or mvd. correlations between worsening renal function (wrf -scr elevation ≥ . mg/dl or % from baseline), furosemide administration, upc levels, radiographic parameters of heart enlargement and echocardiographic parameter were investigated. only a statistically significant difference in iris class between the groups according to wrf and in the echocardiographic parameter left atrium to aortic root (la/ao) according to furosemide amount were observed. both these results were expected. none of the cases included experienced renal damage (wrf or iris class change or upc change) concomitant to episodes of chf. the persistence of normal renal condition regardless of chf events and therapy administration was unexpected. in conclusion, experiencing chf seems not to directly affect renal function. to authors' opinion, the use of wrf, better than single scr and urea levels, may be useful in the long term management of aged patients affected by mvd. however, the small number of cases included in this study represents a great limit. we consider this work a pilot study. disclosures: no disclosures to report. hypertrophic cardiomyopathy (hcm) is a primary myocardial disease characterized by inappropriate thickening of the myocardium in absence of other causes of hypertrophy including hypertension, hyperthyroidism, aortic stenosis and acromegaly. it is also the most common heart disease in cats. hcm presents a wide variety of clinical sings depending on the severity and location of the hypertrophy. cats affected with hcm have a mean age of . - . years old at the time of the diagnosis however this disease can affect cats as young as months although this later age is unusual hcm is a heterogeneous disease both in terms of phenotypic degree of hypertrophy and clinical outcome. hallmark histopathological hallmarks lesions of hcm are myocyte disarray, small coronary arteriosclerosis and interstitial fibrosis replacement in order to confirm hcm echocardiography has to be made. primary hypertrophy diagnosis is made based on the presence of ventricular hypertrophy, symmetric or asymmetric, in the absence of systemic disorders. the purpose of this study was to assess the prevalence of hcm in a feline population. in order to achieve this goal echocardiograms were made in all cats older of years clinically asymptomatic with or without cardiac murmur. all echocardiograms were made according to the guidelines of the acvim published in . diagnosis of ventricular hypertrophy was made from the right parasternal window using the b mode to measure the diameter of the lvfw and the ivs in diastole. cats with more than mm of wall thickness measured t , bun, crea, blood pressure. only cats within the normal limits of the later parameters were considered hcm positive. total number of cats in this study was cats male and female. from this population had no defined breed, were persian, maine coon, norwegian woods, siamese, chartreaux. no murmour was detected in ( . %) cats, s or s was detected in ( %) cats and differente degree of murmour was detected in ( . %) cats. hypertrophy was detected in cats. from this cats ( . %) were diagnosed as hcm, ( . %) cats were excluded either because of lack of values of t and or because they had high values of blood pressure, t levels or crea. in this study . % of the population had hcm. the epidemiological and phenotype distribution is highly variable. the average age at diagnosis of hcm in this study was . years. disclosures: no disclosures to report. mildly increased concentrations of crp are associated with cardiovascular disease in humans and dogs. it is not known whether increased concentrations of crp are associated with myxomatous mitral valve disease (mmvd) in dogs, or rather its sequel, congestive heart failure (chf). the aim of this study was to investigate whether serum concentrations of crp, determined using a novel automated canine-specific high-sensitivity crp assay (gentian hscrp), were associated with severity of mmvd and certain clinical variables in dogs. the study included client-owned dogs with different severities of mmvd. disease severity was determined by medical history, physical examination, echocardiography and response to diuretic therapy. dogs were allocated into groups based on acvim consensus statement guidelines (group a (n = ), group b (n = ), group b (n = ) and group c (n = )). data were analysed using descriptive statistics and multiple regression analysis. dogs with chf (group c) had significantly higher serum crp concentrations ( . mg/l, [ . ; . ]) (median, [quartile ; quartile ]) compared to dogs in groups a ( . mg/l, [< . ; . ]) (p = . ), b ( . mg/l, [< . ; . ]) (p < . ) and b ( . mg/l, [< . ; . ]) (p < . ). other measures of disease severity including left atrial to aortic root ratio and left ventricular end-diastolic diameter normalized for body weight were positively correlated with serum crp concentration. in conclusion, slightly higher serum crp concentrations were found in dogs with chf whereas the severity of asymptomatic mmvd showed limited association with serum crp concentrations. disclosures: no disclosures to report. medetomidine is a a -agonist widely employed for sedation in dogs but its use is discouraged in cardiac patients even those suffering from myxomatous mitral valve disease (mmvd). however, only one investigation was previously conducted in a wide rangeregarding the class of the disease -of mmvd patients, reporting a general safety of that protocol. the present study was focused just on class b of mmvd, with the aim to provide more detailed information on the cardiovascular effects of medetomidine in such patients, by the analysis of clinical and instrumental parameters suggestive of disease severity or congestive heart failure. dogs weighing < kg, needing a soft clinical procedure and showing a systolic apical heart murmur were screened and selected for the study if la/ao < . . the sedative protocol consisted in an iv injection of lg/kg medetomidine antagonized, after the clinical procedure, by an im injection of the recommended dose of atipamezole. clinical parameters, echocardiographic variables, thoracic radiographs and oscillometric blood pressure measurements were collected at baseline (t ), minutes after medetomidine administration (t ) and hours after atipamezole injection (t ). of dogs screened, were definitively enrolled. at t a significative decrease in the right parasternal regurgitant jet area (rp-arj/laa), peak velocity of mitral regurgitation and shortening fraction was observed along with an increase in lvids (p < . ). left parasternal arj/laa decreased without reaching statistical significance but showing a high correlation with rp-arj/laa (r = . ). interestingly, la/ao changed only mildly and never reached a value > . . the other echocardiographic variables did not show a particular trend. systolic blood pressure showed values at the upper physiologic limit at t , lower values than t at t , and an increase above the initial value at t but without significance. thoracic radiographs were evocative of heart enlargement without pulmonary venous congestion or pulmonary oedema both at t and t . respiratory rate did not change between t and t . the degree of sedation was optimal during the clinical procedure in all cases. sedation with lg/kg medetomidine is safe in dogs suffering from mmvd in stage b (la/ao < . ). the decrease observed in peak velocity and color-doppler appearance of mitral regurgitation at t could be due to a reduction of both myocardial contractility and systolic blood pressure, by a lowering of sympathetic activity via baroreceptors stimulation. disclosures: no disclosures to report. in this retrospective study were included a total of clientowned dogs, undergoing transarterial occlusion of pda with mreye Ò flipper detachable embolization coil (n = ), amplatz â canine duct occluder (acdo; n = ) and amplatzer â vascular plug (n = ). device size selection was based on pda dimensions assessed by transesophageal echocardiography (tee) in cases and transthoracic echocardiography (tte) in cases. angiography was performed during the procedure to assess the success of the occlusion, and it confirmed complete occlusion in dogs and a trivial residual flow in dogs. the following day, transthoracic color-doppler echocardiography revealed that complete ductal closure was achieved in all dogs. the procedure was hemodynamically successful, as evidenced, by a reduction in indexed left ventricular internal diameter in diastole (lvidd; p < . ), fractional shortening (fs; p < . ) and left atrial to aortic ratio (la: ao; p < . ) within hours after procedure. four months after surgery, indexed lvidd was significantly reduced (p = . ) and la:ao remained constant. secondary complications included pulmonary arterial embolization of an acdo and a late rotation of an amplatzer â vascular plug resulting in an increased flow through the pda. the dog with the rotated device required subsequent surgical ligation of the pda. at this time, dogs were reported to be alive and the other dogs were lost to follow up. only one dog remained on congestive heart failure therapy after the pda occlusion. we can conclude that pda occlusion using an acdo for dogs with more than kg and a transarterial coil embolization for dogs with < kg had a high rate of immediate complete occlusion. pda occlusion using those devices proved to be a safe and effective therapeutic method for pda in dogs. disclosures: no disclosures to report. echocardiographic evaluation of the right pulmonary artery distensibility index (rpad index) was recently described as a valuable method for early detection and severity evaluation of pulmonary arterial hypertension in dogs. rpad index is calculated as the percentage change in diameter of the right pulmonary artery (rpa) between systole and diastole, obtained by m-mode echocardiography from the right parasternal long axis view. the aim of this study was to compare the rpad index obtained by different echocardiographic views in dogs. the study design was a prospective, multicenter, observational study. forty-five clientowned dogs from different breeds were included: dogs with heart disease and healthy dogs. two different right parasternal views, long axis (rpla) and short axis (rpsa), were used to measure the rpad index. from the rpla view (method ) and rpsa view (method ) a short axis and a long axis image were respectively optimized for the right pulmonary artery. the rpad index was calculated by m-mode as the percentage change in diameter of the right pulmonary artery: [(systolic diameter -diastolic diameter)/ systolic diameter]* . measurements were done off-line as an average of consecutive cardiac cycles by a single investigator blinded to the dogs' diagnosis. a pearson and a bland-altman test were used to assess correlation and agreement between the methods, respectively. intra-and inter-observer measurement variability was quantified by average coefficient of variation (cv). level of significance was set at p < . . m-mode evaluation of the rpad index was satisfactorily obtained by both methods in all dogs. pearson test showed a strong positive linear correlation between the values of rpad index obtained from both methods (r = . , p < . ). bland-altman test showed a good agreement between the methods in estimating rpad index (bias = . %, sd = . %, % limits of agreement = À . , . %). the mean difference between the methods was . % ( % confidence interval = À . ; . ). intra-and inter-observer measurement variability was clinically acceptable (cv< %).the study showed a good agreement between short axis and long axis m-mode evaluation of rpa. both methods can be used interchangeably to evaluate rpad index. further studies are needed to evaluate the rpad index in a larger population of healthy dogs and the diagnostic and prognostic role of this echocardiographic parameter in dogs with different types of pulmonary hypertension. disclosures: no disclosures to report. this retrospective study (march to october ) includes client-owned great pyrenees diagnosed with hypoadrenocorticism. the medical records of dogs with a diagnosis of naturally occurring hypoadrenocorticism were reviewed, with an emphasis on great pyrenees' record. the prevalence of hypoadrenocorticism in the studied population, as well as the prevalence per breed, was calculated. data collected included breed, clinical signs, laboratory findings, age at diagnosis, treatment, and cause of the death. one hundred dogs were diagnosed with naturally occurring hypoadrenocorticism, representing . % of the overall canine population studied. thirty-five breeds were represented, with a prevalence per breed varying between . % and . %. a high prevalence was observed in west highland white terriers ( . %), great danes ( . %), standard poodles ( . %), saint-bernards ( . %) and jack russell terriers ( . %). out of gp presented during that period of time, . % (n = ) were diagnosed with hypoadrenocorticism. median age at diagnosis was . years (range: . to . ) in dogs with hypoadrenocorticism, and . years ( . to . ) in gp. the main reason for presentation of the addisonian gp was lethargy (n = ) and anorexia (n = ). clinical findings included hypotension (n = ), poor body condition (n = ), and heart murmur (n = ). the majority (n = ) had serum electrolytes abnormalities, with a na:k ratio ranging from . to . . other major laboratory findings included azotemia (n = ), anemia (n = ) and the absence of a stress leukogram (n = ). the majority (n = ) received fludrocortisone, with prednisone as needed. one gp was euthanized at time of diagnosis. great pyrenees diagnosed with hypoadrenocortism were overrepresented in the studied population, with a prevalence of hypoadrenocorticism in our gp population of . %. therefore, an inherited susceptibility can be suspected. reason for presentation and clinical signs were nonspecific, and similar to what is reported in other breed. in human medicine, haemoglobin a c (hba c), a form of glycosylated haemoglobin, is used as the standard measure of average glycaemic control over to months. the measurement of hba c in dogs has been previously demonstrated however high pressure liquid chromatography techniques are too technically difficult for routine use and other methods are no longer available. the objective of this study was to assess the use of latex immunoagglutination inhibition using a monoclonal antibody for the measurement of hba c in dogs, using the siemens dca vantage Ò . repeatability was assessed by measuring samples times within minutes. the effect of storage on edta anticoagulated samples was examined by measuring samples stored at °c every day for up to days. storage was further assessed by freezing samples and measuring them at , and weeks. the machine was then used to compare the hba c values in groups of dogs with diabetes mellitus (group , n = ), hyperadrenocorticism (group , n = ) or non-diabetic/cushingoid hospitalised patients (group , n = ). differences in the groups were examined for significance using a kruskal-wallis analysis of variance. the reference range of hba c has been previously calculated to be . - . % ( - mmol/mol) and values of . -> % ( -> mmol/mol) are seen in diabetic animals using high pressure liquid chromatography. the median coefficient of variation for the repeatability study was found to be % (range % to %). it was possible to store samples at °c for up to days (median cv% = %, range % to %) and at À °c for at least weeks (median cv% = %, range % to %). the median hba c concentrations were group ; . % ( mmol/mol), group ; . % ( . mmol/mol) and group ; . % ( mmol/mol). group was significantly different from the other groups using kruskal-wallis analysis of variance. in conclusion, the latex immunoagglutination method was repeatable for the measurement of hba c in dogs. in addition, hba c in canine edta anticoagulated samples were stable at °c for up to days and, if frozen, could be stored for at least weeks without significant sample deterioration. the assay provides the expected results in dogs with and without abnormalities of glycaemic control. disclosures: the siemens dca vantage was provided on loan from siemens, as well as the cartridges used on this machine to run all samples in the study. a. wehner , r. anderson , s. reese , k. hartmann . clinic of small animal medicine, munich, germany, institute of veterinary anatomy, histology and embryology, munich, germany measurement of total thyroxine (t ) is often the first diagnostic step in the work up of thyroid disease in dogs and cats. blood samples are routinely sent to a reference laboratory causing a delay in testing which might impact the results. the aim of this study was to validate an enzyme fluorescence assay (elfa) as an inhouse method to measure t in dogs and cats. t was measured in sera of dogs and cats by methods, an enzyme immunoassay (eia) and an enzyme fluorescence assay (elfa). the eia served as the standard method to which the elfa results were compared. the elfa was performed with the minividas automated analyser (biom erieux, craponne, france) according to the manufactors instructions. coefficient of variation (cv) of the elfa in dogs sera was . % and of the eia - . %, respectively. cv of the elfa in cats sera was . - . % and of the eia . - . %, respectively. overall bias of the elfa in dogs was . %; however up to À . % in lower t ranges. maximal bias of the elfa in cats was . %. correlation of both methods was linear only in cats. using bland altman plots limits of agreement were - - % in dogs and - - % in cats. cohen s kappa revealed only slight agreement between both methods in dogs, but a good to very good agreement in cats. the elfa is a fast method with a high precision and can be recommended to measure t in cats, but cannot be recommended for dogs. disclosures: no disclosures to report. dysfunctions of the central nervous system (cns) are the most frequent causes of neurological disorders in dogs. our study aimed to find ( ) if some cns diseases could be associated with a selected group of common microbial or viral pathogens in dogs and ( ) if cns diseases have any characteristic profile with regard to parameters, c-reactive protein (crp) and iga, that are reported to be potentially useful but unspecific markers of cns diseases. we analysed cerebrospinal fluid samples obtained from dogs with varying neurological signs between june and november . real-time pcr was employed with probes for toxoplasma gondii, neospora caninum, anaplasma phagocytophilum and canine distemper virus. igg-antibodies to tickborne encephalitis virus (tbe) were assayed and iga titres were measured using elisa, while crp concentrations were determined by immunoturbidimetric assay. the dogs had a median age of years (range: . - ) and comprised breeds most frequently involving chihuahuas, labrador retrievers, bernese mountain dogs and boxers. gender distribution was . % female, . % spayed bitches, . % male, . % neutered male, and . % non-identified. none of the cases were pcr-positive for toxoplasma gondii or canine distemper virus. one dog was positive for anaplasma phagocytophilum and another for neospora caninum. antibodies to tbe virus were within the borderline range in / dogs. the dogs could be divided into age groups: (= . %) for young dogs (< years, median year) and (= . %) for older dogs (≥ years, median years). igahigh (> . mg/dl) cases represented % and % for young and older dogs, respectively. crp-high (> . mg/l) cases were almost half and equal: . % and . % in young and older dogs, respectively. compared with older dogs, young dogs had higher levels of crp (p = . ) and iga (p = . ). within the iga-high cohorts, crp-high and crp-low cases distributed almost equally ( . % versus . %) in young dogs but disproportionate ( . % versus . %) in older dogs. there were no [iga-low/crp-low] cases in young dogs but . % in older dogs. our present data suggest that ( ) canine cns disorders were largely characterized by high iga and particularly in young dogs ( ) inflammatory types (crp-high) were almost equal in both groups and ( ) although the significance remains yet to be determined, pathogens like anaplasma phagocytophilum and neospora caninum could be detected in a few cases of canine cns disorders. disclosures: the authors breu d and guthardt j are employed at laboklin gmbh & co kg, germany. m€ uller, e is owner/manager of the laboklin gmbh & co kg. internet is a potential source of medical information for pet owners. therefore, it could play an indirect but important role in the veterinary practice. this survey assesses the online search behaviour of french pet owners for their pets' health and its influence on a veterinary consultation. in april , french pet owners coming in a veterinary teaching hospital for a medical or a surgical consultation were surveyed. ( . %) owners fulfilled the questionnaire on a voluntary basis. the survey contained questions dealing with topics: the online search behaviour of owners for their pets' health, their perception of the information found online and the internet's influence on a consultation and on the veterinarian/client relationship. . % of owners use the internet to obtain information on their pets' health. among them, . % use it rarely and . % occasionally. they mainly look for information on a disease ( . %), a symptom ( . %), a breed ( %) or a nutritional advice ( . %). . % of owners try to verify the accuracy of the information found, most often by questioning their veterinarian ( . %). few owners ( . %) think that online information is always trustworthy. most of the research ( . %) is randomly made, websites being found through search engines. the majority of pet owners ( . %) aren't aware of any health certification label for websites. internet enables certain pet owners to feel more at ease with their pets' health care: they ask more questions to their veterinarian ( . %) and feel more involved in medical choices ( . %). . % of owners consider that the internet can positively impact their relationship with the veterinarian. relief is the most common ( . %) emotional response to online research for medical information. however, . % of owners feel overwhelmed by the amount of information found, % are confused and . % frightened by the serious or graphic nature of the information found online. this study emphasizes the frequent but measured use of the internet by french pet owners for their pets' health. they seem to consider information found on the net with a critical mind. unexpectedly, it appears that the internet could be an ally for veterinarians by promoting exchanges between the clients and the veterinarian and by improving compliance with the care project. disclosures: no disclosures to report. sinonasal aspergillosis is a well-known and described fungal infection of the sinonasal cavities in dogs. topical treatment either with enilconazole or itraconazole infusion administered surgically or endoscopically are effective. the use of % clotrimazole cream have been described in a surgical setting after itraconazole infusion by sissener et al. in . the aim of the work was to report the effectiveness of the use of topical % clotrimazole cream as the only treatment for sinonasal aspergillosis in dogs. inclusion criteria were a full medical record with radiological and endoscopical imaging, record of clotrimazole discharge after instillation and endoscopic control between and days after procedure. the % clotrimazole cream was applied through catheters placed under direct endoscopic vision after fungal plaques removal. nine dogs were included. three dogs were mixed breed dogs, dogs golden retriever, one dog a german shepherd and one old english sheepdog, one bull terrier and one cane corso. six dogs were male (one neutered) and female (one intact). mean age was . years. main clinical signs were muco-purulent discharge ( ), pain at sinonasal palpation ( ), nasal planum alterations ( ), epistaxis ( ) . nasal discharge was bilateral in dogs. mean duration of clinical signs was . months. main radiological findings were turbinates lysis ( ), frontal sinus empyema ( ), frontal bone thickening ( ) and frontal bone lysis ( ). rhinoscopy disclosed lysis and remodelling of the nasal turbinates ( ), easy access to the frontal sinus ( ), septum lysis ( ), bilateral sinonasal aspergillosis ( ), monolateral nasal aspergillosis ( ), monolateral sinonasal aspergillosis and contralateral nasal aspergillosis ( ), monolateral frontal aspergillosis ( ) . main duration of nasal cream discharge was . days. all dogs underwent endoscopic control between and days after the procedure. seven dogs were disease free; dogs had persistent fungal plaques and underwent a second treatment. success rate was . %. success rate of this study is comparable to other studies with larger and smaller case series. endoscopic removal of the fungal plaques can be time consuming and topical administration of either enilconazole or itraconazole require an additional hour. the catheter placement and the % clotrimazole cream application lasted minutes for each cavity in the dogs here reported. the use of % clotrimazole cream as the only treatment for sinonasal aspergillosis needs further evaluation on a larger case series. disclosures: no disclosures to report. few studies exist on euthanasia in small animal practice. however, such an act belongs to veterinary procedures, more or less frequently depending of the kind of practice, and may deeply impact both owners and veterinarians. we intended to study practical, ethical and psychological aspects of euthanasia of dogs and cats among french veterinary practitioners. from october to february , an on-line -item questionnaire on small animals' euthanasia, addressing practical aspects of euthanasia, communication with the owners, ethical problems, owners' and veterinarians' perceptions, was emailed via professional associations and networks. results were analyzed using commercial software (sphinx iq Ò and excel Ò ). french veterinarians practicing small animal medicine completed the questionnaire, representing > % of this population. euthanasia occurs rarely at home. over % of veterinarians propose the owners some time alone with their pet, and to stay during euthanasia, performed most commonly by intravenous injection ( . %) mainly after sedation/anesthesia ( . %). ninety nine percent of veterinarians consider communication, including description of events' sequence, and disposal of the body, as important. estimated minimum communication time required varies from - to - minutes. following euthanasia, . % are often thanked by the owners. most veterinarians (> %) have refused a euthanasia, considered unjustified, or had their own suggestion of euthanasia rejected. reasons for such a suggestion include intractable pain ( . %), non-acceptable complications ( . %), financial considerations ( . %) or animal considered dangerous ( . %). veterinarians think most owners ( . %) experience some sense of guilt during euthanasia. themselves perceive euthanasia most commonly as relief of animal's suffering ( . %) and part of veterinary practice ( %), less frequently as a defeat ( . %). almost all veterinarians have experienced emotionally challenging euthanasia, and . % estimate that practicing euthanasia influences their perception of death. practical ( %) and psychological ( . %) aspects of euthanasia have been discussed in most teams. veterinarians' gender influences euthanasia management, mostly concerning some communicational and practical aspects. euthanasia is definitely not an ordinary veterinary act, neither for the owner nor for the veterinarian. therefore, this act must be performed with as much care and communication as possible. disclosures: no disclosures to report. canine pancytopenia is associated with a range of intra-marrow or extra-marrow causes, including though not limited to, infectious agents, drugs, toxins and neoplasms. there is currently limited information regarding the clinicopathological features of the underlying causes or the prognosis in pancytopenic dogs. the objective of this retrospective study was to better define the spectrum of diseases associated with canine pancytopenia, to establish possible clinicopathological discriminators for the common causes and to investigate if the severity of pancytopenia or the underlying disease were associated with the clinical outcome (death or survival). medical records of dogs with a comprehensive diagnostic investigation admitted in a veterinary teaching hospital were retrospectively reviewed. pancytopenia was defined by a hematocrit (hct) < % (< % for dogs < months of age), white blood cell counts (wbc) < , /ll and platelet counts (plt) < , /ll. a control group of dogs without any evidence of blood cytopenia(s) was also established. in total, pancytopenic dogs were studied. bone marrow aspiration cytology was examined in cases and aplasia of all hematopoietic lineages was observed in ( . %) dogs. the most common diagnoses included monocytic ehrlichiosis (cme) (n = ), leishmaniosis (canl) (n = ), parvoviral enteritis (pe) (n = ), and concurrent cme and canl (n = ). mixed breed dogs were more likely to develop pancytopenia as compared to purebreds and pancytopenic dogs tended to be younger than the controls (conditional dependent logistic regression model, p = . and p = . , respectively). among the most common diseases associated with pancytopenia, the mean wbc counts were significantly lower in dogs with cme and pe compared to dogs with canl (one way anova with bonferroni test for multiple comparisons, p = . and p = . , respectively), while plt counts were significantly lower in cme compared to canl (p< . ) or pe (p < . ). total protein concentrations were significantly lower in dogs with pe compared to cme (p < . ) and canl (p < . ). using a univariable logistic regression analysis model, no association was established between the underlying disease and the final outcome. however, higher hct (by at least one percentage unit), wbc (by at least , /ll) and plt (by at least , /ll) values tended to significantly increase the odds of survival (p = . , p < . and p = . , respectively). in the present study, cme, canl and pe were the major causes of canine pancytopenia. potentially useful diagnostic indicators included severe leucopenia (cme, pe), thrombocytopenia (cme) and hypoproteinemia (pe). disclosures: no disclosures to report. laryngeal masses (lm) usually produce air flow limitation during inspiration, expiration or transiently during subsegments of both breathing phases. feline bronchial diseases (fbd) have predominantly expiratory flow restrictions. barometric whole-body plethysmography (bwbp) is a non-invasive pulmonary function test (pft) that allows a dynamic study of breathing patterns widely used to evaluate lower airway responsiveness. the objective of this preliminary study was to evaluate if there were significant differences in respiratory rate [rr ( bwbp detects both upper and lower airways diseases because any site of airway obstruction will result in increased pressure changes associated with breathing. nevertheless our results suggest that there are significant differences in tv(p = . ), ti (p = . ), pef(p = . ), eep(p = . ), penh(p = . ) and pau(p = . ) between lm and fbd cats. no other significant difference in bwbp parameters was found. upper airway obstructions have been previously evaluated in cats by using pft (mckieman, , lin, but in authors' knowledgement this is the first study designed to compare upper and lower airway obstructions by using bwbp. attending our results, there is the evidence that bwbp can help characterize mechanical dysfunction of the airways in cats with lm obstruction. however we must keep in mind some limits of this study as the low number of animals, individual variability in breathing pattern and to have the chance of doing bronchoreactivity tests. disclosures: no disclosures to report. the median lifespan of domestic dogs has been estimated to - years, but little is known about risk factors for mortality in aged dogs: most mortality studies in dogs have been carried out among diseased dogs (renal or heart diseases, cancers, post-operative death). to determine which characteristics are associated with mortality in a priori healthy aged dogs, a prospective cohort study has been conducted in guide dogs, followed from a systematic geriatric examination (ge) to either (all-cause) death or cut-off date (july th , ) . survival analyses (kaplan-meier estimators, log-rank tests, and multivariate cox proportional hazards models) were used to assess the associations with time to death. median age at ge was . years, all dogs were neutered, and % were female. the majority of dogs were golden retriever (n = ) and labrador retriever (n = ). among these dogs, % were obese, % presented skin nodules and % used bus as transportation. a total of dogs died during follow-up, leading to a median survival time from ge of . years. after adjustment for demographic and biochemical variables (age, sex, total proteins, cholesterol and alp), an increased alanine amionotransferase level (≥ ui/l; adjusted hazard ratio [ahr], . ), presenting skin nodules (ahr, . ), and not being a labrador (ahr, . ) were independently associated with a shorter time to death (p < . ). public transportation tended to be associated with mortality (ahr, . ; p = . ), highlighting the importance of environment in mortality. neither sex nor other biochemical parameters were significantly associated with mortality. the alanine amionotransferase level and the presence of skin nodules seem predictors of mortality in senior guide dogs, mostly labrador, golden, or mixed breed of labrador/golden. the impact of environment, in particular urban environment, on mortality needs further investigation. studies in other breeds and pets are also necessary to generalize these results. disclosures: sara hoummady received a grant from mp labo for his phd work about dog aging and marc blondot work at the paris guide dog school. laryngeal dysfunction is most commonly associated with aspiration pneumonia, however its role in other lower airway diseases has not been investigated. laryngoscopic and bronchoscopic findings in dogs examined by the author between and were evaluated for the presence or absence of laryngeal abnormalities. dogs that presented for evaluation of inspiratory difficulty or panting were excluded from analysis. clinical diagnoses of inflammatory airway disease, airway collapse, airway infection, eosinophilic bronchopneumopathy or a combination of these disorders were obtained through bronchoscopy and bronchoalveolar lavage fluid analysis. detection rates for laryngeal abnormalities were compared among disease groups using chi square analysis and fisher's exact test, with significance set at p < . . a total of dogs were evaluated and varied in age between months to . years (median years). weight ranged from . - . kg (median kg), with dogs < kg, dogs from . - . kg, dogs from - kg, dogs from . - kg, and dogs > kg. laryngeal hyperemia or swelling was found in / dogs ( %), and detection rate did not differ among disease processes. laryngeal function was considered suspect in / cases, prompting administration of doxapram, which normalized function in / dogs. laryngeal paresis or paralysis was reported in a total of / dogs ( %). a substantial number of dogs with chronic cough displayed evidence of abnormal laryngeal structure or function, suggesting that a complete laryngoscopic examination should be performed in all dogs evaluated for cough. disclosures: member of the feline advisory board, speaker honoraria for international, national, and regional continuing education meetings. bronchiectasis is a poorly characterized disease in dogs identified by airway dilatation on radiographs, computed tomography, bronchoscopy, or histopathology. little is known about underlying disease processes associated with bronchiectasis in dogs. medical records from dogs presented to uc davis were searched for identification of bronchiectasis. underlying disease processes and clinical diagnoses were obtained through review of the history, physical examination, respiratory endoscopy and bronchoalveolar lavage fluid analysis and microbiology. historical reports, results of imaging, bronchoscopy and fluid analysis, and scrutiny of pathologic and clinical diagnoses were comprehensively evaluated to identify the most likely underlying disease process associated with bronchiectasis. between and , bronchiectasis was diagnosed in / dogs ( %) that had bronchoscopy performed. dogs ranged in age from . to years (median years) with / dogs < months, / dogs ( %) - years, / dogs ( %) . - years of age and / dogs ( %) over years of age. dog breeds affected more than once included labrador retrievers, cocker spaniels, golden retrievers and standard poodles. duration of cough ranged from days to years (median months). underlying disease processes included pneumonia in / ( %) dogs, inflammatory airway disease in / ( %) dogs, and eosinophilic bronchopneumopathy in / ( %) dogs. twenty-three of dogs ( %) had positive bacterial cultures, with isolation of streptococcus (n = ) and enteric species (n = ) most commonly. this study found that bronchiectasis often occurs in older, large breed dogs with infectious or inflammatory pneumonia. disclosures: johnson: feline advisory board, speaker honoraria. chronic respiratory disease, often characterized by a chronic cough, is common in dogs. the purpose of this study was to determine the etiology of chronic respiratory disease in dogs that were presented with persistent and chronic coughing. a retrospective study of client-owned dogs with signs of persistent and chronic lower respiratory disease, that underwent bronchoscopy together with either an endotracheal wash (etw) or broncho-alveolar lavage (bal), was performed. all dogs were evaluated by means of full clinical examination, hematology and serum biochemistry analyses, survey thoracic radiographs, echocardiography and ecg (if indicated), and bronchoscopy with cytological analysis and aerobic culture of etw or bal fluid. an etw was performed in / ( %) dogs while a bal was performed in / ( %) dogs. a positive aerobic bacterial culture was identified in / ( %) of submitted etw/bal fluid samples. most commonly isolated bacteria included mycoplasma sp. ( %), bordetella bronchiseptica ( %) and pseudomonas aeruginosa ( %). a definitive diagnosis was made in / cases ( . %). chronic bronchitis was the most common diagnosis ( . %), median age years; followed by airway tracheal collapse or bronchomalacia ( %), median age years,; and primary bacterial infections ( . %), median age years. less common etiologies identified included neoplasia ( . %), median age years; parasitic infections ( . %), median age years; and eosinophilic bronchopneumopathy ( . %), median age years. rare etiologies identified included primary pulmonary hypertension, primary ciliary dyskinesia, excitement-induced cough, and obesity. myxodematous mitral valve disease was found concurrently in / ( . %) dogs. this study concluded that by using a structured combination of survey thoracic radiography, bronchoscopy and etw or bal with cytology and culture, a diagnosis could be made in the majority of dogs with chronic respiratory disease. disclosures: no disclosures to report. canine sterile steroid-responsive lymphadenitis (cssrl) is an uncommon cause of lymphadenomegaly. diagnosis is one of exclusion after extensive investigations exclude infectious, inflammatory or neoplastic aetiologies. resolution of clinical signs occurs with corticosteroids. this retrospective study aimed to further define characteristics, progression and treatment regimens. cases were recruited from uk referral centres between - . diagnostic investigations in each case excluded other potential causes of lymphadenomegaly. thirty-six dogs were diagnosed with cssrl from lymph node cytology and/or histopathology. eighteen breeds were represented, of which were spaniels. english springer spaniels (ess) accounted for cases along with cocker spaniels ( ), cavalier king charles spaniels ( ) and border collies ( ) clinical presentation varied between dogs. clinical signs of pyrexia ( %), lethargy ( %) and anorexia ( . %) were the most common. other signs included cough, tachypnoea, dyspnoea, dysphagia, vomiting, diarrhoea, neck or spinal pain, abdominal pain, joint effusion or dermatologic signs. median referral time was days (ess , cockers and border collies days). twenty-two animals were pyrexic at presentation (mean . °c, range . - . °c). thirty-one animals presented with peripheral lymphadenomegaly, but animals displayed only internal lymph node enlargement. cytology was performed in / cases; neutrophilic lymphadenitis ( ), followed by granulomatous ( ), pyogranulomatous ( ) and reactive hyperplasia ( ). histopathology was performed in / cases documenting neutrophilic ( ), pyogranulomatous ( ) or granulomatous ( ) lymphadenitis. lymph node culture or staining (gram, pas, zn) was performed in and animals respectively, all of which were negative. prednisolone was administered in all cases (dose range . - mg/kg daily). animals were initiated therapy at mg/kg q hours. mean treatment length was weeks. dogs relapsed throughout the study period ( ess, cockers, ckcs, border collie, lurcher and beagle). ess relapsed within months of diagnosis. median relapse time was weeks. this study documents dogs with cssrl in the uk suggesting an over-representation in spaniel breeds (particularly ess), with females and young dogs typically affected. cytologic and histopathologic examination confirmed sterile lymphadenitis with animals showing marked and rapid clinical improvement with corticosteroids. disclosures: no disclosures to report. brachycephalic dogs have unique upper respiratory anatomy with abnormal breathing patterns similar to those in humans with obstructive sleep apnea syndrome (osas). oxidative stress in the body represents the imbalance between the production of reactive oxidative species and the ability of antioxidant defense mechanisms to detoxify the reactive intermediates. oxidative stress is involved in the pathogenesis of many diseases, including hemolytic anemia, atherosclerosis, tissue reperfusion injury and has also carcinogenic potential. several studies have clearly shown an association between obstructive sleep apnea syndrome in humans and oxidative stress, but detailed underlying pathomechanism remains unclear. due to the consideration of brachycephalic dogs as an animal model of human osas, this study was designed to evaluate oxidative stress (paraoxonase type- activity; pon and total antioxidant status; tac) in brachycephalic dogs with brachycephalic airways obstructive syndrome (baos) before and after surgery compared to control dogs. this study was conducted on dogs with baos and control dogs. twenty out of baos dogs were evaluated - months after surgical correction. mean values of tac and pon in different studied groups were as follows: control dogs (tac: . ; pon : . ), baos dogs (tac: . ; pon : . ), baos dogs post-surgery (tac: . ; pon : . ) a statistically significant difference on tac levels is observed between dogs with baos and control dogs (p < . ). no statistically significant differences were observed in pon and tac levels before and after surgery. on the other hand, no differences have been observed between pon and tac levels in baos dogs according type of brachycephalic breed, grade of respiratory and digestive signs or presence of everted ventricular laryngeals. the results of our study showed a statistically significant difference in tac values between control and dogs with baos, confirming the oxidative stress previously described in humans. even that human patients with osas can partially reverse their increase in oxidative stress by using a nasal continuous positive airway pressure treatment, in dogs with baos no differences were observed before and month after surgical treatment. baos surgical treatment is not useful to reduce pon- or tac levels, probably because baos does not induce such an evident inflammatory process in dogs as in human patients with osas. disclosures: no disclosures to report. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease mainly affecting west highland white terriers (whwts). the cipf course varies greatly among dogs from rapid deterioration to slowly progressive forms and the survival time from onset of clinical signs ranges from a few months to several years. in human ipf, increased chemokine (cc-motif) ligand (ccl ) concentrations in bronchoalveolar lavage fluid (balf) are indicative of a poor outcome and serum concentrations are correlated with clinical parameters of lung function. in dogs, serum and balf ccl concentrations were shown to be elevated in whwts with cipf compared with healthy whwts. the aim of the present study was to investigate whether serum ccl concentrations measured in whwts with cipf at diagnosis ( ) can be used as an indicator of prognosis and ( ) correlate with lung function parameters. ccl concentrations were determined by elisa (canine ccl quantikine elisa kit, r&d systems) in the serum of whwts suspected of cipf (median age . years, range . - . ), for which a follow-up was available (median follow-up time . months, range - . ). serum sampling extended from may to january . cipf diagnosis was confirmed by thoracic high resolution computed tomography, lung histopathology, or both examinations in , and whwts respectively. kaplan-meier analysis was conducted to investigate differences in survival times according to serum ccl concentrations at diagnosis. spearman analysis was used to assess correlations between serum ccl concentrations and lung function parameters, namely the distance walked in the -minute walking test ( mwd) and the arterial partial pressure of oxygen (po ). among the cipf whwts included, died or were euthanized for cipf-related reason, died or were euthanized for non-cipf-related reason and were still alive at the end of the study. the median survival of whwts with cipf-related death or euthanasia was . (range . - . ) months from diagnosis. serum ccl concentrations above pg/ml were significantly associated with a shorter survival time in whwts affected with cipf (p = . ). a weak negative correlation was found between serum ccl concentrations and the mwd (r = À . , p = . , n = ), while no correlation was observed with arterial po values (n = ). in conclusion, serum ccl concentration provides prognostic information in whwts suffering from cipf, while this marker is weakly correlated with the clinically lung function parameters available in the present study. disclosures: no disclosures to report. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease mainly affecting west highland white terriers (whwts). this study was intended to ( ) describe thoracic high-resolution computed tomography (t-hrct) findings obtained in cipf dogs under general anesthesia (ga) using the glossary of the fleischner society and ( ) compare images obtained under ga (t-hrct ga ) with those obtained under sedation (t-hrct s ). t-hrct images from whwts with cipf and control whwts were retrospectively reviewed by observers in consensus. specific t-hrct features were assessed and graded for each lung lobe ( = absence, = mild, = moderate and = severe). a global score was then calculated. the khi² test with the threshold % was used for the statistical analysis. ground glass opacity (ggo) was observed in all cipf whwts and in / of controls (p = . ). in controls, ggo was mild and localised mainly in cranial lobes. in cipf whwts, ggo was mild, moderate or severe in , and dogs respectively, without lobe predilection. consolidation was observed in / cipf whwts but not in controls (p = . ) and was mild ( / ) to moderate ( / ). a mosaic pattern, suggestive of air trapping, was noticed in / cipf whwts but not in controls (p = . ) and was mild, moderate or severe in , and whwts respectively, without lobe predilection. nodules were present in / cipf whwts but not in controls. reticulation, subpleural bands and parenchymal bands were noticed in , , and / cipf whwts respectively. honeycombing, emphysema, pleural effusion and pleural thickening were never observed. bronchial wall thickening and mild bronchiectasis were present in / and / cipf whwts respectively but not in controls (p = . and p = . ). the overall t-hrct s quality was good in / examinations compared with / for t-hrct ga (p = . ). the presence of motion artefacts was higher for t-hrct s (p < . ), but were most frequently graded as mild (p < . ). t-hrct s allowed identification of a mosaic pattern in additional cipf whwts, while consolidation could not be identified in others. there was no difference in identification or gradation for the other features between t-hrct ga and t-hrct s . in conclusion, ggo, consolidation, mosaic pattern and bronchial wall thickening are the main t-hrct features of cipf in whwts. honeycombing, the major feature of ipf in humans, was never observed, which suggests a different pathophysiology between the entities. t-hrct s images are in accordance with t-hrct ga and can be used for cipf diagnosis. disclosures: no disclosures to report. literature on mesenterial lymphadenitis (lad) or mesenterial lymph node abscesses (lab) in small animals is scarce. case files from to were searched for dogs with the diagnosis of mesenterial lad/lab based on cytology and/or histopathology. of cases identified, had to be excluded due to incomplete data. the remaining dogs were of mixed breed (n = ), small munsterlander (n = ) and one each of other breeds. nine dogs were male and female. median age was months (range . diagnosis was based on fine needle aspiration (fna; n = ), histopathology (n = ) or both (n = ). significant findings in the dogs' history included gastrointestinal signs (n = ), puppy whose mother had mastitis, bite wounds/abscesses of the skin (n = ), pulmonic stenosis (n = ) and orthopaedic diseases (n = ). most common presenting complaints were lethargy (n = ), hyperthermia (n = ), diarrhoea (n = ), vomiting/nausea (n = ), inappetence/anorexia (n = ), back/abdominal pain (n = ) and lameness (n = ). diagnostic tests performed included haematology/serum biochemistry (n = ), thoracic (n = ) and abdominal (n = ) radiographs, abdominal ultrasound (n = ), ct/mri (n = ), fnas of other organs (n = ), urinalysis (n = ) with culture (n = ), coagulation profiles (n = ), orthopaedic radiographs (n = ), cpl (n = ), blood cultures (n = ), and csf/joint taps (n = ). dogs were retrospectively divided into group a (n = ): dogs with no other disease than lad/lab ("idiopathic") and group b (n = ): dogs with other diseases diagnosed simultaneously. these included neoplasia (carcinoma n = , lymphoma n = , leiomysarcoma n = , histiocytic neoplasia n = , prostate carcinoma n = ), gastroenteritis (n = ), presumed pancreatitis (n = ), purulent monoarthritis (n = ), purulent hepatitis/ splenitis (n = ), fungal infection at a distant site (n = ), and mycobacteriosis (n = ). eleven dogs received surgical treatment and antibiotics, and dogs conservative medical management consisting of supportive treatment and antibiotics. all dogs were discharged alive. dogs in group a were hospitalized longer (mean days, sd . ) than dogs in group b (mean days, sd . ) (p = . ). the median follow-up time was days ( - days). there was no difference in pretreatment with antibiotics or anti-inflammatories between groups. t-tests or kruskall-wallis tests showed that dogs in group a were borderline significantly younger (p = . ), had significantly higher respiratory rate (p = . ), rectal temperature (p = . ), monocyte count (p = . ) and crp concentration (p = . ) than dogs in group b. the small munsterlander had an odds ratio of over other breeds to suffer from lad/lab. in conclusion, idiopathic mesenterial lad/lab was seen in young dogs with hyperthermia and gastrointestinal signs. diagnosis of purulent lad/lab on fna does not exclude the presence of another underlying pathogenesis. disclosures: no disclosures to report. dogs were included, if they showed respiratory signs (< days) consistent with cird and if non-infectious respiratory conditions could be excluded. nasal and pharyngeal swabs were taken from dogs with cird and tested for respiratory pathogens, including canine parainfluenza virus (cpiv), canine adenovirus (cav), canines distemper virus (cdv), canine herpes virus (chv), canine respiratory coronavirus (crcov), canines influenza virus (civ), and bordetella bronchiseptica (b. bronchiseptica) by polymerase chain reaction. results of clinical and laboratory data were correlated with the underlying pathogen using fisher's exact test and chi-square test (p≤ . ). cpiv was detected in , crcov in , and b. bronchiseptica in dogs; patients showed infections with more than one pathogen. there was no significant difference for age and gender distribution between the groups; however, dogs infected with cpiv more likely originated from a shelter (p = . ). when clinical data were compared, there was no significant difference for the parameters depression, fever, cough, nasal discharge, dyspnoea, and abnormal lung sounds. furthermore, there was no significant difference regarding abnormalities of erythrocytes, platelets, leukocytes, and differential count between groups. the study shows that in dogs with cird clinical and laboratory parameters cannot indicate the underlying pathogen. furthermore, diseases severity does not seem to depend on the infectious organism involved. disclosures: no disclosures to report. breed related predisposition to bacterial bronchopneumonia (bp) has been reported in irish wolfhounds (iwhs). underlying factors are unknown, however immune deficit, ciliary dysfunction and aspiration have been suggested as predisposing factors. the purpose of this prospective study was to evaluate lymphocyte subpopulations in iwhs with one or more previous bp and compare results to elderly iwhs without any previous bacterial respiratory infections. additionally, healthy dogs of other breeds were included as controls. previous bp was confirmed in iwhs (median age . years, interquartile range . - . years). healthy iwhs (n = , . , . - . years) or dogs of other breeds (n = , . , . - . years) had no history or findings suggestive of previous bp. edta blood samples, collected from all dogs when they were healthy, were stained with fluorescent mouse anti dog cd , cd , cd , cd and mhc class ii antibodies (abd serotec Ò ) and flow cytometry analysis was performed with bd facsaria Ò ii cell sorter and facsdiva Ò software. statistical comparison between groups and the effect of age was studied using analysis of covariance (ancova) models. the number of leucocytes did not differ significantly between groups. the total numbers of lymphocytes and numbers of major lymphocyte subpopulations (b-cells, cd + and cd + t-cells) were significantly lower in healthy iwhs and iwhs with previous bp compared to dogs of other breeds (lymphocytes p < . and p < . ; b-cells p = . and p = . ; cd + t-cells p = . and p = . ; cd + t-cells p < . and p = . respectively). percentage and number of mhc class ii+ non-b lymphocytes was significantly higher in both iwh groups than in dogs of other breeds (p < . in all comparisons). lymphocyte numbers and subpopulations did not differ significantly in healthy iwhs compared to iwhs with previous bp. an age-related decline in the total number of lymphocytes (p = . ), t-cells (p = . ), cd + t-cells (p = . ) and mhc class ii+ non bcells (p < . ) was noticed only in the group of iwhs with previous bp. these preliminary results indicate that iwhs may have significantly lower numbers of lymphocytes, b-cells as well as cd + and cd + t-cells than dogs of other breeds. further studies are needed to determine whether these alterations represent a breed related phenomenon or are connected to the predisposition to bp. an age-related decline in lymphocyte, total t-cell and cd + t-cell numbers was detected in iwhs with previous bp. in humans, age related changes in cd + t-cells have been associated with increased susceptibility to infections. disclosures: no disclosures to report. feline chronic kidney disease (ckd) is a common feature of ageing cats. angiotensin-converting enzyme inhibitors (acei) are recommended to treat hypertension associated with ckd to limit target-organ damage and especially glomerular hypertension. in addition, the international renal interest society (iris) guidelines recommends the prescription of an acei in patients with ckd and proteinuria. to our knowledge no study has demonstrated the effects of long term administration of acei in a client owned population of cats suffering from ckd on glomerular filtration rate (gfr). the aim of the study was to evaluate the effect of an acei (imidapril, prilium Ò , v etoquinol sa) on the gfr of cats suffering from naturally occurring chronic kidney disease (ckd) over months. sixty-six cats presenting with clinical and biological signs of ckd were enrolled by european investigators and followed up till months in this randomized blinded study. thirty-two cats provided suitable data for gfr analysis; animals received imidapril at the dosage of . mg/kg/day and received placebo. animals with no available gfr value on day or with no data after day were excluded as well as animals for which the iohexol clearance could not be determined. follow up was censored after months due to small sample sizes for statistical comparisons. on day , month , month and month , cats were sampled , and minutes after intravenous administration of mg of iohexol. gfr was based on determination of the iohexol clearance which was calculated with the phoenix Ò winnonlin . software. statistical analyses were performed with sas/stat . software. as gfr were not available on each time point for a given animal, the treatment groups were compared on each gfr determination point using an ancova (analysis of covariance) with the gfr determined on day as the covariate. on d , gfr were . ae . and . ae . ml/kg/min in the imidapril and placebo group, respectively. a significant statistical difference ( . ae . versus . ae . ml/kg/min in the imidapril and placebo group respectively, p = . ) was observed in favor of a higher gfr in the imidapril treated animals on month . higher gfr were also observed in the imidapril group on mon-th and month but not significantly different from the placebo group. daily long term imidapril treatment compared to placebo, may be an effective treatment to slow the progression of renal failure in cats with naturally occurring chronic kidney disease. disclosures: authors are employees of vetoquinol. there is a concern over the potential of cytotoxic drugs which could harm exposed workers. the speciality of oncology of the ecvim-ca published guidelines in order to prevent that risk. however few data exist for evaluation of the real risk of occupationnal exposure. this concern was the aim of our study. biomarkers used were vincristine and cyclophosphamide. surface samples were collected in the consultation room and ward dedicated for chemotherapy. samples were collected with wet filter paper on cm surfaces, or objects (computers for instance). samples were analysed by liquid chromatography and mass spectrometric method.the limit of quantification was . ng/ cm (or . g/object) for vincristine and . ng/ cm (or . g/object) for cyclophosphamide. samples in consultation room were collected after treating dogs with vincristine and after cleaning. samples in dedicated ward were collected after cleaning and after -days stay of treated dogs. aeras/objects were tested in consultation room; in dedicated ward for vincristine; in ward for cyclophosphamide. after treatment of dogs in consultation room, traces of vincristine were detected on the floor and the laboratory bench top ( . to . ng/ cm ). moreover, the surface of auscultation table and extern side of gloves were contaminated after preparation and administration of vincristine (respectively . , and ng/objet). after cleaning, % of samples in consultation room were positive. traces of vincristine were detected on the floor or objects (wall otoscope. . .: . to . ng/ cm ). after cleaning, % of samples in ward were positive. traces of vincristine were detected on the floor and objects (boxes, wastebin, bowls. . .: . to . ng/objet) after cleaning and animals treatment. cyclophosphamide were detected on all areas tested ( . a . ng/objet). despite protective guidelines to avoid spread of cytotoxic drug, environemental exposure was demonstrated. however contaminations were limited and show that handling procedures of cytotoxic drugs are well controlled. most-elevated contamination level for vincristin were noticed on extern side of gloves depsite of using appropriate material. workers should pay a particular attention for gloves withdrawal to limit exposure. small amounts of vincristine were found in inapropriate places: computeur mouse,. . . even if there is few of those residues, we thought about people working on a regular basis in this room for other activities than chemotherapy., and we decided to adapt our clinical practices. evaluation of occupational exposure to cytotoxic drugs is an important step to prevent incidents and heigt awarness of nursing staff to apply those good clinical practices. disclosures: no disclosures to report. the tumor-associated inflammatory response had the effect of enhancing mammary tumorigenesis, helping incipient neoplasias to acquire hallmark capabilities, both in human and dogs. t-cells migration to the tumor site and the following activation may be the essential requirement for their promoting effect on tumors. in human breast cancer the signaling pathways of c-kit have been described as possibly being involved in differentiation, migration, survival, and maturation of t-cells and other inflammatory cells into tumor sites. in order to clarify this subject in canine mammary tumors (cmt), malignant neoplasms were studied by using immunohistochemistry comparing the intratumoral cd + tlymphocytes and c-kit expression together with vegf, microvessel density (mvd) and clinicopathological characteristics of tumor aggressiveness. cd + t cells and high c-kit immunoexpression revealed a positive and statistically significant correlation with vegf (r = . , p < . ; r = . , p = . for cd and c-kit respectively) and cd (r = . , p < . ; r = . , p = . for cd and c-kit respectively). a statistically significant association (p = . ) and a positive correlation (r = . , p = . ) between cd + t-lymphocytes and c-kit was also observed. tumors with high c-kit expression showed higher counts of cd + t-cells. the mvd of high cd /vegf tumors was significantly more elevated (p < . ). a similar association was observed for high c-kit/vegf tumors (p < . ). in this study high cd /vegf, high c-kit/vegf and high cd /c-kit tumors were statistically significantly associated with elevated grade of malignancy (p < . for cd /vegf, c-kit/vegf and cd /ckit), presence of neoplastic intravascular emboli (p < . for cd /vegf and cd /c-kit; p = . for c-kit/vegf) and presence of lymph node metastasis (p < . for cd /vegf, c-kit/ vegf and cd /c-kit). tumors with high cd /vegf (p = . ), high c-kit/vegf (p < . ) and high cd /c-kit (p = . ) expression were associated with shorter os time. inter-estingly the group of tumors with high c-kit/vegf retained their significance by multivariate analysis arising as independent predictor of os. results of this study suggest that t-lymphocytes may share common signaling pathways with c-kit and vegf in cmt progression and may contribute to increased angiogenesis, aggression and shorter os in these tumors. disclosures: no disclosures to report. lgl lymphoma, but no comparisons were made with cats with other diseases or other forms of feline lymphoma. therefore, the objective of this study was to assess differences in prevalence, signalment (breed, sex, and age), physical exam findings (body weight, body condition score, body temperature, heart rate, respiratory rate, and systolic blood pressure), and felv/ fiv status between cats with lgl lymphoma (group ) and all other type of feline lymphoma (group ). the electronic data-base of the san marco veterinary clinic was searched between january- and december- for cats with a cytological or histopathological diagnosis of lymphoma. differences between groups were assessed by t-test, mann whitney, pearson-chi square, pearson chi square yates corrected, and fisher's exact test. during the study period out of sick cats seen at the clinic were diagnosed with lymphoma (group : n = ; group : n = ). the prevalence of all type of feline lymphoma between and compared to sick cats did not change over time ranging from . % to . % per year (p = . ; overall prevalence . %, % ci . - . ). the lymphoma lgl prevalence between and compared to all types of lymphoma did not change over time ranging from . % to . % per year (p = . ; overall prevalence . %, % ci . - . ). among the variables studied, only sex (group : [ . %] females, [ . %] males; group : [ . %] females, [ . %] males; p = . ) and age (group : ae months; group : ae months; p = . ) were significantly different between groups. sixteen out of cats with lgl lymphoma were tested for their felv/fiv status resulting all felv-and one ( . %) fiv+. seventy-four out of cats with all other types of lymphoma were tested for their felv/fiv status resulting ( . %) felv+ and ( . %) fiv+. five cats ( . %) were both felv+/fiv+. prevalence of felv infection was significantly lower (p = . ) in group compared to group . there was no difference in prevalence of fiv infection between groups. lymphoma lgl affects more females and older cats compared to all other type of feline lymphoma. opposite to all other type of lymphoma, and in accordance to previous litterature information, felv+ status does not play a role in the pathogenesis of feline lgl lymphoma. disclosures: no disclosures to report. the activity of t regulatory cells (tregs) is known to be closely associated with the expression of foxp transcription factor. foxp regulatory t cells (foxp treg) are a distinct group of t lymphocytes that have immunosuppressive properties. normally this cells work for prevention of harmful autoimmune responses, however can also interfere with beneficial immune responses such as anti-tumor immunity. in human breast cancer these cells play a crucial role in tumor progression. in canine mammary tumors (cmt) there are only a few studies and this topic are not welldocumented. in this study we included malignant cmt and studied, by immunohistochemistry, the intratumoral foxp expression together with vascular endothelial growth factor (vegf), microvessel density (mvd, by cd antibody) and several clinicopathological characteristics. abundant foxp treg cells was statistically associated with presence of tumor necrosis (p = . ), nuclear grade (p = . ), poor differentiation of tumors (p < . ), high mitotic grade (p < . ), high histological grade of malignancy (p < . ), presence of neoplastic intravascular emboli (p < . ) and presence of lymph node metastasis (p < . ). intratumoral foxp levels were correlated with the levels of vegf (r = . ; p < . ) and intratumoral mvd (r = . ; p < . ). additionally tumors with abundant foxp treg cells were associated with shorter overall survival (os) time (p = . ). results suggest that treg cells play a role in cmt progression and may contribute to increased angiogenesis and aggression in these tumors. the association of intratumoral foxp expression with shorter os of animals suggests a utility of treg cells activity as a prognostic marker. disclosures: no disclosures to report. cyprus is an island state in the eastern mediterranean basin. no epidemiological studies have yet been performed on infectious agents in cats from cyprus. the aim of this study was to determine the prevalence of several infectious agents, including some vector-borne infections, in cats from cyprus. surplus edta-blood and serum samples were recruited from cypriot cats, from which signalment and lifestyle characteristics were recorded. dna was extracted and real-time quantitative polymerase chain reaction (qpcr) assays were used to detect haemplasmas(mycoplasma haemofelis, 'candidatus mycoplasma haemominutum' and 'candidatus mycoplasma turicensis'), leishmania spp.and bartonella henselae. conventional pcr assays were used to detect ehrlichia/anaplasma spp. samples yielding positive results for leishmania spp. or ehrlichia/anaplasma spp. underwent further characterisation (sequencing). elisas were performed for the detection of l. infantum antibodies, feline leukaemia virus (felv) antigen and feline immunodeficiency virus (fiv) antibodies. statistical analysis was performed using spss for the assessment of any associations between variables and infectious agents. of the samples extracted, were excluded due to failure of ≥ one internal control pcr. of the remaining samples, ( . %) were positive by pcr for haemoplasma including ( . %) for m. haemofelis, ( . %) for 'ca. m. haemominutum' and ( . %) for 'ca. m. turicensis'. nineteen ( . %) were positive for b. henselae. one cat ( . %) was pcr positive for ehrlichia/anaplasma spp. and sequencing revealed identity with anaplasma platys. leishmania spp. dna was detected in of the ( . %) cats; sequencing revealed l. infantum in of these cases. l. infantum serology was positive in of the cats tested ( . %). only one cat was positive for both leishmania pcr and serology. of the cats that underwent retroviral serology, ( . %) were felv and ( . %) were fiv positive. statistical analysis identified several significant associations (p < . ) including the following; haemoplasma infection and both outdoor access and feral-shelter cat origin, felv or fiv infection and both anaemia and feral-shelter cat origin. this study documents, for the first time, the presence of haemoplasmas, l. infantum, b. henselae, a. platys, felv and fiv in the feline population of cyprus. the prevalence of haemoplasma, fiv and b. henselae infections were among the highest reported in cats from mediterranean countries, while that of leishmania spp. was similar. this is the second report of a. platys infection in a cat from a mediterranean country. disclosures: no disclosures to report. bartonella species (spp.) are zoonotic pathogens, and infections in cats are common. prevalence in cats from southern germany is still unknown. the aim of this study was to determine the prevalence of bartonella spp. dna in blood of cats in southern germany and to evaluate associations between bartonella bacteremia, housing conditions, feline immunodeficiency virus (fiv), and feline leukemia virus (felv) status, including progressive, regressive, and abortive felv infection. blood samples of cats that were presented to different veterinary clinics in southern germany for various reasons were tested for bartonella spp. dna using a previously published conventional polymerase chain reaction (pcr) targeting a fragment of the s- s rrna intergenic spacer region. for statistical analysis, fisher's exact test was used. prevalence rate of bartonella spp. bacteremia was . % ( / ; ci: . % - . %). b. henselae was amplified in of these cats. one cat was positive for b. clarridgeiae dna. most of the infected cats were clinically healthy, but half of the cats had thrombocytopenia, potentially caused by their bartonella spp. infection. there was no significantly higher risk to be infected with bartonella spp. when living mainly outdoors or being fiv-or felv-infected. prevalence of bartonella spp. bacteremia is low in southern german cats, but there is still a risk of human bartonella infection associated with cat ownership. most clinical changes of the bartonella spp.-infected cats were related to other diseases. however, thrombocytopenia was common and further studies are required to define its potential clinical relevance. disclosures: no disclosures to report. ante-mortem diagnosis of feline infectious peritonitis (fip) is still challenging. the aim of this study was to evaluate sensitivity and specificity of a 'combined reverse transcription nested polymerase chain reaction (rt-npcr) and sequencing approach', detecting mutations at different nucleotide positions within the spike gene, that previously were shown to correlate with the fip phenotype. the study population consisted of cats with confirmed fip and a defined control group of cats for which fip was considered an important differential diagnosis, but that were definitively diagnosed with other diseases. blood and/or effusion samples were examined for feline coronavirus (fcov) rna by rt-npcr and, if positive, nucleotide positions and were sequenced for nucleotide transitions. sensitivity, specificity, negative and posi-tive predictive values were determined and % confidence intervals ( % ci) calculated. rt-npcr detected fcov in cats in blood (n = ) and/or effusion (n = ); all of them had fip. one of the mutations of interest was found in / of the pcr-positive blood samples and in / of the pcr-positive effusion samples. diagnostic specificity of the 'combined rt-npcr and sequencing approach' was % in blood ( % ci . - . ) and effusion ( % ci . - . ). diagnostic sensitivity was . % ( % ci . - . ) in blood and . % ( % ci . - . ) in effusion. a positive test result therefore confirms a suspicion of fip. a negative result, however, cannot be used to rule out fip, especially if only blood is available. therefore, if no effusion is present, diagnosis of fip still remains challenging. disclosures: dr. elisabeth mueller is the managing director of laboklin gmbh & co.kg. dr. karola weider is employed at laboklin gmbh & co.kg. this laboratory offered the 'combined rt-npcr and sequencing approach' on a commercial basis and performed the testing in this study. feline infectious peritonitis (fip) is a viral disease caused by the virulent strain of feline coronavirus (fipv). the disease can appear under clinical forms, dry or effusive, both leading to a fatal outcome. diagnosis was based on histopathologic lesions on necropsy until the recent discovery of mutations associated with the fipv strain in the c and spike (s) genes. our main goal was to detail the distribution of c or s gene mutations in different biological samples of cats suffering from fip. this was a retrospective, observational study of cats showing clinical signs compatible with fip. ten out of cats were of pure breed. . % were males and . % females. median age was . months at presentation. the clinical presentation, pathologic findings and virologic data were reviewed. according to clinical signs, cats were classified with a dry form and with a wet form of fip. the main clinical signs included dehydration, hyperthermia, icterus, abnormal abdominal palpation, neurological and ocular disorders. when possible blood, fecal material, effusion, fine needle aspiration (fna) from relevant organs or a combination of these, was recovered from each cat. feline coronavirus (fcov) was first researched by rt-pcr, then the c and part of the s genes were sequenced to determine the eventual presence of mutations. among the dry cases, fcov was detected in / blood samples, / fecal samples and fna ( / ). among the wet cases, / blood samples, / fecal samples and all effusion samples ( / ) were positive for the presence of fcov. c mutations were never found in fecal samples but were found in / effusion samples and in / fna. s mutations were detected in / fecal samples, / fna and / effusion samples. for cats, no mutation, neither in c or s genes was identified despite the confirmation of fip by necropsy. s gene mutation is more frequently observed than c gene despite in cases where only c mutations were identified. moreover the presence of strains harbouring s mutation in feces has never been described before and could suggest the possible diffusion of fipv among feline population. in conclusion, viral diagnosis of fip based on rt-pcr sequencing in effusion and fna samples is essential. rt-pcr resulting from blood samples should be carefully interpreted because of high risk of missing fipv. finally, searching for mutations in both s and c genes is recommended. disclosures: no disclosures to report. methicillin resistant staphylococcus aureus (mrsa) has recently become a great concern for pet animals' disease and zoonotic infection. mrsa strains transfer between pet animals and humans could occur. the aim of the present study was to determine the occurrence of mrsa in household dogs. from january to june , clinical samples were collected from dogs, patients of the veterinary teaching hospital of the department of veterinary sciences of messina (italy), affected by several diseases of various origins. all samples were processed by bacteriological conventional methods for isolation and identification. all strains were tested for phenotypic susceptibility to oxacillin and were subjected to a pcr protocol for the detection of meca gene. strains carrying the gene were considered methicillin resistant (mrs). lastly, on both mrs and methicillin sensitive (mss) strains, kirby-bauer disk diffusion susceptibility testing were performed to highlight resistance profiles using molecules belonging to the main classes of antimicrobials used in veterinary practice. strains resistant to at least one molecule of or more classes of antibiotics were considered multidrug-resistant (mdr). the statistical analysis of the results was made using the z-test by a primer Ò software. forty staphylococcus spp. strains were isolated, belonging to species. the most frequently isolated microorganisms were staphylococcus aureus with isolations ( %) and staphylococcus pseudintermedius with isolations ( . %), followed by staphylococcus epidermidis with isolations ( %) and staphylococcus cohnii and staphylococcus warneri, both with isolations ( %). a single isolation ( . %) was obtained for each of the species staphylococcus chromogenes, staphylococcus haemolyticus, staphylococcus lentus, staphylococcus lugdunensis, staphylococcus saprophyticus, staphylococcus simulans and staphylococcus sciuri. thirteen ( . %) strains of staphylococcus spp. were phenotypically resistant to oxacillin and staphylococcus aureus ( . %; n. from pyoderma, n. from exudative pleural effusion) were positive for the meca gene. all strains of staphylococcus spp. were mdr. our results showed the presence of mrsa and multidrug-resistant staphylococcal strains in household dogs. a lack of correspondence between antimicrobial susceptibility tests and molecular methods was found in the present study. disclosures: no disclosures to report. a fourth haemoplasma called 'candidatus m. haematoparvun-like' (cmhp) was latter identified in cats. the only published study in chile was carried out in cats, with prevalences by pcr of . % to mhf, and % to cmhm.the aim of this study was to perform molecular detection of haemoplasmas in cats from valdivia, southern chile. blood samples were taken from cats and used for haemoplasmas dna detection by quantitative real time pcr (qpcr) at universidad austral de chile. qpcr protocol was based on detection of feline dna targeting s housekeeping gene and mycoplasma spp. s rrna gene (universal primers, my sf forward, my sr y my sr , both reverse) by sybr green method. the melting temperature (tm) analysis allowed identifying the infecting mycoplasma species (mhf, cmhm, cmt). it was not posible to identify haemoplasmas species on co infected cats, so a second qpcr specie specific protocol was applied on these samples. second qpcr protocol was based on s rrna gene, with specific primers to detect mhf, cmhm, cmt and cmhp. all samples ( / ) were positive to s gene, proving presence of cat dna. from the cats, . % ( / ) were positive to haemoplasmas, where . % ( / ) corresponded to cmhm (tm . - . °c), . % ( / ) to mhf (tm . - . °c), % ( / ) to cmt (tm . - . °c) and . % ( / ) to co infections. associations between cmhm+mhf, cmhm+cmt, cmhm+cmhp and cmhm+mhf+ cmhp were detected on co infected animals. these results agree with those found in previous reports from chile, europe, eua and brazil, where cmhm is the most prevalent species and co infections are less frequent. valdivia cats are infected by different haemoplasma species and cmt and cmhp are reported for the first time in chile.founding: did uach, project s- - disclosures: no disclosures to report. clinical manifestations of canine visceral leishmaniasis (canl) are non-specific and include progressive weight loss, anemia, lymphadenomegaly, hepatosplenomegaly, dermatological, renal and ocular alterations. cardiac lesions resulting in clinical signs has been scarcely described in dogs with vl, and the presence of the parasite in the cardiac tissue has been involved in few reports. accordingly, the present study aimed to evaluate histopathological abnormalities in cardiac tissue from dogs naturally infected by leishmania infantum chagasi. a total of dogs were evaluated. all dogs were symptomatic but no one presented clinical signs of cardiac involvement. in compliance with a federal law and under the owners' signed consent, all dogs were submitted to euthanasia and comprehensive post-mortem evaluation. samples from right atrium free wall (ra), right ventricle free wall (rv), interventricular septum (ivs) and left ventricle free wall (lv) were collected and evaluated. tissue samples were fixed in formalin, embedded in paraffin, sectioned at mm, and stained with hematoxylin and eosin (he) and anti-leishmania immunohistochemistry was also performed. the study was approved by the ethics committee in animal experimentation and animal welfare (protocol number / ). histopathological changes were observed in at least one of the evaluated cardiac regions in % ( / ) of the dogs. the most frequent cardiac injury was an inflammatory reaction, characterized by the presence of mononuclear cell infiltrate in different degrees. of the evaluated regions, ra was the one with the highest incidence of histopathological changes, observed in % ( / ) of the animals, followed by rv, lv and siv, affected in . % ( / ), . % ( / ) and . % ( / ) of the dogs, respectively. immunohistochemistry revealed amastigotes in the cardiac tissue in % ( / ) of the dogs. a positive correlation was found between cardiac lesions and the presence of amastigotes in the myocardium (p < . ). disclosures: no disclosures to report. canine leishmaniosis is a life threatening zoonotic disease. the combination of meglumine antimoniate and allopurinol is consid-ered the most effective therapy for canine leishmaniosis and constitutes the first line protocol against this disease. allopurinol is a parasitostatic drug used in long-term to maintain low parasite loads and to avoid clinical relapses. traditionally, allopurinol is considered a very safe drug in the dog. however, some reports indicate that xanthinuria and xanthine urolithiasis is produced after prolonged therapy with allopurinol in the dog. the aim of this prospective study was to evaluate the prevalence of urinary adverse effects of allopurinol treatment ( mg/kg/bid/po) in dogs with leishmaniosis. diagnosis was made by compatible clinicopathological abnormalitites with leishmaniosis and high leishmania infantum-specific antibody levels assessed by quantitative elisa. once leishmaniosis was diagnosed, a close follow-up (day , , , and during treatment) including physical examination, baseline laboratory tests (cbc, biochemistry profile, serum electrophoresis, urinalysis, urinary protein/creatinine ratio) and abdominal ultrasound was performed. in our preliminary results, dogs were included. dogs did not present any urinary abnormalities based on biochemistry profile, urinalysis and abdominal ultrasound at the time of diagnosis. four out of presented xanthinuria (day- (n = ), day- (n = ), and day- (n = )). two out of dogs presented renal mineralization at day- of treatment. two out of dogs presented bladder urolithiasis since day- of treatment. xanthinuria was presented initially in all dogs that developed renal mineralization or bladder urolithiasis. dogs with renal mineralization and urolithiasis were treated with a restricted protein diet and, so far, they did not develop renal disease. the present study describes early xanthinuria, renal mineralization and urolithiasis as adverse effects due to chronic allopurinol treatment in dogs with leishmaniosis. neither mineral analysis nor renal biopsy was performed to confirm the origin of these lesions, but no urinary abnormality was present before allopurinol treatment was instituted. a thorough monitoring of dogs treated against leishmaniosis combined with urinalysis and abdominal ultrasound should be performed to evaluate urinary adverse effects and to help in the clinical management of these adverse effects. disclosures: no disclosures to report. giardia duodenalis is one of the most important gastrointestinal parasites in dogs and cats with a zoonotic potential. in germany the prevalence in dogs and cats reaches up to % and %, respectively. genotypes of genetic assemblages of the parasites infect humans (assemblages a and b) and other mammals including small animals. in contrast, parasites of the assemblages c and d are specific for dogs, assemblage f for cats. objectives of the study were to analyse the prevalence, potential epidemiological risk factors and symptoms of g. duodenalis infections in dogs and cats. to detect g. duodenalis, feces from dogs and cats was analysed with an elisa technique. after dna extraction real time pcr as well as multi-locus sequence typing was performed for the following gene loci: triosephosphate isomerase-, glutamate dehydrogenase-, beta-giardin-gene, ssu rrna. with a questionnaire possible epidemiological risk factors were evaluated. statistical analyses were performed using spss (odds ratio, kolmogorow-smirnow test, spearman correlation). fecal samples of dogs and cats were collected over a time period of months. the elisa test was positive in / dogs and / cats. of giardia positive dogs and of positive cats had gastrointestinal signs. genotyping was successful in of dog samples and were assigned to assemblages as follows: assemblage a (n = ), a/c (n = ), a/d (n = ), b (n = ), b/d (n = ), c (n = ), c/d (n = ), d (n = ). only one of positive cat samples could be genotyped and was atypically identified as assemblage d. significant correlations between giardia infection and age, clinical signs, deworming status and staying abroad were found. in this monocenter study a prevalence rate of . % in dogs and . % in cats was detected, which is in good accordance with previous studies. the study further highlights a high rate ( %) of asymptomatically g. duodenalis infected animals. as potential zoonotic assemblages were detected, transmission of giardia from small animals to humans (and vice versa) cannot be excluded. especially young and not dewormed animals had a higher prevalence. disclosures: no disclosures to report. canine parvoviral enteritis remains a common cause of morbidity and mortality in young dogs. the goal of this study was to document a large cohort of affected dogs and analyze several factors as possible predictors of fatal outcome. medical records were retrospectively searched for dogs with parvoviral enteritis diagnosed with a positive fecal antigenic test or a fecal pcr. dogs were included only if the medical records were complete. the population was compared to the reference population of the hospital on the same time period with chi square tests and several factors were analyzed as possible predictors of death with a logistic regression. one hundred and fourty seven cases were included. seventy percent of the dogs were non vaccinated puppies under the age of months. intact females and rottweiler, american staffordshire terrier and french beauce shepherd dogs were over-represented. clinical signs such as vomiting, diarrhea and dehydration were present in . %, . % and . % of the dogs respectively. hyperthermia, anemia and leucopenia were observed in . %, . % and . % of dogs respectively. the majority of the affected dogs were hospitalized for to days and the mortality rate was . % ( / dogs). hypoglycemia at admission was observed in / ( . %) dogs in which blood glucose was measured and was the only risk factor associated with death (p < . ). in this study, a predisposition of rottweiler, american staffordshire terrier and french beauce shepherd dogs was observed and hypoglycemia at admission was the only predictor of fatal outcome. disclosures: no disclosures to report. canine parvovirus (cpv) infections in dogs remain widespread around the world and still represent a major health threat in puppies. all vaccine manufacturers include this component in their core vaccination package, recommending injections at - weeks interval from to weeks of age. despite broad vaccination coverage, number of reports suggesting lack of efficacy in vaccinated dogs have been reported, which implicate vaccines belonging to all major manufacturers. these cases are usually considered as being linked to the interference with maternal antibodies (matab), able to persist beyond weeks of age, which has led most expert groups to recommend a third vaccination around weeks of age. persistence of matab actually represents a major issue when immunizing puppies against parvovirosis. indeed, matab titres higher than / in the haemaglutination inhibition (hi) test can still inhibit vaccine uptake whereas such titres do not prevent field virus infection. in contrast, hi titres higher than / to / are usually considered as protective against disease and virus excretion. this "immunity gap"is therefore a critical period for the puppy and the outcome of the vaccination. in order to evaluate the impact of residual mda on the efficacy of a standard primary vaccination protocol, we performed a vaccination field trial with serological follow-up. puppies from to weeks of age presented at veterinary practice received injections at weeks interval. serology was performed by elisa before (at d /v ), during (at d /v ) and after (d ) vaccination. average maternal antibodies titres were strongly correlated with the age of the puppy at primary vaccination, remaining at vaccine inhibiting level until~ week of age. average titres increased significantly after st injection of primary vaccination in most groups and in all groups after the nd injection of primary vaccination. individual variability remained significant: vaccine uptake was inversely and strongly correlated to the pre-vaccinal matab titre at vaccination. out of puppies ( %) didn't seroconvert, despite vaccination complying to the recommended schedule. vaccination was started in such dogs between . and . weeks and completed between . and . weeks and average initial matab titre was . log compared to . for the general population. in conclusion, this trial supports the recommendation of an additional injection of primary vaccination at weeks, especially in areas of high parvovirus prevalence / pressure, where high levels of matab are likely to be transferred to puppies. disclosures: all authors are employees of merial. ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . . % of the dogs were pcr positive. the aim of our study was to evaluate the usefulness of determining serum level of c-reactive protein (crp) in dogs naturally infected with bacterium a. phagocytophilum as a possible indicator of the clinical phase of the disease. pcr and/or ifa positive dogs with clinical presentation and/or thrombocytopenia were included in the study. based on the results, the dogs were divided into groups: pcr positive dogs; ifa positive (subdivided according to titer level from : -> : ) and pcr negative dogs; positive control group -pcr and ifa negative dogs with clinical signs and/or thrombocytopenia; negative control group -clinically healthy, pcr and ifa negative dogs. serum level of crp was determined using lifeassays Ò canine crp (lifeassays, lund, sveden) . an elevated concentration of crp (> mg/l) was determined in pcr positive and ifa positive dogs with an ifa titer ≥ : and coincides with the presence of clinical signs (most commonly general clinical signs, elevated body temperature, gastrointestinal problems) and/or mild ( . %) or severe ( %) thrombocytopenia. the assessment of crp concentration, in correlation with certain clinical alterations and thrombocytopenia, suggests that crp concentration is elevated in the acute phase of the disease and is in correlation with the aforementioned changes therefore can serve as an additional diagnostic parameter. the crp concentration in ifa positive dogs, regardless of ifa titer levels, and with present clinical signs and thrombocytopenia is higher (above detectable level, > mg/l) than in dogs without clinical signs or laboratory alterations, which may speak in favor of reinfection or reactivation of a persistent infection at least in cases when no other cause of inflammation can be found. specific treatment would therefore be reasonable in such cases, especially in cases of rising crp concentration. disclosures: no disclosures to report. gallbladder agenesis is a very rare cause of elevated liver enzymes in dogs. in this study, we evaluated the features of dogs [ males ( castrated) and females ( spayed)] with suspected gallbladder agenesis on ultrasonography. five different breeds were included: chihuahua (n = ), toy poodle ( ), german shepherd ( ), jack russell terrier ( ), and shiba dog ( ). the median age was . ( . - . ) years. ten dogs were asymptomatic, while the other dogs showed decreased appetite ( ), vomiting ( ), ascites ( ), seizure ( ), and diarrhea ( ). all dogs showed elevated liver enzymes, with high alanine aminotransferase levels (median, u/l; - u/l) in dogs and high gamma-glutamyl transpeptidase levels (median, u/l; - u/l) in . gallbladder agenesis was confirmed using laparoscopy in dogs and laparotomy in . liver biopsy samples were obtained from all dogs. additional computed tomography cholangiography was performed for dogs using a -slice multidetector computed tomography (mdct) scanner following the intravenous administration of contrast medium (meglumine iotroxate). the obtained images were analyzed on a workstation, and they revealed an absent gallbladder in dogs and a vestigial gallbladder in . the common bile duct was dilated in dogs. for all dogs, laparotomy or laparoscopy was used to visualize the gallbladder and liver abnormalities, including malformed lobes and surface irregularities. acquired portal systemic collaterals were visually confirmed in dogs, who also exhibited hypoplasia of the portal vein on histological examination. in conclusion, most animals with gallbladder agenesis were asymptomatic in our study, indicating a good long-term prognosis. however, symptoms associated with portal hypertension must be monitored in animals with primary portal vein hypoplasia. disclosures: no disclosures to report. assessment of systolic arterial blood pressure (sap) is an important tool in small animal internal medicine practice, especially with diseases or clinical conditions that can cause hypertension or hypotension. the doppler method is noninvasive and has several advantages compared to oscillometric method. there are few studies about the effect of body position on sap in conscious dogs. the hypothesis was that animal positioning during measurement alters sap values. the study design was prospective and randomized regarding order of positioning measurements. one hundred and twenty client-owned, conscious, healthy or sick adult dogs, weighing up to kg were included. sap was recorded by doppler ultrasonography following american college of veterinary internal medicine consensus statement with animals positioned in sternal recumbency, right lateral recumbency and with the dog laying down on owner s lap. the order of body position was raffled at the time of measurement. five consecutive measurements on each body position were performed always on left forelimb and the average was calculated. sap values were higher in sternal recumbency ( mmhg, p %- % = - . ; p < . ) compared to those obtained on the owner s lap ( mmhg, p %- % = . - . ), and both were similar to right lateral recumbency ( mmhg, p %- % = - ). these results suggest that sap measurement obtained on owner s lap or right lateral recumbency can be used on clinic routine, but sap measurement obtained on sternal recumbency should be avoided, because such measures may be overestimated. disclosures: no disclosures to report. canine patients may be presented for blood pressure (bp) assessment when clinical diseases associated with systemic hypertension (ht) are suspected but not confirmed; this population may encompass patients that have normal bp, true ht or situational ht. the clinician's aim is to identify animals with ht reliably, while minimizing false positives. this prospective study investigated the repeatability of duplicate within-visit systolic bp assessments (sbp and sbp ) in consecutive canine patients presented for bp assessment in the small animal clinic ( duplicate sbp recorded from dogs) and in a control group of healthy dogs ( duplicate sbp obtained from control dogs), resulting in duplicate measurements for analysis. doppler methods were used for duplicate assessments and oscillometric methods were used for duplicate assessments; cuff size/location were consistent within any dog. sbp ≤ mmhg was considered normal (nml); sbp > mmhg was considered abnormal (abn). median (range) elapsed time between duplicate readings was ( - ) minutes; % of sbp were obtained within minutes of sbp . there was no correlation between elapsed time and change in sbp (p = . ). % of sbp were equal to or lower than sbp ; median decrease was ( - no dog with sbp > mmhg (n = ) had nml sbp . more dogs with abn sbp were panting ( / scored, %) compared to the group with dogs with nml sbp ( / scored, %, p = . ). sbp of dogs that stopped panting ( / , %) tended to decrease (p = . ). within-visit repeatability of bp diagnosis was good in dogs with nml sbp , but apparent false positive diagnoses of ht occurred in % of dogs with abn sbp . sbp > mmhg was repeatable in all dogs. panting may be associated with increased measured sbp by these methods. duplicate within-day measurements may help identify false positive ht diagnoses in dogs with initial sbp measurements > mmhg. disclosures: no disclosures to report. hypovitaminosis d has previously been shown to be prevalent amongst dogs with protein losing enteropathy (ple). outcome is generally poor in canine ple, and there is a lack of studies identifying underlying risk factors. the hypothesis of this study was that low vitamin d serum concentrations could be a risk factor for bad outcome in such patients. medical records for dogs seen at the royal veterinary college between and were reviewed to identify dogs with a diagnosis of ple confirmed by histopathology. dogs were included in the study if they had serum samples frozen within minute after sampling, had been kept at À degrees c until analysis, and if clinical activity scoring (cce-cai) had been recorded at the time of diagnosis. forty-three dogs were included in the study. follow-up with referring veterinarians was made to determine outcome of patients. patients were divided into two groups: patients deceased due to ple (poor outcome group, n = ) and patients alive or deceased due to another disease (good outcome group, n = ). treatments for patients were allocated to two groups: one group consisted of patients who were prescribed diet only and the other group received diet and immunosuppressive agents. samples were sent on dry ice to michigan state university's diagnostic center for population and animal health. ionised calcium (ica) was measured using an ion specific electrode and (oh)d was measured using a commercially available radio-immunoassay that has been validated for use in veterinary medicine. comparisons of outcome groups for age, ccecai, treatment, serum (oh)d and ica were performed using a mann-whitney u test or chi . logistic regression analysis was performed to determine possible risk factors for poor outcome. results: ccecai scores, age, and ica concentrations between the two groups were not significantly different. there was a significantly greater number of dogs treated with food alone in the group with good outcome ( / ) than in the poor outcome group ( / , p = . ). furthermore, median serum (oh)d concentration was significantly lower in patients with poor outcomes ( . nmol/l, range - nmol/l) compared to patients with good outcomes ( nmol/l, range - nmol/l, p = . ). using logistical regression, (oh)d serum concentration was a statistically significant factor for poor outcome (p = . ), with an increase of (oh)d serum concentration reducing the odds of having a poor outcome (odds ratio = . , % ci: . - . ). further studies are required to investigate vitamin d as a potential adjuvant therapeutic agent in ple patients. disclosures: no disclosures to report. campylobacter jejuni (cj), c. upsaliensis (cu) and c. helveticus (ch) are commonly isolated from dog and cat faeces but association with clinical signs is discordant or lacking. cj is a recognized human pathogen, cu is considered an "emerging" pathogen and ch is not considered pathogenic despite a high level of genetic similarity. recently, the greater wax moth, galleria mellonella, was described as an animal model of disease; these invertebrates have a high degree of functional and structural homology with the mammalian innate immune system. this study aimed to evaluate the pathogenic potential of cj, cu and ch using the galleria mellonella larvae model. twelve isolates of cj, of cu and of ch from dogs and cats were used for the inoculation of larvae. inocula were prepared by suspending isolates in phosphate-buffered saline (pbs) from which three -fold dilutions were made. each dilution was tested in duplicate sets of larvae. each larva was injected with - ll into the haemocoel via the last left pro-leg using g insulin syringes. controls consisted of pbs inoculated larvae and un-inoculated larvae. survival of larvae at °c in a h enriched microaerobic atmosphere was monitored for days postinjection. one subset of isolates was grown in mueller-hinton broth and used for the preparation of secretory products, and another grown on blood-agar and suspended in pbs for heat inactivation of minutes at °c for testing of whole-cell lysates and heat-stable insoluble and soluble components. the overall median survival of larvae was % with cj [iqr - ], % with cu [iqr - ], % with ch [iqr - ], % with pbs [iqr - ] and % for un-inoculated larvae [iqr - ]. a dose-dependent association was evident for each species with larval survival being similar between a low bacterial dose and pbs. larval survival presented a consistent pattern between species for medium and high bacterial loads; cj had a higher and faster larval death rate than cu and ch (p < . ), but no difference was observed between cu and ch (p = . ). there were no significant differences between species in any of the assays with secretory products, inactivated cells and soluble/insoluble cellular components. the observations within this invertebrate disease model support a varying pathogenic potential between the species studied that appears related to the (patho)biology of the species rather than their cellular components or metabolic products. the invertebrate animal model is promising in comparative pathogenicity studies. disclosures: no disclosures to report. a. grellet , s. dubois , a. feugier , c. girardet , s. magnan , v. andr eo , g. trombini , c.a. boehringer , j. suchodolski , j. steiner . royal canin, aimargues, france, veterinary department of the french army health service, suippes, france, gastrointestinal laboratory, texas a&m university, college station, tx, usa acute stress from medium or high duration high-intensity exercise has been reported to be associated with an increase in serum c-reactive protein (crp) concentrations, an important acute-phase reactant in dogs. however, the effect of exercise on fecal s a concentration, a biomarker of intestinal inflammation has not previously been evaluated in dogs. the goal of this study was to determine if moderate intensity short duration exercise causes an increase in crp and/or s a concentrations in dogs, potentially leading to misinterpretation of their results. thirty-seven adult military working dogs (german and belgian shepherd dogs; males; mean age = years [ . - . ]) were included in the study. fecal quality, fecal s a , and serum crp concentrations were evaluated just before and after standardized exercise ( minutes of bikejoring at a speed of km/h). fecal quality was evaluated based on a -point scale (from : liquid to : dry and hard feces). fecal s a and crp concentrations were assayed with previously validated elisa tests. data were analyzed with an anova test for repeated measurements (sas software). results are presented as medians and ranges. serum crp concentrations increased significantly after exercise (median before and after excercise mg/l [ - ] and mg/l [ - ] (p = . ). also, fecal s a concentrations were significantly higher after exercise compared with baseline concentrations ( ng/g [ - ] vs. ng/g [ - ], p = . ). no significant effect of exercise on fecal score was observed ( [ . - . ] before and after the exercise; p = . ). our study demonstrates that a moderate-intensity, short-duration effort performed by healthy army dogs causes significant increases in fecal s a and serum crp concentrations, as compared with baseline values, but within the respective reference intervals. therefore, a moderate exercise does not present a confounding variable in the interpretation of fecal s a or serum crp concentrations in healthy dogs. disclosures: this study was performed thanks the financial support of royal canin. imaging is an integral part of the work-up of canine gastrointestinal (gi) disease. radiography and ultrasonography are noninvasive modalities that can evaluate the bowel, but many findings lack desirable sensitivity or specificity. endoscopy directly visualizes gi mucosa, but is limited by the length of the endoscope and the need for general anesthesia, advanced training and expensive equipment. ambulatory light-based imaging (ali) is a new imaging modality that utilizes high-resolution cameras, a microprocessor, and led illumination to non-invasively visualize the gastrointestinal mucosa. ali is performed by oral administration of a fully automated device the size of a pill that is propelled by peristalsis. the aim of this study was to analyze image quality and gi transit times in a series of five client owned dogs undergoing ali. dogs were food-restricted for hour before and hour after capsule administration. capsules were retrieved and images were downloaded and analyzed. video clips of frames duration were obtained from the stomach; proximal, middle and distal small intestine; and proximal colon for assessment of image quality. internists rated the images on a scale of - ( = poor, = ex-cellent) based on clarity and resolution of images, and obscuration of the mucosa by fluid, bubbles or debris. scores for each region were compared using general estimating equation analysis. gastric and small intestine transit time were calculated based on visualization of passage of the capsule from the stomach to duodenum, and ileum to colon. clinical analysis of the entire video was performed by one of the authors. ali was successfully performed in / patients, with no adverse effects. average study duration was . ae . hour and mean image acquisition count was . ae . . gastric and small intestinal transit times were . ae . minute and . ae . minute, respectively. median (range) image quality scores were ( - ), ( - ) and ( - ), for the stomach, si and colon, respectively. image quality scores were significantly higher in the stomach and si than in the colon (p < . ). visualized lesions were consistent with gi ulcers ( dogs), inflammatory bowel disease ( dog), and bilious vomiting syndrome ( dog). one dog receiving chronic nsaids had a normal study. ambulatory light-based imaging resulted in good to excellent image quality throughout most of the gi tract. bowel preparation should be considered to enhance visualization of the colon. ali was safe and easy to perform in ambulatory dogs, and should therefore be considered in the work-up of canine gi disease. disclosures: drs. hardy and solomon are employed by infiniti medical. escg-p- establishment of a severity scoring system for outcome prediction in dogs with pancre-atitis. p.c. liu , f.r. wu , y.j. lee , b.l. su . graduate institute of veterinary medicine, national taiwan university, taipei, taiwan, national taiwan university veterinary hospital, national taiwan university, taipei, taiwan, institute of veterinary clinical sciences, national taiwan university, taipei, taiwan canine pancreatitis is the most common exocrine pancreatic disorder. the prognosis of canine pancreatitis is variably and no logistic regression constructed severity scoring systems are available. four hundred and thirty nine dogs diagnosed as pancreatitis with acute onset of compatible clinical signs, a positive snap Ò cpl[trademark] test, and/or associated abdominal ultrasonographic abnormalities between january and december were presented at national taiwan university veterinary hospital (ntuvh). one hundred and three dogs hospitalized with complete medical therapy and outcomes were selected for further analysis. the dogs were divided into survival (n = ) and non-survival (n = ) groups. forty-seven parameters including signalment, clinical signs, physical examinations, clinicopathological examination, complications and concurrent diseases were analyzed and compared between the two groups. logistic regression analyses were performed in this study. variables with p ≤ . were considered for further analyses. the mortality in this study was . %. age, heart rate, respiratory rate, white blood cell count, albumin, bun, creatinine, potassium, presence of systemic inflammatory response syndrome (sirs) and presence of oliguria or anuria were selected for constructing the scores. continuous variables outside the reference interval were separated into quartiles to yield quartile-specific odds ratios (ors) for survival. based on the integer value of the or, the scoring system was then developed by incorporating weighting factors assigned to each quartile. a predictive total score was calculated for each dog by summing all weighting factors. the total scores of each dog ranged from to . the severity scores in this study achieved an area under the receiver operating characteristic (auroc) of . . the optimal cut-off point for discriminating outcome was . with a sensitivity of . % and specificity of . %, respectively. the mortality was . % with a score ≥ , whereas . % with a score ≤ . there was a significant difference (p < . ) between the two groups seperated by the cut-off point. the severity scoring system of this study provides a reliable and clinical applicable method to predict clinical outcome in dogs with pancreatitis. disclosures: no disclosures to report. glucocorticoids (gcs) are known for their anti-inflammatory and immunmodulatory properties and are therefore often used in the therapy of canine inflammatory bowel disease (ibd). it was recently shown that endogenous gcs are also produced in the intestinal epithelium of men and mice and influence the gastrointestinal immune system in case of inflammatory or neoplastic conditions. thus, the aim of this project was to prove that gcs can be produced or metabolized in the canine intestinal epithelium. five healthy beagle dogs were included into this prospective study. all dogs were clinically examined, given a clinical score using the canine ibd activity index (cibdai) scoring system, also gastrointestinal endoscopy was performed. mucosal biopsy specimens from duodenum were examined histologically from a board certified pathologist using the wsava grading. biopsy incubation of - endoscopical mucosal biopsies in tissue culture medium with h-labeled progesterone in the absence of any stimulation was performed. the mean age of the included dogs was . + . years, the mean weight was . + . kg. all beagle dogs had a mean clinical score of + . the mean wsava scoring was + . . after hours, supernatant was harvested and radioactive progesterone metabolites formed were detected using high performance liquid chromatography plus liquid scintillation counting. in all dogs the h-progesterone was metabolized into various steroid species, nevertheless a local production of cortisol could not be proven. in summary, it could be shown that precursors of gcs can be metabolized by healthy canine intestinal mucosal tissue. disclosures: no disclosures to report. we studied the relationship between pancreatitis and cardiac injury in dogs and cats. previously, we validated a cardiac troponin i (ctni; vet j : - , ) assay for sensitive and specific detection of cardiac injury in domestic animals. we found various non-cardiac diseases of dogs and cats were associated with cardiac injury detected by serum cardiac troponin i, including some cases of pancreatitis. also, we validated the dggr-lipase assay for cost-effective, sensitive and specific detection of pancreatitis in dogs and cats (vet clin path :e - , ; :e - , ). herein, we tested the hypothesis that pancreatitis was associated with cardiac injury. ctni was measured by advia centaur tni-ultra assay; dggr-lipase by the randox colourimetric assay. we retrospectively analysed data from dogs and cats admitted to ucd veterinary hospital in which both ctn and lipase had been measured. upper limit of reference range for lipase in dogs is u/l; we consider - indicative of mild pancreatitis, - moderate, and > as marked. upper limit of reference range for lipase in cats is . reference range for ctni is < . ug/l for dogs and cats. we consider . - . indicative of mild cardiac injury, . - as moderate, and > . as marked. dogs and cats had both lipase and ctni measured. seventy-eight dogs had normal troponin; had normal lipase and had normal lipase and normal ctni. dogs ( %) had pancreatitis as indicated by increased lipase. in ( %), pancreatitis was mild, in ( %) it was moderate, and in ( %) it was marked. sixty-seven of dogs had increased ctni: mild in ( %), moderate in ( %), and marked in ( %). cardiac injury in dogs with pancreatitis was absent in %, mild in %, moderate in %, and marked in %. of cats had normal ctn; had normal lipase. of cats had pancreatitis, severely in . lipase and ctni was correlated (r = . ) for dogs and cats. we conclude that both pancreatitis and cardiac injury, as indicated by high-sensitivity and high-specificity assays randox-dggr-lipase and centaur-ctni, respectively, are not uncommon in veterinary hospital cases. we confirm and extend our previous work. pancreatitis in dogs and cats is typically associated with cardiac injury. severities of pancreatitis and cardiac injury are correlated. for~ % of dogs and cats with pancreatitis, cardiac injury is moderate to marked. disclosures: no disclosures to report. intracellular colonization may serve as a protected niche where helicobacter spporganisms evade effective treatment, contributing to recolonization. confocal endomicroscopy (cem) is an endoscopic modality allowing in vivo gastrointestinal imaging at high resolution; and has aided real-time identification of helicobacter pylori and intracellular and mucosally associated bacterial. in dogs, non-helicobacter pylori-helicobacter (nhph) are described intracellularly. the objective of this study was to determine the utility of cem to identify nhph in dogs compared with other diagnostic modalities; and to assess its ability to identify intracellular organisms. fourteen clinically healthy dogs underwent standard gastroduodenoscopy followed by cem using topical acriflavine. images were obtained using cem at a minimum of five sites within the stomach. endoscopic pinch biopsies were obtained for histopathology, polymerase chain reaction (pcr) and fluorescence in situ hybridisation (fish). methodologies were compared for their sensitivity in detecting the presence and distribution of nhph and their ability to identify intracellular organisms. cem provided high quality images allowing in vivo identification ofnhph in dogs, as did fish post-procedure analysis. standard histopathology identified nhph in only . nhph were identified within the superficial gastric mucus, and gastric pits. distribution throughout the stomach was diffuse and multi-focal. cem findings correlated with fish and pcr, however only fish enabled identification of intracellular nhph which were present in of dogs. cem provides in vivo histology images and is capable of identifying nhph during gastroscopy, but is unable to identify intracellular organisms using the current fluorophore protocol. nhph in the canine stomach are commonly identified intracellularly. disclosures: dr sharman has shares in optiscan imaging pty ltd. chronic enteropathies (ce) and exocrine pancreatic insufficiency (epi) can both cause hypocobalaminemia in cats. current supplementation protocols for cobalamin in cats call for repeated parenteral injections. in humans, several studies have reported equal efficacy of oral administration of cobalamin. there is also evidence that oral supplementation is effective in dogs with hypocobalaminemia. recently, it has also been reported that oral cobalamin substitution restores normocobalaminemia in healthy elderly cats. the purpose of this retrospective case series was to evaluate whether oral cobalamin supplementation can restore normocobalaminemia in hypocobalaminemic cats with chronic enteropathies. a computerized database search for cats treated at evidensia specialist animal hospital, helsingborg, sweden during - was performed. inclusion criteria were cats with symptoms of ce, an initial serum cobalamin concentration below pmol/l (reference interval: - pmol/l) and daily oral treatment with cyanocobalamin ( mg/tablet; ⅛-¼ tablet/cat daily). follow-up serum cobalamin concentration was measured - days after initiation of daily oral cobalamin supplementation. thirteen cats aged - years (median ) of different breeds met the inclusion criteria. presenting complaints included vomiting ( / ), anorexia ( / ), diarrhea ( / ), weight loss ( / ), and lethargy ( / ). increased pancreas specific lipase (spec fpl Ò ) serum concentrations were reported in / cats and / had increased serum alanine transaminase activity. feline serum trypsin like immunoreactivity (ftli) was determined in / cats revealing results within the reference interval. all cats had an abdominal ultrasound, / had changes related to the gastrointestinal tract such as mild-moderate thickening of the small intestinal wall, thickening of the muscularis layer, poor definition of intestinal wall layers, and/or enlargement of the mesenterial lymph nodes, histopathology was performed in / cats, revealing small intestinal inflammation in five cats and small intestinal lymphoma in one. serum cobalamin increased in all cats with treatment. the concentration difference ranged from to pmol/l (mean: pmol/l). mean (aestandard deviation) serum cobalamin concentrations were (ae ) pmol/l before and (ae ) pmol/l after supplementation. this difference was statistically significant (p < . , paired t-test). our results suggest that oral cobalamin supplementation is effective in normalizing serum cobalamin concentrations in cats with various enteropathies. prospective studies are warranted comparing cellular cobalamin status in cats being treated with parenteral or oral cobalamin supplementation. disclosures: no disclosures to report. pulmonary thromboembolism (pte) is observed in dogs with idiopathic-inflammatory-bowel disease (ibd) and particularly with protein-losing enteropathy (ple). hypercoagulability has been attributed to antithrombin (at) loss although the pathogenesis is likely to be more complex. in humans, where venous thromboembolism (te) is a wellrecognised complication of crohn's disease and ulcerative colitis, the pathogenesis of te is still not completely understood. derangements in procoagulant and anticoagulant factors have been demonstrated, including increased circulating procoagulant microparticles (mps). the aim of this pilot study was to evaluate mp-procoagulant activity in the plasma of dogs with ibd and ple using a functional elisa assay (zymuphen-mp-activity, aniara). we hypothesised that all dogs with ple and a subset of dogs with ibd but without ple would have increased levels of circulating mps. the study group consisted of dogs with ibd, including with ple. diagnosis was based on compatible clinical and histopathology and exclusion of other causes of chronic gastrointestinal disease. ple was defined as ibd plus hypoproteinaemia (serum total protein < g/l) and hypoalbuminaemia (serum albumin < g/l). pte was diagnosed in one dog with ple, and suspected in a second. a control group comprised healthy dogs undergoing blood sampling for reasons unrelated to the study including blood donor screening (n = ) and health assessment (n = ). dogs were considered healthy based on owner evaluation, physical examination, haematology and serum biochemistry. median mp procoagulant activity in dogs with ibd was . nm (range . - . ) compared with . nm (range . - . ) in the control group. median mp activity in ple dogs was . nm (range . - . ) compared with . nm (range . - . ) in non-ple ibd dogs. using kruskal-wallis test for nonparametric data and dunn's multiple comparisons test the groups were not statistically different. interestingly, mp-procoagulant activity value in the dog with documented pte was . nm; in the dog with high clinical suspicion for pte, mp-procoagulant activity was . nm. the highest mp-procoagulant activity was detected in a healthy control dog, raising concerns for pre-analytical or sampling error. removing this measurement had no impact on statistical analysis, which remained nonsignificant. mp-procoagulant activity > nm is considered clinically relevant in humans. employing a similar cut-off, / of controls, / of ibd and / of ple group would be defined as having increased levels of circulating mps. further studies are required to fully evaluate the clinical relevance and diagnostic potential of mp evaluation. disclosures: no disclosures to report. the intestinal microbiota is increasingly linked to the pathogenesis of chronic enteropathies (ce) in dogs. while imbalances in duodenal and fecal microbial communities have been associated with mucosal inflammation, relatively little is known about alterations in mucosal bacteria seen with ce involving the ileum and colon. the aim of the present study was to use fluorescence in situ hybridization (fish) techniques to investigate the composition and spatial organization of mucosal microbiota in endoscopic biopsies obtained from dogs with ce and controls. tissue sections from the ileum and colon from dogs with inflammatory bowel disease (ibd), dogs with granulomatous colitis (gc), dogs with intestinal neoplasia, and controls were studied by fish targeting the s rrna genes of total bacteria, group-specific organisms, and individual bacterial species shown to be relevant in human ibd. the numbers of mucosal bacteria were analyzed using generalized linear models for each of the colon and ileum tissues, with spearman's rank correlation coefficients used to test the correlation between mucosal microbiota and inflammatory (cib-dai score, histopathology) indices. the ileal and colonic mucosa of healthy dogs and dogs with ce was predominantly colonized by bacteria localized to free and adherent mucus compartments. dogs with ce harbored more (p < . ) mucosal bacteria belonging to the clostridium-coccoides/eubacterium rectale group, bacteroides, enterobacteriaceae, and escherichia coli versus controls. within the ce group, ibd dogs had increased (p < . ) enterobacteriaceae and e. coli bacteria attached onto surface epithelia or invading within the intestinal mucosa. bacterial invasion with e. coli was present in the ileal and colonic mucosa of dogs with gc (p < . ). dogs with intestinal neoplasia had increased (p < . ) adherent (total bacteria, enterobacteriaceae, e. coli) and invasive (enterobacteriaceae, e. coli, and bacteroides) bacteria in biopsy specimens versus all other groups. increased numbers of total bacteria adherent to the colonic mucosa were associated with clinical disease severity (cibdai score) in ibd dogs (p < . ). these results indicate that histopathologic lesions of canine ce are associated with different populations in ileal and colonic mucosal microbiota. these spatial, segment-specific structure and differential response of select bacterial groups to intestinal inflammation may be pivotal regarding the functional consequences of these alterations in the pathogenesis of canine ce. disclosures: no disclosures to report. abdominal girth is used as an indicator of human adiposity, with such measurements being made by tape measure. given concerns in precision and accuracy of repeat measurements, some tape measure designs have inbuilt mechanisms to improve consistency. although body condition scoring is the most common method of assessing adiposity in dogs, zoometric systems have also been developed requiring the use of a tape measure. however, the precision and accuracy of such zoometric measurements are not known. the aim of this study was to determine the precision and accuracy of different types of tape measure for a variety of dimensional measurements. a variety of length (head, forelimb, hindlimb) and circumferential (neck, thorax, and abdomen) were made using three different tape measures, two of which were designed to improve precision (standard tape; myotape tm and gulick ii tm ). to assess intra-operator variability, measurements were taken for consecutive days from healthy dogs; to assess inter-operator variability, operators independently took measurements from a group of dogs of various breeds and sizes. for intra-operator comparisons, precision was good overall (coefficient of variation [cv] ≤ % for all measurements). for interoperator comparisons, precision was more variable and, although reasonable on average (mean cv - %), it varied depending upon tape measure type (p = . ; greatest for standard tape measure, least for gulich ii tm ), and could be highly variable for some measurements in individuals dogs (maximum cv % for head measurements with standard tape measure). significant differences also existed in the absolute results of circumferential measurements taken by the different tape measure types (neck p = . ; thorax p < . ; abdomen p < . ). finally, significant operator differences were also evident for some measurements (head p = . ; hindlimb p = . ), but not for others (forelimb p = . ; neck p = . ; thorax p = . ; abdomen p = . ). in summary, although precision for individual operators making zoometric measurements is good, significant inter-operator and tape type differences exist. these results have implications for systems using a range of zoometric measures to assess adiposity. in order to ensure precision and accuracy, it is recommended that the same operator take all measurements with the same type of tape. disclosures: the study conducted was not supported by a research grant. ajg's readership is funded by royal canin; ajg has also received financial remuneration and gifts for providing educational material, speaking at conferences, and consultancy work; slh's post at the university of liverpool is also funded by royal canin. koizumi , m. noda , c. shimokawa , a. kusumi , t. kobayashi , t. watari , k. otsuji . teikyo university of science, tokyo, japan, grace animal hospital, tokyo, japan, nihon university, fujisawa, japan body condition score (bcs) is a method that is commonly used in the diagnosis of nutritional status in small animals. however, this method is subjective due to its sensory evaluation. therefore, the improvement of the precision of the bcs diagnosis is expected. our previous study has shown that the bcs model that we created improved the precision of the bcs diagnosis ( ). however, a palpation site was not identified. a palpation site must be the site where thickness of subcutaneous fat is able to capture for measuring animal's obesity status. therefore the objective of this study was to find a remarkable body site of the changes with obesity status using ultrasonic diagnostic equipment. nine dogs which varied in the percent of body fat were used in this study. the percent of body fat was measured by a body fat analyzer for dog (kao). the image analysis of a palpation site was evaluated using echo, xario ssa- a (toshiba) which attached to a linear probe. the measurement points were , and o'clock positions on the ribs of t , t and t . the distance (d) from skin surface to the rib was measured in the echogram. the distance (l) from scapula to ilium was measured to offset the difference in physique by dog breeds. the d/l was used to compare relative value of the quantity of fat at each measurement point. bcs of dogs which used in this study were from bcs of to bcs of . there were no dogs in bcs of and bcs of . a statistically significant correlation was found between bcs and d/l value. the d/l value increased in order of t , t and t in bcs of and . this suggests that the thickness of subcutaneous fat in the chest is thicker at the head side than the tail side. also, as for the d/ l value from back to abdomen, the highest value was found at the position of : and : . this tendency was the most remarkable in bcs of but no difference in the d/l value was recognized in the dogs in bcs of . in conclusion, the position of : or : on the t is the suitable palpation point at the chest. ( ) k. otsuji, m. suzuki, n. furukawa, n. kobayashi, a. koizumi, a. kusumi, t. kobayashi efficacy of the body condition score (bcs) model in the bcs diagnosis wsava proceeding p , disclosures: no disclosures to report. body condition score (bcs) is a method that is commonly used in the diagnosis of nutritional status in small animals. bcs has been recognized as one of the screen method of nutrition diagnosis by american animal hospital association in . however, this method is subjective due to its sensory evaluation. therefore we made a bcs model to increase the precision of the bcs diagnosis and have shown the efficacy of the bcs model ( ). however, the prototype model which we have reported before tended to have higher bcs than a target bcs. therefore, we improved the bcs model in this study. sixty seven dogs which varied in the bcs were used in this study. body fat percentage was measured by using a body fat analyzer for dogs (kao healthlab bif- ). the bcs model was improved by using several rubber sheets. relative hardness of stacking rubber sheets in each bcs was measured by durometer mj-dua-c (satotec tokyo, japan). bcs diagnosis of dogs was performed by pet owner by using the bcs model. bcs of represents the most hard in the bcs model and the hardness decreased linearly and it was the lowest in of bcs. these values were as expected. high correlation was recognized between bcs and body fat percentage. these results suggested the efficacy of bcs model. however, the body fat percentage in the dogs diagnosed as bcs of was higher than body fat percentage which has been reported in the previous paper. there were no dogs with the body fat percentage < % which were diagnosed as bcs of . we need more study in future to make clear the difference of body fat percentage between our data and date of the previous research. the completion of this bcs model will help provide the precision of nutritional diagnosis in dogs. ( in humans the metabolic syndrome (ms) is a well-recognised and extensively studied entity that comprises obesity, hypertension, dyslipidaemia, and glucose intolerance. it is associated with an increased risk of cardiovascular diseases and diabetes. recently, human ms criteria were adapted for dogs to define the condition of obesity-related metabolic dysfunction (ormd). it was observed that ormd was associated with increased circulating insulin and decreased adiponectin concentrations, suggesting that in dogs, as in humans, there are links between obesity, ormd, and associated diseases, although pathogenetic mechanisms and health significance for dogs remain unknown. the main aim of the present study was to compare plasma proteomes of obese dogs with and without ormd, so as to investigate the mechanisms associated with canine ormd and their possible significance in the health status. eight obese dogs referred for weight management at the royal canin weight management clinic, university of liverpool participated in the study. clinical assessments included physical examination, body condition scoring, blood pressure measurement and routine clinicopathological analysis. surplus plasma was used in proteomic analysis. samples were first treated with proteominer for thedepletion of high-abundance proteins and subsequently analysed by using -de dige methodology. of the eight dogs in the study, dogs had ormd and dogs did not. image analysis and further statistical analysis allowed identification of spots with differential expression concentration between dogs with and without ormd. among the spots, were over-expressed and were down-expressed in dogs with ormd than in dogs that did not presented ormd. although the results of the present study are preliminary and still the identification of the spots is up to be performed, the observed datareveal that dogs with ormd present alterations in their plasma proteomes that could be responsible for the development of ormd-related pathologies. disclosures: the study was funded by waltham. ajg's readership is funded by royal canin; ajg has also received financial remuneration and gifts for providing educational material, speaking at conferences, and consultancy work; slh's post at the university of liverpool is also funded by royal canin. vb is an employee of royal canin and pjm is an employee of wal-tham. the aim of the study was to assess the diagnostic value and the discrimination potential between the normal heart size and microcardia or cardiomegaly of a method which calculates the cardiothoracic ratio (ctr) using area measurement, compared to the vertebral heart scale method (vhs) used as reference for the cardiac size, in dogs. one hundred-nine dog x-rays were accepted into study. the patients belonged to small and medium size breeds, fourty-seven were males and sixty-two females with age between and years. the analogic x-rays were scanned and transferred to a computer where the vhs and ctr was calculated for each patient with a commercial software and the data was collected and processed in a statystical analysis software. the patients were distributed into groups by respiratory phase and heart size. there was a low correlation between the vhs and ctr (r = . ), but statistically significant (p? . ). a good correlation was obtained between vhs and ctr in microcardia, normal heart size and cardiomegaly groups (p < . ). furthermore, between the ctr in dogs with microcardia and those with normal cardiac size, as well as between ctr in dogs with normal cardiac size and those with cardiomegaly, a significantly statistic difference (p < . ), respectively (p < . ), was obtained. among the groups distributed by respiratory phase and vhs, a statistically significant difference was obtained only between normal cardiac size and cardiomegaly during inspiratory phase groups (p > . ). for the x-rays taken in inspiratory phase, a cutoff of . had a sensitivity of % and a specificity of % for diagnosing cardiomegaly. the ctr can be considered a valid method being able to discriminate between the patients with microcardia and cardiomegaly from those with normal heart size. moreover, it was found that a ctr over the cutoff of . , mesured during inspiratory phase is a good predictor for cardiomegaly. key words: cardiac, cardio-thoracic ratio, dog, x-ray. disclosures: no disclosures to report. the canine cardiac conduction system is modified by anatomical and functional adaptations of the maternal heart during gestation. however, it is not clear if these changes persist or are modified after parturition. therefore, the aim of this study was to describe canine electrocardiographic features during the course of normal puerperium. twenty healthy pure-bred, - ( . ae . ) year-old, weighing . - kg ( . ae . ) bitches were included in this study. all the animals whelped healthy puppies at term which were weaned on day after parturition (day ). all the dogs were electrochardiographically evaluated on days À , , , , , , and . mean electrical axis (mea; degrees), p wave amplitude (pa; mv) and duration (pd; ms), p-r interval (pr; ms), qrs complex amplitude (qrsa; mv) and duration (qrsd; ms), q-t interval (qt; ms), and s-t segment (st; mv) were calculated at mm/s of velocity. the rr interval immediately preceding each complex was recorded and qt interval was corrected (qtc) by van de water formula [qtc = qt- . (rr- )]. later, lead ii was recorded at mm/s to analyze heart rate (hr; bpm) and cardiac rhythm (cr; normal sinus rhythm or sinus arrythmia). values of hr, mea, pa, pd, pr, qrsa, qrsd, qt, rr and qtc were analyzed by anova for repeated measures followed by tukey test. cardiac rhythm was analyzed by chi square test (spss . , spss inc. chicago, il, usa). p < . was considered significant. during the study period, hr (p < . ) and qtc (p < . ) progressively decreased, while rr (p < . ) and pa increased (p < . ). qrs complex amplitude diminished in the second week after parturition and then increased during the following weeks (p < . ). mean electrical axis shifted to the right during this period (p < . ). on day À , most of the bitches presented normal sinus rhythm in contrast with day , in which most of the bitches presented sinus arrhythmia (p < . ). from day onward, all the bitches showed sinus arrhythmia. p wave duration, pr, qrsd, qt and st remained unchanged during puerperium. it is concluded that most electrophysiological adaptive changes of canine gestation reverted during normal puerperium. the present study contributes to the understanding of canine cardiac physiology during this reproductive stage. disclosures: no disclosures to report. esvc-p- echocardiographic assessment of pregnant queens. p.g. blanco, r. rodr ıguez, a. carranza, a. rube, r. vercellini, p.r. batista, m. t ortora, c. gobello. national university of la plata, la plata, argentina cardiovascular adaptation during gestation guarantees an appropriate development of the fetuses and maternal cardiovascular maladaptation is highly correlated with adverse pregnancy outcome. while, the hemodynamic changes occurring during canine pregnancy have been described there is scarce information concerning maternal cardiac variations during feline gestation. thus, the aim of this study was to describe cardiac morphology and systolic function variations during normal feline pregnancy. eighteen pregnant queens were echocardiographically evaluated (toshiba nemio xg, japan, mhz transducer) every days from day (defined as day of mating) to parturition. left ventricular dimensions were measured in the short axis view, during mmode tracing. shortening fraction was calculated as (lvdd À lvds)/lvdd to assess systolic function. stroke volume (ml) was calculated as the product of the velocity time integral (measured by pulsed-wave doppler) and the cross-sectional area of the aorta. cardiac output (l/minute) was calculated as the product of stroke volume and heart rate (bpm) derived from electrocardiographic monitoring. uterine artery resistance index (ri) was obtained by doppler ultrasound. all the parameters were analyzed by repeated measures anova. all the queens delivered healthy kittens at term. throughout the study period, interventricular septum in diastole (p < . ) and systole (p < . ) and left ventricular diameter in diastole (p < . ) augmented during gestation. shortening fraction (p < . ), cardiac output (p < . ) and maternal heart rate (p < . ) also increased up to parturition. conversely, uterine artery resistance index decreased in the same period (p < . ). it is concluded that cardiac structure and function varied during normal pregnancy in these queens. cardiac eccentric hypertrophy, systolic function and cardiac output increases appear to be the consequences of the hemodynamic modifications occurring during pregnancy. the assessment of maternal cardiovascular function may prove a useful screening tool to detect pregnancy complications in feline reproduction. disclosures: no disclosures to report. tricuspid annular plane systolic excursion (tapse) is an echocardiographic measure that allows to assess right ventricular systolic function. it has been described reference values for tapse in normal adult dogs, but there is no reference to influence of age in tapse in dogs. this influence has been reported in humans. thus, the goal of this study is to determine the reproducibility of the measure tapse in normal dogs and to determine the relationship between tapse and age in healthy beagle dogs. tapse was measured from an m-mode recording of the lateral aspect of the tricuspid valve annulus obtained through a left parasternal apical -chamber view. tapse values were averaged from measurements on consecutive beats during sinus rhythm. the measurements were recorded by two different persons (c-v, a.; m-m f.) with different grade of experience in canine echocardiography studies. all patients had a complete two-dimensional and doppler study using an envisor chd (philips Ò ) ultrasound system. twenty-three healthy beagles were used. the study was approved by the ethical committee of veterinary medicine service of las palmas de gran canaria university (spain) and it was carried out in accordance with the current european legislation on animal protection. these dogs were divided in three different groups according to age: group included twelve dogs under years, group included three dogs between and years, group included eight dogs older than years. we analyzed differences between groups using non-parametric (kruskal wallis and wilcoxon scores [rank sums]) test. there were no differences with respect to sex. dogs in group presented higher tapse values than group or ( . ae . cm vs . ae . cm vs . ae . cm; p < . ). statistic intra-observer and inter-observer agreement using the intraclass correlation coefficient was . (p < . ). this study showed that tapse measurement is easily obtainable with a standard echocardiography system, and has adequate interobserver agreement. this study showed higher values of tapse in normal young dogs with respect to older dogs. these results are similar to the results obtained in humans, and could reflect a less effective right ventricle with age. the values presented should be taken with caution due to the relatively small number of patients included. it may also be necessary to validate results in future studies with a second independent sample of dogs of other races. disclosures: no disclosures to report. tetralogy of fallot (tof) is a congenital heart disease characterized by abnormalities, i.e., pulmonic stenosis, ventricular septal defect (vsd), aortic overriding and secondary right ventricular hypertrophy, caused by anterior deviation and abnormal septation of the conal septum during the embryonic period. few studies have reported the hemodynamic consequences and clinical outcome of tof in small animals. the objective of this retrospective study was therefore to document the epidemiological, clinical, echo-doppler findings, and survival, in a canine and feline population with tof. the case records of animals diagnosed with tof by combined use of echocardiography and doppler examination were reviewed ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . tetralogy of fallot was identified in animals ( dogs, cats). the most commonly represented breeds were terriers for dogs ( / , . %), and domestic shorthair for cats ( / , . %). most included animals ( / , . %) were clinically affected at the time of diagnosis. pulmonic stenosis was characterized by a variable systolic doppler-derived pressure gradient both in dogs (median [range] mmhg ) and cats ( mmhg ), and associated with hypoplasia of the pulmonary trunk in one third of the cases ( . %). most vsd were large, with a median vsd: aorta ratio of . [ . - . ] in dogs and . [ . - . ] in cats. median age at death from cardiac cause was . months [ . - . ] without significant difference between dogs and cats (p = . ). these results suggest that in both cats and dogs tof-related death occurs predominantly in young adult animals with major hemodynamic consequences at the time of diagnosis. disclosures: no disclosures to report. the aim of this study was to assess whether and how radiographic and echocardiographic cardiovascular variables differ across age bands of healthy cats. a cohort of clinically healthy cats were categorized into three groups: adolescent-adult ( . - years; n = ), middle-aged ( - years; n = ), and geriatric ( - years; n = ). all cats underwent a full physical examination, a complete blood count, routine biochemical profile, a baseline serum total thyroxine concentration, auscultation, noninvasive blood pressure measurements, thoracic radiography, electrocardiography, and echocardiography. cats with hypertension, hyperthyroidism, cardiac, or renal disease were excluded from the study. body weight, body condition score, systolic blood pressure, heart rate, and all echocardiographic indices were similar across the three groups. the mean (aestandard deviation [sd] ) vertebral heart scale (vhs) value obtained for the geriatric group ( . ae . ) was significantly greater than that obtained for the adolescent-adult group ( . ae . ; p = . ). the mean ratio of the distance between the cardiac base and dorsal sternum to thoracic cavity height at the point of the cardiac base was significantly less in the middle-aged ( . ae . ) and geriatric ( . ae . ) groups than in the adolescent-adult group ( . ae . ; both p < . ). the mean angle between the cardiac long axis and the body axis was significantly smaller in middle-aged ( . ae . °) and geriatric cats ( . ae . °) than in adolescent-adult cats ( . ae . °; both p < . ). the mean angle between the cardiac long axis and the sternum of middle-aged ( . ae . °) and geriatric cats ( . ae . °) was significantly smaller than that in adolescent-adult cats ( . ae . °; p = . and p = . , respectively). additionally, the degree of undulation of the thoracic aorta correlated positively with age (r = . , p = . ). these findings suggest that differences in the horizontal alignment of the heart, thoracic-aorta undulation, and vhs in healthy geriatric cats, relative to observations in younger cats, can be considered to be age-related. disclosures: no disclosures to report. the aim of this study was to investigate the presence of pulmonary hypertension (ph) in young cats affected by single or mixed lungworm infections. twenty-three cats infected with lungworms were examined at the veterinary teaching hospital of teramo, italy, in - . animals underwent to a complete physical examination and to two-or three-views radiographic analysis of the thorax. a minimum database (i.e. cbc, serum biochemistry, serology for fiv antibody and felv antigen) was obtained for each patient. nine cats were excluded for concomitant diseases, while cats were included in the study. microscopic identification of parasites was confirmed by molecular tests and all cats received an anthelmintic treatment. a single infection by aelurostrongylus abstrusus was diagnosed in eleven cats, while three cats had a troglostrongylus brevior infection either alone or in combination with a. abstrusus. transthoracic echocardiography was performed using an ultrasound unit with a mhz phased array transducer. no structural abnormalities of the tricuspid valve and sign of pulmonary stenosis were detected. the two-dimensional and m-mode echocardiography showed a cardiac involvement in three cats. one cat, infected by a. abstrusus and t. brevior showed a mild systolic tricuspid regurgitant jet with color doppler of . m/s, while another a. abstrusus-infected cat, had mild tr of . m/s with a mean paps of mmhg which resolved within weeks after therapy. one cat diagnosed with troglostrongylosis, showed a marked right-sided cardiac enlargement of mm, and a large systolic tricuspid regurgitant jet with a tr peak velocity of . m/s recorded at continuous-wave doppler via a color doppler echocardiography. the minimum pressure difference between the right ventricle and the right atrium was estimated mmhg and the paps was at least mmhg. the echocardiographic and doppler evidence of mild ph persisted at further examination performed until months after diagnosis. ph is rare in cats, despite cases of reversible ph are known in cat aelurostrongylosis. in this study the first case of irreversible ph infection in a cat affected by t. brevior is presented and this finding further supports the high pathogenicity of troglostrongylosis, especially in young patients. in cats with lungworm infection, possible cardiovascular complications must be taken into account and these infections should be always considered in the differential diagnosis in cats with cardiorespiratory signs. disclosures: no disclosures to report. ventricular function by conventional echocardiography and speckle tracking imag-ing although uncommonly assessed in veterinary cardiology, a right ventricular (rv) function has been shown to be an important prognostic determinant of many congenital and acquired heart diseases in human patients. our group has already demonstrated that two-dimensional ( d) color tissue doppler imaging provides a non-invasive evaluation of systolic and diastolic rv function in the awake dog with adequate repeatability and reproducibility. b however, other noninvasive ultrasound imaging variables reflecting rv function need to be further investigated, particularly in correlation with pulmonary arterial pressure (pap) values and left ventricular (lv) function. the aim of this prospective study was therefore to assess several indices of systolic and diastolic rv function using conventional echocardiography and speckle tracking echocardiography (ste) in healthy awake dogs of different breeds with documented systolic pap (spap) and lv function (lv ejection fraction and global lv systolic strain assessed using the simpson's derived method of disks and ste, respectively). imaging rv tested variables included tricuspid annular plane systolic excursion (tapse), right fractional area change (rfac, %), ste longitudinal systolic strain of the rv free wall (rvfw, %) and of the whole rv (i.e., global rv strain, %), ste longitudinal systolic strain rate (sr, s À ) and diastolic early:late sr ratio. additionally, d-guided m-mode ventricular measurements included the end-diastolic rv:lv diameter ratio (rvdd: lvdd) and the end-systolic rvfw:lvfw ratio. correlations between imaging variables were calculated by using spearman's correlation coefficients. means of age and body weight (aesd; range) of the study population were . years (ae . ; . - . ) and . kg (ae . ; . - . ), respectively. no correlations were found between rv morphological variables (i.e., rvdd:lvdd and rvfw:lvfw ratios) and all indices of systolic and diastolic rv function. global rv strain (mean ae sd = . ae . %) and rvfw strain ( . ae . %) were positively correlated (p < . ) with rfac ( . ae . %, r = . and r = . , respectively), and negatively correlated (p < . ) with spap ( . ae . mmhg [ . - . ], r = À . and r = À . , respectively). spap was also negatively correlated with the tapse:body weight ratio and systolic sr (r = À . and À . respectively, p < . ). there was no correlation between indices of lv function and ste indices of rv function, and no correlation either between ste rv indices of systolic function and the diastolic early:late sr ratio. in conclusion, ste provides a rapid and non-invasive evaluation of rv function that may be used for clinical investigations in canine cardiology. doppler-derived +dp/dt and -dp/dt from mitralregurgitation are considered indexes for assessment of systolic and diastolicfunction respectively, that have less load dependence than the ejection phaseindexes. this study aimed to determine correlation between doppler-deriveddp/dt and other systolic and diastolic echocardiographic indexes, and if theycan be used to identify dogs with and without remodeling, with or withoutcongestive heart failure (chf) and for evaluation of chronic mitral valvedisease (cmvd) severity. fifty-seven dogs with cmvd (stages b , b , c+d) wereincluded prospectively in an observational cross-sectional clinical study anddistributed in groups regarding the presence of remodeling and chf, to evaluate+dp/dt and -dp/dt, and distributed according to tdi-diastolic pattern tocompare Àdp/dt. group c+d ( mmhg/s, p -p = - ) had +dp/dt significantly lower compared to b ( mmhg/s, p -p = - ) and b ( mmhg/s, p -p = - ) (p = . ). groupc+d also had lower Àdp/dt, compared to b ( . mmhg/s ae . and mmhg/s ae . ; p = . ). dogs with chf compared to those without chf, presented lower +dp/dt ( mmhg/s, p -p = - ; mmhg/s, p -p = - ; p = . ) and Àdp/dt ( . mmhg/ s ae . ; mmhg/s ae . ; p = . ). regardingdiastolic function, Àdp/dt was lower for the restrictive pattern group ( . mmhg/s ae . ) compared to those without diastolic dysfunction, ( mmhg ae . ), delayedrelaxation ( mmhg ae . ) and pseudonormal patterns ( mmhg ae . ) (p < . ).when +dp/dt< mmhg/s, the post-test chance for the dog with cmvd to havechf is twice the chance than not having it. for Àdp/dt< mmhg/s theposttest chance of having chf is eight times higher than not having it. in conclusion, doppler-derived +dp/dt and Àdp/dt may contribute respectively, for systolic and diastolic assessment ofdogs with cmvd. disclosures: no disclosures to report. pulmonic stenosis (ps) is one of the most common congenital heart defects seen in veterinarycardiology practice. pulmonary balloon valvuloplasty (pbv) is considered to be the treatment of choice for dogs withsevere stenosis. whether dogs with moderate stenosis benefit from pbv remains unclear, and variables such as degree of hypertrophy, valve morphology, amount of tricuspid insufficiency and presence or absence of clinical signs aregenerally used when recommendations are made to pet owners. in this study we report the effect of valve type on pbv outcome in dogs treated at three different academic speciality cardiology practices. baseline echocardiographic images were evaluated at each institution and valve morphology was classified as either type a (n = , . mmhg, range - ) or type b (n = , . mmhg, range - ) and "no" (n = , mmhg, range - ) or "yes" (n = , mmhg, range - ) for presence of pulmonary annular hypoplasia when diameter was compared to aortic annulus. twenty-four hours following pbv both type a ( mmhg, range - ) and type b ( mmhg, range - ) valves had significant reduction in gradient compared to baseline (p < . ). this reduction remained significant at days (a: mmhg, range - ; b: mmhg, range - ; p < . for both). dogs with annular hypoplasia ( mmhg, and without annular hypoplasia ( mmhg range - ) had a significant reduction in gradient hours post pbv. it remained significant at days (with annular hypoplasia: mmhg, range - ; without annular hypoplasia: mmhg, range - ; p < . for both). when comparing to baseline, considering valve type, there was no significant difference in percent reduction in gradient for type a versus type b valves at both the -hour (a: %, range - ; b: %, range - ; p = . ) and -day (a: %, range - ; b: %, range - ; p = . ) recheck evaluation time points. additionally, there was no significant difference in gradient reduction when looking only at whether or not there was annular hypoplasia at hours (yes: %, range - ; no: %, range - ; p = . ) and days (yes: %, range - ; no: %, range - ; p = . ). in conclusion, classification of dogs with ps according to valve type and annulus morphology did not help predict the -day response to pbv. disclosures: no disclosures to report. hypertrophic cardiomyopathy (hcm) is the most common feline inherited cardiac disease and it is a major cause of morbidity and mortality. the osservatorio italiano hcm felina was formed in by a network of clinicians, geneticists and breeders, to monitor and study hcm in italian cats. since april , adult cats, belonging to various breeds, including maine coon, siberian, norwegian forest cats, ragdoll, sphynx, british sh, birmans and others have been prospectively enrolled. recheck evaluations were performed in cats. each cat underwent a clinical examination, echocardiography, and blood collection for genetic testing (when appropriate) and storage in the italian feline bio-bank. the disease status was defined by echocardiography according to established guidelines (left ventricular diastolic wall thickness < . mm = hcm negative, = . but < mm = hcm equivocal; = mm = hcm positive). the prevalence of hcm in the population was % ( cats); equivocal diagnoses were conferred on % ( cats). these prevalences did not differ between breeds. the prevalence of hcm in the italian feline population was lower compared to those reported by other investigators. evaluation of data from the entire population demonstrated that left ventricular end-diastolic wall thicknesses and aortic diameter showed a weak positive correlation with body weight (p < . , r < . for all variables), suggesting that weight-dependent limits on wall thickness should be considered in cats as is currently practiced in dogs. the lower prevalence of hcm in italian cat breeds compared with those examined elsewhere might be explained by different criteria for determining presence or absence of disease, differences in ages at which the subjects were examined, or a selection bias by breeders in presenting cats they consider "normal". disclosures: no disclosures to report. chronic mitral valve disease is by far the most common cardiovascular disease in dogs. the disease is caused by myxomatous degeneration of the mitral valve leaflets and, in approximately % of cases, it's accompanied by degeneration of the tricuspid valve. it is also described in previous studies that approximately % of affected dogs also have evidence of associated pulmonary arterial hypertension. the prevalence of the disease is higher in small breed dogs (under kg), although large breeds can also be affected and it occurs more frequently in males than in females. the present study aims to characterize the disease in a population of dogs in portugal. we retrospectively reviewed the medical records of dogs presented to hospital veterin ario do porto, with an echocardiographic diagnosis of canine chronic mitral valve disease, during a period of years. from this records, cases were identified, from which ( . %) were males and ( . %) were females. most of the dogs were mixed breed ( ) and different breeds of dogs were represented. the poodle was by far the most represented breed (n = ; . %), followed by english cocker spaniel ( . %), yorkshire terrier ( . %), boxer ( . %), epagneul breton ( . %), dalmatian ( . %), pekingese ( . %), labrador retriever ( %) and portuguese podengo ( . %). all other breeds represented . % of the population. regarding weight, . % of the dogs (n = ) weighted < kg, with a mean body weight of . kg (range . - kg). the mean age at diagnosis was . years old. we also observed that . % of the dogs (n = ) had concomitant degeneration of the tricuspid valve and . % (n = ) pulmonary arterial hypertension (ph). we categorized these dogs according to the severity of ph, in mild ph if they had a doppler echocardiography derived systolic pulmonary arterial pressure (spap) of - mm/hg, moderate ph (spap - mm/hg) and severe ph (spap > mm/hg). we found that . % (n = ) of dogs had mild ph; . % (n = ) moderate ph and . % (n = ) severe ph. as described in previous studies, the disease affects mainly males and small breed dogs, with a breed distribution that reflects the canine population in the country, including very including very popular large breed dogs in portugal, as the boxer and labrador. both the presence of concomitant tricuspid valve disease and ph had a higher prevalence in our study than previously described. disclosures: no disclosures to report. in people anemia is frequent in patients with heart failure (hf) and it is associated with poor outcomes. the most likely pathogenic factors include iron deficiency, chronic kidney disease (ckd), and cytokine production, although other factors may contribute. little is known about the prevalence of anemia in dog with cardiovascular disease. the aim of this retrospective study was to define the prevalence of anemia (hct ≤ %) in dogs with mitral valve disease (mvd) and to investigate associated risk factors (age, weight, azotemia, hf, iris/acvim class). medical records of dogs presented at the cardiology service, divet, university of milan (january -march ) were retrospectively evaluated. dogs with mvd with complete physical, thoracic and echocardiographic examinations, and serum biochemical panel, including serum creatinine (scr), were included in the study. dogs with other heart or systemic diseases, except ckd, or neoplasm were excluded. statistical analysis was performed using jmp . (sas institute). a p value < . was considered significant. two hundred and ninety dogs ( males/ females), . ae . years of age, . ae . kg of body weight fulfilled the inclusion criteria. the % of males and the % of females were neutered. the most represented breeds were mongrel ( %), miniature poodle ( %), york shire terrier ( %), and cavalier king charles ( %). dogs were % b , % b , % c and % d acvim class. while the % of the dogs were normoazotemic (scr < . mg/dl), . % were staged in iris , % in iris and . % in iris . the prevalence of anemia in dogs with mvd was % ( / ): showed mild ( ≤ hct ≤ %) and moderate ( ≤ hct ≤ %) anemia. sixteen dogs were in b , in b , in c and in d acvim class; thirty-four were normoazotemic ( %). anemic dogs showed a significant higher scr. normoazotemic dog showed significant higher hb, hct and rbc both in the overall population and in the anemic group. in the overall population dogs in different iris class showed statistically different hb, hct and rbc and hb was significantly lower in decompensated hf dogs. in conclusion although a relationship between anemia and azotemia/ckd was documented in our study, it is important to emphasize that most of the anemic dog were normoazotemic: anemia is not an exclusive finding of cardiorenal syndrome and should be considered as possible complication in dogs with mvd alone. disclosures: no disclosures to report. the objective of this study was to evaluate left atrial (la) function by left atrial total fractional area change (la-factotal) and left atrial ejection fraction (laef) in dogs affected with chronic mitral valve disease (cmvd) naturally acquired with and without congestive heart failure (chf). our hypothesis was that la-factotal and laef decrease with severity of cmvd. eighty dogs were included in a prospective observational cross-section clinical study, grouped according to cmvd severity based on echocardiographic evaluation and clinical signs. the dogs were equally distributed in each group: a, b , b and c, according to american college of veterinary internal medicine staging system. indicators of la function were calculated with the following equations: la-factotal = (lamaximum area Àlaminimum area)/lamaximum area, measured by apical four view; and laef = (lamaximum volume À laminimum volume)/ lamaximum volume, by biplane area-length method from the left apical four and two-chamber views. la-factotal showed lower values (p < . ) in group c ( . %, p %- % = . - . ) compared with groups a ( . %, p %- % = . - . ), b ( . %, p %- % = . - . ) and b ( . %, p %- % = . - ). group c had lower laef ( . %, p %- % = . - . ) than groups a ( . %, p %- % = . - . ), b ( . %, p %- % = . - . ) and b ( . %, p %- % = . - ) (p < . ). left atrial function, assessed by la-factotal and laef, was reduced in dogs with cmvd and chf compared with healthy and asymptomatic cmvd groups. disclosures: no disclosures to report. recurrent episodes of heart and/or kidney failure are considered one of the causes leading to worsening heart/renal functions in human patients. the aim of this prospective study was to assess the influence of heart/kidney worsening on elected parameters of heart/kidney function in dogs affected by mitral valve disease (mvd). between july and may , dogs affected by mvd in acvim class b and without comorbidities were included in the study group. the control group was constituted by healthy dogs, matched with the cases for age (older than years) and gender. all the dogs underwent physical examination, thorax radiography, ecg, echocardiography, systemic blood pressure assessment, a complete blood count, serum biochemical analysis, including assessment of serum creatinine (scr), serum urea nitrogen (urea) and glycaemia (gly) and urine analysis with urine protein/creatinine ratio (upc). dogs were re-evaluated every -month until october . statistical analysis was performed using ibm spss statistics (p value significant if < . ). twenty-one dogs affected by mvd (cases) were included and healthy dogs (controls) were randomly selected among the eligible population. the % of cases experienced at least one episode of congestive heart failure (chf), but none of these patients developed chronic kidney disease (ckd). the % of cases developed ckd while remaining in acvim class b . no dogs in the control group developed ckd or mvd. correlations between worsening renal function (wrf -scr elevation ≥ . mg/dl or % from baseline), furosemide administration, upc levels, radiographic parameters of heart enlargement and echocardiographic parameter were investigated. only a statistically significant difference in iris class between the groups according to wrf and in the echocardiographic parameter left atrium to aortic root (la/ao) according to furosemide amount were observed. both these results were expected. none of the cases included experienced renal damage (wrf or iris class change or upc change) concomitant to episodes of chf. the persistence of normal renal condition regardless of chf events and therapy administration was unexpected. in conclusion, experiencing chf seems not to directly affect renal function. to authors' opinion, the use of wrf, better than single scr and urea levels, may be useful in the long term management of aged patients affected by mvd. however, the small number of cases included in this study represents a great limit. we consider this work a pilot study. disclosures: no disclosures to report. hypertrophic cardiomyopathy (hcm) is a primary myocardial disease characterized by inappropriate thickening of the myocardium in absence of other causes of hypertrophy including hypertension, hyperthyroidism, aortic stenosis and acromegaly. it is also the most common heart disease in cats. hcm presents a wide variety of clinical sings depending on the severity and location of the hypertrophy. cats affected with hcm have a mean age of . - . years old at the time of the diagnosis however this disease can affect cats as young as months although this later age is unusual hcm is a heterogeneous disease both in terms of phenotypic degree of hypertrophy and clinical outcome. hallmark histopathological hallmarks lesions of hcm are myocyte disarray, small coronary arteriosclerosis and interstitial fibrosis replacement in order to confirm hcm echocardiography has to be made. primary hypertrophy diagnosis is made based on the presence of ventricular hypertrophy, symmetric or asymmetric, in the absence of systemic disorders. the purpose of this study was to assess the prevalence of hcm in a feline population. in order to achieve this goal echocardiograms were made in all cats older of years clinically asymptomatic with or without cardiac murmur. all echocardiograms were made according to the guidelines of the acvim published in . diagnosis of ventricular hypertrophy was made from the right parasternal window using the b mode to measure the diameter of the lvfw and the ivs in diastole. cats with more than mm of wall thickness measured t , bun, crea, blood pressure. only cats within the normal limits of the later parameters were considered hcm positive. total number of cats in this study was cats male and female. from this population had no defined breed, were persian, maine coon, norwegian woods, siamese, chartreaux. no murmour was detected in ( . %) cats, s or s was detected in ( %) cats and differente degree of murmour was detected in ( . %) cats. hypertrophy was detected in cats. from this cats ( . %) were diagnosed as hcm, ( . %) cats were excluded either because of lack of values of t and or because they had high values of blood pressure, t levels or crea. in this study . % of the population had hcm. the epidemiological and phenotype distribution is highly variable. the average age at diagnosis of hcm in this study was . years. disclosures: no disclosures to report. mildly increased concentrations of crp are associated with cardiovascular disease in humans and dogs. it is not known whether increased concentrations of crp are associated with myxomatous mitral valve disease (mmvd) in dogs, or rather its sequel, congestive heart failure (chf). the aim of this study was to investigate whether serum concentrations of crp, determined using a novel automated canine-specific high-sensitivity crp assay (gentian hscrp), were associated with severity of mmvd and certain clinical variables in dogs. the study included client-owned dogs with different severities of mmvd. disease severity was determined by medical history, physical examination, echocardiography and response to diuretic therapy. dogs were allocated into groups based on acvim consensus statement guidelines ( in conclusion, slightly higher serum crp concentrations were found in dogs with chf whereas the severity of asymptomatic mmvd showed limited association with serum crp concentrations. disclosures: no disclosures to report. medetomidine is a a -agonist widely employed for sedation in dogs but its use is discouraged in cardiac patients even those suffering from myxomatous mitral valve disease (mmvd). however, only one investigation was previously conducted in a wide rangeregarding the class of the diseaseof mmvd patients, reporting a general safety of that protocol. the present study was focused just on class b of mmvd, with the aim to provide more detailed information on the cardiovascular effects of medetomidine in such patients, by the analysis of clinical and instrumental parameters suggestive of disease severity or congestive heart failure. dogs weighing < kg, needing a soft clinical procedure and showing a systolic apical heart murmur were screened and selected for the study if la/ao < . . the sedative protocol consisted in an iv injection of lg/kg medetomidine antagonized, after the clinical procedure, by an im injection of the recommended dose of atipamezole. clinical parameters, echocardiographic variables, thoracic radiographs and oscillometric blood pressure measurements were collected at baseline (t ), minutes after medeto-midine administration (t ) and hours after atipamezole injection (t ). of dogs screened, were definitively enrolled. at t a significative decrease in the right parasternal regurgitant jet area (rp-arj/laa), peak velocity of mitral regurgitation and shortening fraction was observed along with an increase in lvids (p < . ). left parasternal arj/laa decreased without reaching statistical significance but showing a high correlation with rp-arj/laa (r = . ). interestingly, la/ao changed only mildly and never reached a value > . . the other echocardiographic variables did not show a particular trend. systolic blood pressure showed values at the upper physiologic limit at t , lower values than t at t , and an increase above the initial value at t but without significance. thoracic radiographs were evocative of heart enlargement without pulmonary venous congestion or pulmonary oedema both at t and t . respiratory rate did not change between t and t . the degree of sedation was optimal during the clinical procedure in all cases. sedation with lg/kg medetomidine is safe in dogs suffering from mmvd in stage b (la/ao < . ). the decrease observed in peak velocity and color-doppler appearance of mitral regurgitation at t could be due to a reduction of both myocardial contractility and systolic blood pressure, by a lowering of sympathetic activity via baroreceptors stimulation. disclosures: no disclosures to report. in this retrospective study were included a total of clientowned dogs, undergoing transarterial occlusion of pda with mreye Ò flipper detachable embolization coil (n = ), amplatz â canine duct occluder (acdo; n = ) and amplatzer â vascular plug (n = ). device size selection was based on pda dimensions assessed by transesophageal echocardiography (tee) in cases and transthoracic echocardiography (tte) in cases. angiography was performed during the procedure to assess the success of the occlusion, and it confirmed complete occlusion in dogs and a trivial residual flow in dogs. the following day, transthoracic color-doppler echocardiography revealed that complete ductal closure was achieved in all dogs. the procedure was hemodynamically successful, as evidenced, by a reduction in indexed left ventricular internal diameter in diastole (lvidd; p < . ), fractional shortening (fs; p < . ) and left atrial to aortic ratio (la: ao; p < . ) within hours after procedure. four months after surgery, indexed lvidd was significantly reduced (p = . ) and la:ao remained constant. secondary complications included pulmonary arterial embolization of an acdo and a late rotation of an amplatzer â vascular plug resulting in an increased flow through the pda. the dog with the rotated device required subsequent surgical ligation of the pda. at this time, dogs were reported to be alive and the other dogs were lost to follow up. only one dog remained on congestive heart failure therapy after the pda occlusion. we can conclude that pda occlusion using an acdo for dogs with more than kg and a transarterial coil embolization for dogs with < kg had a high rate of immediate complete occlusion. pda occlusion using those devices proved to be a safe and effective therapeutic method for pda in dogs. disclosures: no disclosures to report. echocardiographic evaluation of the right pulmonary artery distensibility index (rpad index) was recently described as a valuable method for early detection and severity evaluation of pulmonary arterial hypertension in dogs. rpad index is calculated as the percentage change in diameter of the right pulmonary artery (rpa) between systole and diastole, obtained by m-mode echocardiography from the right parasternal long axis view. the aim of this study was to compare the rpad index obtained by two different echocardiographic views in dogs. the study design was a prospective, multicenter, observational study. forty-five client-owned dogs from different breeds were included: dogs with heart disease and healthy dogs. two different right parasternal views, long axis (rpla) and short axis (rpsa), were used to measure the rpad index. from the rpla view (method ) and rpsa view (method ) a short axis and a long axis image were respectively optimized for the right pulmonary artery. the rpad index was calculated by m-mode as the percentage change in diameter of the right pulmonary artery: [(systolic diameter À diastolic diameter)/systolic diameter]* . measurements were done off-line as an average of consecutive cardiac cycles by a single investigator blinded to the dogs' diagnosis. a pearson and a bland-altman test were used to assess correlation and agreement between the methods, respectively. intra-and inter-observer measurement variability was quantified by average coefficient of variation (cv). level of significance was set at p < . . m-mode evaluation of the rpad index was satisfactorily obtained by both methods in all dogs. pearson test showed a strong positive linear correlation between the values of rpad index obtained from both methods (r = . , p < . ). bland-altman test showed a good agreement between the methods in estimating rpad index (bias = . %, sd = . %, % limits of agreement = À . , . %). the mean difference between the two methods was . % ( % confidence interval = À . ; . ). intra-and inter-observer measurement variability was clinically acceptable (cv< %).the study showed a good agreement between short axis and long axis m-mode evaluation of rpa. both methods can be used interchangeably to evaluate rpad index. further studies are needed to evaluate the rpad index in a larger population of healthy dogs and the diagnostic and prognostic role of this echocardiographic parameter in dogs with different types of pulmonary hypertension. disclosures: no disclosures to report. naturally occurring hypoadrenocorticism (addison's disease) is an uncommon illness. its prevalence in the general canine population is estimated between . and . %. certain breeds appear to have an increased risk for developing hypoadrenocorticism, including bearded collie, standard poodle, portuguese water dog and nova scotia duck tolling retriever, with reported prevalence of . , . , . and . %, respectively. the objective is to evaluate the prevalence and clinical features of naturally occurring hypoadrenocorticism in great pyrenees (gp) presented at the centre hospitalier universitaire v et erinaire (chuv) of the university of montreal. this retrospective study (march to october ) includes client-owned great pyrenees diagnosed with hypoadrenocorticism. the medical records of dogs with a diagnosis of naturally occurring hypoadrenocorticism were reviewed, with an emphasis on great pyrenees' record. the prevalence of hypoad-renocorticism in the studied population, as well as the prevalence per breed, was calculated. data collected included breed, clinical signs, laboratory findings, age at diagnosis, treatment, and cause of the death. one hundred dogs were diagnosed with naturally occurring hypoadrenocorticism, representing . % of the overall canine population studied. thirty-five breeds were represented, with a prevalence per breed varying between . % and . %. a high prevalence was observed in west highland white terriers ( . %), great danes ( . %), standard poodles ( . %), saint-bernards ( . %) and jack russell terriers ( . %). out of gp presented during that period of time, . % (n = ) were diagnosed with hypoadrenocorticism. median age at diagnosis was . years (range: . to . ) in dogs with hypoadrenocorticism, and . years ( . - . ) in gp. the main reason for presentation of the addisonian gp was lethargy (n = ) and anorexia (n = ). clinical findings included hypotension (n = ), poor body condition (n = ), and heart murmur (n = ). the majority (n = ) had serum electrolytes abnormalities, with a na:k ratio ranging from . to . . other major laboratory findings included azotemia (n = ), anemia (n = ) and the absence of a stress leukogram (n = ). the majority (n = ) received fludrocortisone, with prednisone as needed. one gp was euthanized at time of diagnosis. great pyrenees diagnosed with hypoadrenocortism were overrepresented in the studied population, with a prevalence of hypoadrenocorticism in our gp population of . %. therefore, an inherited susceptibility can be suspected. reason for presentation and clinical signs were nonspecific, and similar to what is reported in other breed. in human medicine, haemoglobin a c (hba c), a form of glycosylated haemoglobin, is used as the standard measure of average glycaemic control over - months. the measurement of hba c in dogs has been previously demonstrated however high pressure liquid chromatography techniques are too technically difficult for routine use and other methods are no longer available. the objective of this study was to assess the use of latex immunoagglutination inhibition using a monoclonal antibody for the measurement of hba c in dogs, using the siemens dca vantage Ò . repeatability was assessed by measuring samples times within minutes. the effect of storage on edta anticoagulated samples was examined by measuring samples stored at °c every day for up to days. storage was further assessed by freezing samples and measuring them at , and weeks. the machine was then used to compare the hba c values in groups of dogs with diabetes mellitus (group , n = ), hyperadrenocorticism (group , n = ) or non-diabetic/cushingoid hospitalised patients (group , n = ). differences in the groups were examined for significance using a kruskal-wallis analysis of variance. the reference range of hba c has been previously calculated to be . - . % ( - mmol/mol) and values of . to > % ( to > mmol/mol) are seen in diabetic animals using high pressure liquid chromatography. the median coefficient of variation for the repeatability study was found to be % (range - %). it was possible to store samples at °c for up to days (median cv% = %, range - %) and at À °c for at least weeks (median cv% = %, range - %). the median hba c concentrations were group ; . % ( mmol/mol), group ; . % ( . mmol/mol) and group ; . % ( mmol/mol). group was significantly different from the other two groups using kruskal-wallis analysis of variance. in conclusion, the latex immunoagglutination method was repeatable for the measurement of hba c in dogs. in addition, hba c in canine edta anticoagulated samples were stable at °c for up to days and, if frozen, could be stored for at least weeks without significant sample deterioration. the assay provides the expected results in dogs with and without abnormalities of glycaemic control. disclosures: the siemens dca vantage was provided on loan from siemens, as well as the cartridges used on this machine to run all samples in the study. a. wehner , r. anderson , s. reese , k. hartmann . clinic of small animal medicine, munich, germany, institute of veterinary anatomy, histology and embryology, munich, germany measurement of total thyroxine (t ) is often the first diagnostic step in the work up of thyroid disease in dogs and cats. blood samples are routinely sent to a reference laboratory causing a delay in testing which might impact the results. the aim of this study was to validate an enzyme fluorescence assay (elfa) as an inhouse method to measure t in dogs and cats. t was measured in sera of dogs and cats by two methods, an enzyme immunoassay (eia) and an enzyme fluorescence assay (elfa). the eia served as the standard method to which the elfa results were compared. the elfa was performed with the minividas automated analyser (biom erieux, craponne, france) according to the manufactors instructions. coefficient of variation (cv) of the elfa in dogs sera was . % and of the eia - . %, respectively. cv of the elfa in cats sera was . - . % and of the eia . - . %, respectively. overall bias of the elfa in dogs was . %; however up to À . % in lower t ranges. maximal bias of the elfa in cats was . %. correlation of both methods was linear only in cats. using bland altman plots limits of agreement were À to % in dogs and À to % in cats. cohen s kappa revealed only slight agreement between both methods in dogs, but a good to very good agreement in cats. the elfa is a fast method with a high precision and can be recommended to measure t in cats, but cannot be recommended for dogs. disclosures: no disclosures to report. dysfunctions of the central nervous system (cns) are the most frequent causes of neurological disorders in dogs. our study aimed to find ( ) if some cns diseases could be associated with a selected group of common microbial or viral pathogens in dogs and ( ) if cns diseases have any characteristic profile with regard to two parameters, c-reactive protein (crp) and iga, that are reported to be potentially useful but unspecific markers of cns diseases. we analysed cerebrospinal fluid samples obtained from dogs with varying neurological signs between june and november . real-time pcr was employed with probes for toxoplasma gondii, neospora caninum, anaplasma phagocytophilum and canine distemper virus. igg-antibodies to tickborne encephalitis virus (tbe) were assayed and iga titres were measured using elisa, while crp concentrations were determined by immunoturbidimetric assay. the dogs had a median age of years (range: . - ) and comprised breeds most frequently involving chihuahuas, labrador retrievers, bernese mountain dogs and boxers. gender distribution was . % female, . % spayed bitches, . % male, . % neutered male, and . % non-identified. none of the cases were pcr-positive for toxoplasma gondii or canine distemper virus. one dog was positive for anaplasma phagocytophilum and another for neospora caninum. antibodies to tbe virus were within the borderline range in / dogs. the dogs could be divided into two age groups: (= . %) for young dogs (< years, median year) and (= . %) for older dogs (≥ years, median years). igahigh (> . mg/dl) cases represented % and % for young and older dogs, respectively. crp-high (> . mg/l) cases were almost half and equal: . % and . % in young and older dogs, respectively. compared with older dogs, young dogs had higher levels of crp (p = . ) and iga (p = . ). within the iga-high cohorts, crp-high and crp-low cases distributed almost equally ( . % versus . %) in young dogs but disproportionate ( . % versus . %) in older dogs. there were no [iga-low/crp-low] cases in young dogs but . % in older dogs. our present data sug-gest that ( ) canine cns disorders were largely characterized by high iga and particularly in young dogs ( ) inflammatory types (crp-high) were almost equal in both groups and ( ) internet is a potential source of medical information for pet owners. therefore, it could play an indirect but important role in the veterinary practice. this survey assesses the online search behaviour of french pet owners for their pets' health and its influence on a veterinary consultation. in april , french pet owners coming in a veterinary teaching hospital for a medical or a surgical consultation were surveyed. ( . %) owners fulfilled the questionnaire on a voluntary basis. the survey contained questions dealing with three topics: the online search behaviour of owners for their pets' health, their perception of the information found online and the internet's influence on a consultation and on the veterinarian/client relationship. . % of owners use the internet to obtain information on their pets' health. among them, . % use it rarely and . % occasionally. they mainly look for information on a disease ( . %), a symptom ( . %), a breed ( %) or a nutritional advice ( . %). . % of owners try to verify the accuracy of the information found, most often by questioning their veterinarian ( . %). few owners ( . %) think that online information is always trustworthy. most of the research ( . %) is randomly made, websites being found through search engines. the majority of pet owners ( . %) aren't aware of any health certification label for websites. internet enables certain pet owners to feel more at ease with their pets' health care: they ask more questions to their veterinarian ( . %) and feel more involved in medical choices ( . %). . % of owners consider that the internet can positively impact their relationship with the veterinarian. relief is the most common ( . %) emotional response to online research for medical information. however, . % of owners feel overwhelmed by the amount of information found, % are confused and . % frightened by the serious or graphic nature of the information found online. this study emphasizes the frequent but measured use of the internet by french pet owners for their pets' health. they seem to consider information found on the net with a critical mind. unexpectedly, it appears that the internet could be an ally for veterinarians by promoting exchanges between the clients and the veterinarian and by improving compliance with the care project. disclosures: no disclosures to report. sinonasal aspergillosis is a well-known and described fungal infection of the sinonasal cavities in dogs. topical treatment either with enilconazole or itraconazole infusion administered surgically or endoscopically are effective. the use of % clotrimazole cream have been described in a surgical setting after itraconazole infusion by sissener et al. in . the aim of the work was to report the effectiveness of the use of topical % clotrimazole cream as the only treatment for sinonasal aspergillosis in dogs. inclusion criteria were a full medical record with radiological and endoscopical imaging, record of clotrimazole discharge after instil-lation and endoscopic control between and days after procedure. the % clotrimazole cream was applied through catheters placed under direct endoscopic vision after fungal plaques removal. nine dogs were included. three dogs were mixed breed dogs, two dogs golden retriever, one dog a german shepherd and one old english sheepdog, one bull terrier and one cane corso. six dogs were male (one neutered) and three female (one intact). mean age was . years. main clinical signs were muco-purulent discharge ( ), pain at sinonasal palpation ( ), nasal planum alterations ( ), epistaxis ( ) . nasal discharge was bilateral in dogs. mean duration of clinical signs was . months. main radiological findings were turbinates lysis ( ), frontal sinus empyema ( ), frontal bone thickening ( ) and frontal bone lysis ( ). rhinoscopy disclosed lysis and remodelling of the nasal turbinates ( ), easy access to the frontal sinus ( ), septum lysis ( ), bilateral sinonasal aspergillosis ( ), monolateral nasal aspergillosis ( ), monolateral sinonasal aspergillosis and contralateral nasal aspergillosis ( ), monolateral frontal aspergillosis ( ). main duration of nasal cream discharge was . days. all dogs underwent endoscopic control between and days after the procedure. seven dogs were disease free; two dogs had persistent fungal plaques and underwent a second treatment. success rate was . %. success rate of this study is comparable to other studies with larger and smaller case series. endoscopic removal of the fungal plaques can be time consuming and topical administration of either enilconazole or itraconazole require an additional hour. the catheter placement and the % clotrimazole cream application lasted minutes for each cavity in the dogs here reported. the use of % clotrimazole cream as the only treatment for sinonasal aspergillosis needs further evaluation on a larger case series. disclosures: no disclosures to report. few studies exist on euthanasia in small animal practice. however, such an act belongs to veterinary procedures, more or less frequently depending of the kind of practice, and may deeply impact both owners and veterinarians. we intended to study practical, ethical and psychological aspects of euthanasia of dogs and cats among french veterinary practitioners. from october to february , an on-line -item questionnaire on small animals' euthanasia, addressing practical aspects of euthanasia, communication with the owners, ethical problems, owners' and veterinarians' perceptions, was emailed via professional associations and networks. results were analyzed using commercial software (sphinx iq Ò and excel Ò ). french veterinarians practicing small animal medicine completed the questionnaire, representing > % of this population. euthanasia occurs rarely at home. over % of veterinarians propose the owners some time alone with their pet, and to stay during euthanasia, performed most commonly by intravenous injection ( . %) mainly after sedation/anesthesia ( . %). ninety nine percent of veterinarians consider communication, including description of events' sequence, and disposal of the body, as important. estimated minimum communication time required varies from - to - minute. following euthanasia, . % are often thanked by the owners. most veterinarians (> %) have refused a euthanasia, considered unjustified, or had their own suggestion of euthanasia rejected. reasons for such a suggestion include intractable pain ( . %), non-acceptable complications ( . %), financial considerations ( . %) or animal considered dangerous ( . %). veterinarians think most owners ( . %) experience some sense of guilt during euthanasia. themselves perceive euthanasia most commonly as relief of animal's suffering ( . %) and part of veterinary practice ( %), less frequently as a defeat ( . %). almost all veterinarians have experienced emotionally challenging euthanasia, and . % estimate that practicing euthanasia influences their perception of death. practical ( %) and psychological ( . %) aspects of euthanasia have been discussed in most teams. veterinarians' gender influences euthanasia management, mostly concerning some communicational and practical aspects. euthanasia is definitely not an ordinary veterinary act, neither for the owner nor for the veterinarian. therefore, this act must be performed with as much care and communication as possible. disclosures: no disclosures to report. canine pancytopenia is associated with a range of intra-marrow or extra-marrow causes, including though not limited to, infectious agents, drugs, toxins and neoplasms. there is currently limited information regarding the clinicopathological features of the underlying causes or the prognosis in pancytopenic dogs. the objective of this retrospective study was to better define the spectrum of diseases associated with canine pancytopenia, to establish possible clinicopathological discriminators for the common causes and to investigate if the severity of pancytopenia or the underlying disease were associated with the clinical outcome (death or survival). medical records of dogs with a comprehensive diagnostic investigation admitted in a veterinary teaching hospital were retrospectively reviewed. pancytopenia was defined by a hematocrit (hct) < % (< % for dogs < months of age), white blood cell counts (wbc) < . /ll and platelet counts (plt) < , /ll. a control group of dogs without any evidence of blood cytopenia(s) was also established. in total, pancytopenic dogs were studied. bone marrow aspiration cytology was examined in cases and aplasia of all hematopoietic lineages was observed in ( . %) dogs. the most common diagnoses included monocytic ehrlichiosis (cme) (n = ), leishmaniosis (canl) (n = ), parvoviral enteritis (pe) (n = ), and concurrent cme and canl (n = ). mixed breed dogs were more likely to develop pancytopenia as compared to purebreds and pancytopenic dogs tended to be younger than the controls (conditional dependent logistic regression model, p = . and p = . , respectively). among the most common diseases associated with pancytopenia, the mean wbc counts were significantly lower in dogs with cme and pe compared to dogs with canl (one way anova with bonferroni test for multiple comparisons, p = . and p = . , respectively), while plt counts were significantly lower in cme compared to canl (p < . ) or pe (p < . ). total protein concentrations were significantly lower in dogs with pe compared to cme (p < . ) and canl (p < . ). using a univariable logistic regression analysis model, no association was established between the underlying disease and the final outcome. however, higher hct (by at least one percentage unit), wbc (by at least /ll) and plt (by at least , /ll) values tended to significantly increase the odds of survival (p = . , p < . and p = . , respectively). in the present study, cme, canl and pe were the major causes of canine pancytopenia. potentially useful diagnostic indicators included severe leucopenia (cme, pe), thrombocytopenia (cme) and hypoproteinemia (pe). disclosures: no disclosures to report. bwbp detects both upper and lower airways diseases because any site of airway obstruction will result in increased pressure changes associated with breathing. nevertheless our results suggest that there are significant differences in tv (p = . ), ti (p = . ), pef (p = . ), eep (p = . ), penh (p = . ) and pau (p = . ) between lm and fbd cats. no other significant difference in bwbp parameters was found. upper airway obstructions have been previously evaluated in cats by using pft (mckieman, , lin, but in authors' knowledgement this is the first study designed to compare upper and lower airway obstructions by using bwbp. attending our results, there is the evidence that bwbp can help characterize mechanical dysfunction of the airways in cats with lm obstruction. however we must keep in mind some limits of this study as the low number of animals, individual variability in breathing pattern and to have the chance of doing bronchoreactivity tests. disclosures: no disclosures to report. the median lifespan of domestic dogs has been estimated to - years, but little is known about risk factors for mortality in aged dogs: most mortality studies in dogs have been carried out among diseased dogs (renal or heart diseases, cancers, post-operative death). to determine which characteristics are associated with mortality in a priori healthy aged dogs, a prospective cohort study has been conducted in guide dogs, followed from a systematic geriatric examination (ge) to either (all-cause) death or cut-off date (july th, ). survival analyses (kaplan-meier estimators, log-rank tests, and multivariate cox proportional hazards models) were used to assess the associations with time to death. median age at ge was . years, all dogs were neutered, and % were female. the majority of dogs were golden retriever (n = ) and labrador retriever (n = ). among these dogs, % were obese, % presented skin nodules and % used bus as transportation. a total of dogs died during follow-up, leading to a median survival time from ge of . years. after adjustment for demographic and biochemical variables (age, sex, total proteins, cholesterol and alp), an increased alanine amionotransferase level (≥ ui/l; adjusted hazard ratio [ahr], . ), presenting skin nodules (ahr, . ), and not being a labrador (ahr, . ) were independently associated with a shorter time to death (p < . ). public transportation tended to be associated with mortality (ahr, . ; p = . ), highlighting the importance of environment in mortality. neither sex nor other biochemical parameters were significantly associated with mortality. the alanine amionotransferase level and the presence of skin nodules seem predictors of mortality in senior guide dogs, mostly labrador, golden, or mixed breed of labrador/golden. the impact of environment, in particular urban environment, on mortality needs further investigation. studies in other breeds and pets are also necessary to generalize these results. disclosures: sara hoummady received a grant from mp labo for his phd work about dog aging and marc blondot work at the paris guide dog school. laryngeal dysfunction is most commonly associated with aspiration pneumonia, however its role in other lower airway diseases has not been investigated. laryngoscopic and bronchoscopic findings in dogs examined by the author between and were evaluated for the presence or absence of laryngeal abnormalities. dogs that presented for evaluation of inspiratory difficulty or panting were excluded from analysis. clinical diagnoses of inflammatory airway disease, airway collapse, airway infection, eosinophilic bronchopneumopathy or a combination of these disorders were obtained through bronchoscopy and bronchoalveolar lavage fluid analysis. detection rates for laryngeal abnormalities were compared among disease groups using chi square analysis and fisher's exact test, with significance set at p < . . a total of dogs were evaluated and varied in age between months to . years (median years). weight ranged from . to . kg (median kg), with dogs < kg, dogs from . - . kg, dogs from - kg, dogs from . - kg, and dogs > kg. laryngeal hyperemia or swelling was found in / dogs ( %), and detection rate did not differ among disease processes. laryngeal function was considered suspect in / cases, prompting administration of doxapram, which normalized function in / dogs. laryngeal paresis or paralysis was reported in a total of / dogs ( %). a substantial number of dogs with chronic cough displayed evidence of abnormal laryngeal structure or function, suggesting that a complete laryngoscopic examination should be performed in all dogs evaluated for cough. disclosures: member of the feline advisory board, speaker honoraria for international, national, and regional continuing education meetings. bronchiectasis is a poorly characterized disease in dogs identified by airway dilatation on radiographs, computed tomography, bronchoscopy, or histopathology. little is known about underlying disease processes associated with bronchiectasis in dogs. medical records from dogs presented to uc davis were searched for identification of bronchiectasis. underlying disease processes and clinical diagnoses were obtained through review of the history, physical examination, respiratory endoscopy and bronchoalveolar lavage fluid analysis and microbiology. historical reports, results of imaging, bronchoscopy and fluid analysis, and scrutiny of pathologic and clinical diagnoses were comprehensively evaluated to identify the most likely underlying disease process associated with bronchiectasis. between and , bronchiectasis was diagnosed in / dogs ( %) that had bronchoscopy performed. dogs ranged in age from . to years (median years) with / dogs < months, / dogs ( %) - years, / dogs ( %) . - years of age and / dogs ( %) over years of age. dog breeds affected more than once included labrador retrievers, cocker spaniels, golden retrievers and standard poodles. duration of cough ranged from days to years (median months). underlying disease processes included pneumonia in / ( %) dogs, inflammatory airway disease in / ( %) dogs, and eosinophilic bronchopneumopathy in / ( %) dogs. twenty-three of dogs ( %) had positive bacterial cultures, with isolation of streptococcus (n = ) and enteric species (n = ) most commonly. this study found that bronchiectasis often occurs in older, large breed dogs with infectious or inflammatory pneumonia. disclosures: johnson: feline advisory board, speaker honoraria. chronic respiratory disease, often characterized by a chronic cough, is common in dogs. the purpose of this study was to determine the etiology of chronic respiratory disease in dogs that were presented with persistent and chronic coughing. a retrospective study of client-owned dogs with signs of persistent and chronic lower respiratory disease, that underwent bronchoscopy together with either an endotracheal wash (etw) or broncho-alveolar lavage (bal), was performed. all dogs were evaluated by means of full clinical examination, hematology and serum biochemistry analyses, survey thoracic radiographs, echocardiography and ecg (if indicated), and bronchoscopy with cytological analysis and aerobic culture of etw or bal fluid. an etw was performed in / ( %) dogs while a bal was performed in / ( %) dogs. a positive aerobic bacterial culture was identified in / ( %) of submitted etw/bal fluid samples. most commonly isolated bacteria included mycoplasma sp. ( %), bordetella bronchiseptica ( %) and pseudomonas aeruginosa ( %). a definitive diagnosis was made in / cases ( . %). chronic bronchitis was the most common diagnosis ( . %), median age years; followed by airway tracheal collapse or bronchomalacia ( %), median age years,; and primary bacterial infections ( . %), median age years. less common etiologies identified included neoplasia ( . %), median age years; parasitic infections ( . %), median age years; and eosinophilic bronchopneumopathy ( . %), median age years. rare etiologies identified included primary pulmonary hypertension, primary ciliary dyskinesia, excitement-induced cough, and obesity. myxodematous mitral valve disease was found concurrently in / ( . %) dogs. this study concluded that by using a structured combination of survey thoracic radiography, bronchoscopy and etw or bal with cytology and culture, a diagnosis could be made in the majority of dogs with chronic respiratory disease. disclosures: no disclosures to report. canine sterile steroid-responsive lymphadenitis (cssrl) is an uncommon cause of lymphadenomegaly. diagnosis is one of exclu-brachycephalic dogs have unique upper respiratory anatomy with abnormal breathing patterns similar to those in humans with obstructive sleep apnea syndrome (osas). oxidative stress in the body represents the imbalance between the production of reactive oxidative species and the ability of antioxidant defense mechanisms to detoxify the reactive intermediates. oxidative stress is involved in the pathogenesis of many diseases, including hemolytic anemia, atherosclerosis, tissue reperfusion injury and has also carcinogenic potential. several studies have clearly shown an association between obstructive sleep apnea syndrome in humans and oxidative stress, but detailed underlying pathomechanism remains unclear. due to the consideration of brachycephalic dogs as an animal model of human osas, this study was designed to evaluate oxidative stress (paraoxonase type- activity; pon and total antioxidant status; tac) in brachycephalic dogs with brachycephalic airways obstructive syndrome (baos) before and after surgery compared to control dogs. this study was conducted on dogs with baos and control dogs. twenty out of baos dogs were evaluated - months after surgical correction. mean values of tac and pon in different studied groups were as follows: control dogs (tac: . ; pon : . ), baos dogs (tac: . ; pon : . ), baos dogs post-surgery (tac: . ; pon : . ) a statistically significant difference on tac levels is observed between dogs with baos and control dogs (p < . ). no statistically significant differences were observed in pon and tac levels before and after surgery. on the other hand, no differences have been observed between pon and tac levels in baos dogs according type of brachycephalic breed, grade of respiratory and digestive signs or presence of everted ventricular laryngeals. the results of our study showed a statistically significant difference in tac values between control and dogs with baos, confirming the oxidative stress previously described in humans. even that human patients with osas can partially reverse their increase in oxidative stress by using a nasal continuous positive airway pressure treatment, in dogs with baos no differences were observed before and month after surgical treatment. baos surgical treatment is not useful to reduce pon- or tac levels, probably because baos does not induce such an evident inflammatory process in dogs as in human patients with osas. disclosures: no disclosures to report. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease mainly affecting west highland white terriers (whwts). the cipf course varies greatly among dogs from rapid deterioration to slowly progressive forms and the survival time from onset of clinical signs ranges from a few months to several years. in human ipf, increased chemokine (cc-motif) ligand (ccl ) concentrations in bronchoalveolar lavage fluid (balf) are indicative of a poor outcome and serum concentrations are correlated with clinical parameters of lung function. in dogs, serum and balf ccl concentrations were shown to be elevated in whwts with cipf compared with healthy whwts. the aim of the present study was to investigate whether serum ccl concentrations measured in whwts with cipf at diagnosis ( ) can be used as an indicator of prognosis and ( ) correlate with lung function parameters. ccl concentrations were determined by elisa (canine ccl quantikine elisa kit, r&d systems) in the serum of whwts suspected of cipf (median age . years, range . - . ), for which a follow-up was available (median follow-up time . months, range - . ). serum sampling extended from may to january . cipf diagnosis was confirmed by thoracic high resolution computed tomography, lung histopathology, or both examinations in , and whwts respectively. kaplan-meier analysis was conducted to investigate differences in survival times according to serum ccl concentrations at diagnosis. spearman analysis was used to assess correlations between serum ccl concentrations and lung function parameters, namely the distance walked in the -minute walking test ( mwd) and the arterial partial pressure of oxygen (po ). among the cipf whwts included, died or were euthanized for cipf-related reason, died or were euthanized for non-cipf-related reason and were still alive at the end of the study. the median survival of whwts with cipf-related death or euthanasia was . (range . - . ) months from diagnosis. serum ccl concentrations above pg/ml were significantly associated with a shorter survival time in whwts affected with cipf (p = . ). a weak negative correlation was found between serum ccl concentrations and the mwd (r = À . , p = . , n = ), while no correlation was observed with arterial po values (n = ). in conclusion, serum ccl concentration provides prognostic information in whwts suffering from cipf, while this marker is weakly correlated with the clinically lung function parameters available in the present study. disclosures: no disclosures to report. canine idiopathic pulmonary fibrosis (cipf) is a progressive interstitial lung disease mainly affecting west highland white terriers (whwts). this study was intended to ( ) describe thoracic high-resolution computed tomography (t-hrct) findings obtained in cipf dogs under general anesthesia (ga) using the glossary of the fleischner society and ( ) compare images obtained under ga (t-hrct ga ) with those obtained under sedation (t-hrct s ). t-hrct images from whwts with cipf and control whwts were retrospectively reviewed by three observers in consensus. specific t-hrct features were assessed and graded for each lung lobe ( = absence, = mild, = moderate and = severe). a global score was then calculated. the khi² test with the threshold % was used for the statistical analysis. ground glass opacity (ggo) was observed in all cipf whwts and in / of controls (p = . ). in controls, ggo was mild and localised mainly in cranial lobes. in cipf whwts, ggo was mild, moderate or severe in , and dogs respectively, without lobe predilection. consolidation was observed in / cipf whwts but not in controls (p = . ) and was mild ( / ) to moderate ( / ). a mosaic pattern, suggestive of air trapping, was noticed in / cipf whwts but not in controls (p = . ) and was mild, moderate or severe in , and whwts respectively, without lobe predilection. nodules were present in / cipf whwts but not in controls. reticulation, subpleural bands and parenchymal bands were noticed in , , and / cipf whwts respectively. honeycombing, emphysema, pleural effusion and pleural thickening were never observed. bronchial wall thickening and mild bronchiectasis were present in / and / cipf whwts respectively but not in controls (p = . and p = . ). the overall t-hrct s quality was good in / examinations compared with / for t-hrct ga (p = . ). the presence of motion artefacts was higher for t-hrct s (p < . ), but were most frequently graded as mild (p < . ). t-hrct s allowed identification of a mosaic pattern in additional cipf whwts, while consolidation could not be identified in others. there was no difference in identification or gradation for the other features between t-hrct ga and t-hrct s . in conclusion, ggo, consolidation, mosaic pattern and bronchial wall thickening are the main t-hrct features of cipf in whwts. honeycombing, the major feature of ipf in humans, was never observed, which suggests a different pathophysiology between the two entities. t-hrct s images are in accordance with t-hrct ga and can be used for cipf diagnosis. disclosures: no disclosures to report. literature on mesenterial lymphadenitis (lad) or mesenterial lymph node abscesses (lab) in small animals is scarce. case files from to were searched for dogs with the diagnosis of mesenterial lad/lab based on cytology and/or histopathology. of cases identified, had to be excluded due to incomplete data. the remaining dogs were of mixed breed (n = ), small munsterlander (n = ) and one each of other breeds. nine dogs were male and female. median age was months (range . diagnosis was based on fine needle aspiration (fna; n = ), histopathology (n = ) or both (n = ). significant findings in the dogs' history included gastrointestinal signs (n = ), puppy whose mother had mastitis, bite wounds/abscesses of the skin (n = ), pulmonic stenosis (n = ) and orthopaedic diseases (n = ). most common presenting complaints were lethargy (n = ), hyperthermia (n = ), diarrhoea (n = ), vomiting/nausea (n = ), inappetence/anorexia (n = ), back/abdominal pain (n = ) and lameness (n = ). diagnostic tests performed included haematology/serum biochemistry (n = ), thoracic (n = ) and abdominal (n = ) radiographs, abdominal ultrasound (n = ), ct/mri (n = ), fnas of other organs (n = ), urinalysis (n = ) with culture (n = ), coagulation profiles (n = ), orthopaedic radiographs (n = ), cpl (n = ), blood cultures (n = ), and csf/joint taps (n = ). dogs were retrospectively divided into group a (n = ): dogs with no other disease than lad/lab ("idiopathic") and group b (n = ): dogs with other diseases diagnosed simultaneously. these included neoplasia (carcinoma n = , lymphoma n = , leiomysarcoma n = , histiocytic neoplasia n = , prostate carcinoma n = ), gastroenteritis (n = ), presumed pancreatitis (n = ), purulent monoarthritis (n = ), purulent hepatitis/ splenitis (n = ), fungal infection at a distant site (n = ), and mycobacteriosis (n = ). eleven dogs received surgical treatment and antibiotics, and dogs conservative medical management consisting of supportive treatment and antibiotics. all dogs were discharged alive. dogs in group a were hospitalized longer (mean days, sd . ) than dogs in group b (mean days, sd . ) (p = . ). the median follow-up time was days ( - days). there was no difference in pretreatment with antibiotics or anti-inflammatories between groups. t-tests or kruskall-wallis tests showed that dogs in group a were borderline significantly younger (p = . ), had significantly higher respiratory rate (p = . ), rectal temperature (p = . ), monocyte count (p = . ) and crp concentration (p = . ) than dogs in group b. the small munsterlander had an odds ratio of over other breeds to suffer from lad/lab. in conclusion, idiopathic mesenterial lad/lab was seen in young dogs with hyperthermia and gastrointestinal signs. diagnosis of purulent lad/lab on fna does not exclude the presence of another underlying pathogenesis. disclosures: no disclosures to report. canine infectious respiratory disease (cird) is a disease complex caused by different viral and bacterial pathogens. aim of the study was to evaluate clinical and laboratory factors associated with different infectious agents. dogs were included, if they showed respiratory signs (< days) consistent with cird and if non-infectious respiratory conditions could be excluded. nasal and pharyngeal swabs were taken from dogs with cird and tested for respiratory pathogens, including canine parainfluenza virus (cpiv), canine adenovirus (cav), canines distemper virus (cdv), canine herpes virus (chv), canine respiratory coronavirus (crcov), canines influenza virus (civ), and bordetella bronchiseptica (b. bronchiseptica) by polymerase chain reaction. results of clinical and laboratory data were correlated with the underlying pathogen using fisher's exact test and chi-square test (p ≤ . ). cpiv was detected in , crcov in six, and b. bronchiseptica in dogs; patients showed infections with more than one pathogen. there was no significant difference for age and gender distribution between the three groups; however, dogs infected with cpiv more likely originated from a shelter (p = . ). when clinical data were compared, there was no significant difference for the parameters depression, fever, cough, nasal discharge, dyspnoea, and abnormal lung sounds. furthermore, there was no sig-nificant difference regarding abnormalities of erythrocytes, platelets, leukocytes, and differential count between groups. the study shows that in dogs with cird clinical and laboratory parameters cannot indicate the underlying pathogen. furthermore, diseases severity does not seem to depend on the infectious organism involved. disclosures: no disclosures to report. breed related predisposition to bacterial bronchopneumonia (bp) has been reported in irish wolfhounds (iwhs). underlying factors are unknown, however immune deficit, ciliary dysfunction and aspiration have been suggested as predisposing factors. the purpose of this prospective study was to evaluate lymphocyte subpopulations in iwhs with one or more previous bp and compare results to elderly iwhs without any previous bacterial respiratory infections. additionally, healthy dogs of other breeds were included as controls. previous bp was confirmed in iwhs (median age . years, interquartile range . - . years). healthy iwhs (n = , . , . - . years) or dogs of other breeds (n = , . , . - . years) had no history or findings suggestive of previous bp. edta blood samples, collected from all dogs when they were healthy, were stained with fluorescent mouse anti dog cd , cd , cd , cd and mhc class ii antibodies (abd serotec Ò ) and flow cytometry analysis was performed with bd facsaria Ò ii cell sorter and facsdiva Ò software. statistical comparison between groups and the effect of age was studied using analysis of covariance (ancova) models. the number of leucocytes did not differ significantly between groups. the total numbers of lymphocytes and numbers of major lymphocyte subpopulations (b-cells, cd + and cd + t-cells) were significantly lower in healthy iwhs and iwhs with previous bp compared to dogs of other breeds (lymphocytes p < . and p < . ; b-cells p = . and p = . ; cd + t-cells p = . and p = . ; cd + t-cells p < . and p = . respectively). percentage and number of mhc class ii+ non-b lymphocytes was significantly higher in both iwh groups than in dogs of other breeds (p < . in all comparisons). lymphocyte numbers and subpopulations did not differ significantly in healthy iwhs compared to iwhs with previous bp. an age-related decline in the total number of lymphocytes (p = . ), t-cells (p = . ), cd + t-cells (p = . ) and mhc class ii+ non bcells (p < . ) was noticed only in the group of iwhs with previous bp. these preliminary results indicate that iwhs may have significantly lower numbers of lymphocytes, b-cells as well as cd + and cd + t-cells than dogs of other breeds. further studies are needed to determine whether these alterations represent a breed related phenomenon or are connected to the predisposition to bp. an age-related decline in lymphocyte, total t-cell and cd + t-cell numbers was detected in iwhs with previous bp. in humans, age related changes in cd + t-cells have been associated with increased susceptibility to infections. disclosures: no disclosures to report. feline chronic kidney disease (ckd) is a common feature of ageing cats. angiotensin-converting enzyme inhibitors (acei) are recommended to treat hypertension associated with ckd to limit target-organ damage and especially glomerular hypertension. in addition, the international renal interest society (iris) guidelines recommends the prescription of an acei in patients with ckd and proteinuria. to our knowledge no study has demonstrated the effects of long term administration of acei in a client owned population of cats suffering from ckd on glomerular filtration rate (gfr). the aim of the study was to evaluate the effect of an acei (imidapril, prilium Ò , v etoquinol sa) on the gfr of cats suffering from naturally occurring chronic kidney disease (ckd) over months. sixty-six cats presenting with clinical and biological signs of ckd were enrolled by european investigators and followed up till months in this randomized blinded study. thirty-two cats provided suitable data for gfr analysis; animals received imidapril at the dosage of . mg/kg/d and received placebo. animals with no available gfr value on day or with no data after day were excluded as well as animals for which the iohexol clearance could not be determined. follow up was censored after months due to small sample sizes for statistical comparisons. on day , month , month and month , cats were sampled , and minutes after intravenous administration of mg of iohexol. gfr was based on determination of the iohexol clearance which was calculated with the phoenix Ò winnonlin . software. statistical analyses were performed with sas/stat . software. as gfr were not available on each time point for a given animal, the two treatment groups were compared on each gfr determination point using an ancova (analysis of covariance) with the gfr determined on day as the covariate. on d , gfr were . ae . and . ae . ml/kg/min in the imidapril and placebo group, respectively. a significant statistical difference ( . ae . versus . ae . ml/kg/min in the imidapril and placebo group respectively, p = . ) was observed in favor of a higher gfr in the imidapril treated animals on month . higher gfr were also observed in the imidapril group on month and month but not significantly different from the placebo group. daily long term imidapril treatment compared to placebo, may be an effective treatment to slow the progression of renal failure in cats with naturally occurring chronic kidney disease. disclosures: authors are employees of vetoquinol. there is a concern over the potential of cytotoxic drugs which could harm exposed workers. the speciality of oncology of the ecvim-ca published guidelines in order to prevent that risk. however few data exist for evaluation of the real risk of occupationnal exposure. this concern was the aim of our study. biomarkers used were vincristine and cyclophosphamide. surface samples were collected in the consultation room and ward dedicated for chemotherapy. samples were collected with wet filter paper on cm surfaces, or objects (computers for instance). samples were analysed by liquid chromatography and mass spectrometric method.the limit of quantification was . ng/ cm (or . g/object) for vincristine and . ng/ cm (or . g/object) for cyclophosphamide. samples in consultation room were collected after treating dogs with vincristine and after cleaning. samples in dedicated ward were collected after cleaning and after -days stay of treated dogs. aeras/objects were tested in consultation room; in dedicated ward for vincristine; in ward for cyclophosphamide. after treatment of dogs in consultation room, traces of vincristine were detected on the floor and the laboratory bench top ( . - . ng/ cm ). moreover, the surface of auscultation table and extern side of gloves were contaminated after preparation and administration of vincristine (respectively . , and ng/objet). after cleaning, % of samples in consultation room were positive. traces of vincristine were detected on the floor or objects (wall otoscope: . - . ng/ cm ). after cleaning, % of samples in ward were positive. traces of vincristine were detected on the floor and objects (boxes, wastebin, bowls: . - . ng/objet) after cleaning and animals treatment. cyclophosphamide were detected on all areas tested ( . a . ng/objet). despite protective guidelines to avoid spread of cytotoxic drug, environemental exposure was demonstrated. however contaminations were limited and show that handling procedures of cytotoxic drugs are well controlled. most-elevated contamination level for vincristin were noticed on extern side of gloves depsite of using appropriate material. workers should pay a particular attention for gloves withdrawal to limit exposure. small amounts of vincristine were found in inapropriate places: computeur mouse. even if there is few of those residues, we thought about people working on a regular basis in this room for other activities than chemotherapy, and we decided to adapt our clinical practices. evaluation of occupational exposure to cytotoxic drugs is an important step to prevent incidents and heigt awarness of nursing staff to apply those good clinical practices. disclosures: no disclosures to report. the tumor-associated inflammatory response had the effect of enhancing mammary tumorigenesis, helping incipient neoplasias to acquire hallmark capabilities, both in human and dogs. t-cells migration to the tumor site and the following activation may be the essential requirement for their promoting effect on tumors. in human breast cancer the signaling pathways of c-kit have been described as possibly being involved in differentiation, migration, survival, and maturation of t-cells and other inflammatory cells into tumor sites. in order to clarify this subject in canine mammary tumors (cmt), malignant neoplasms were studied by using immunohistochemistry comparing the intratumoral cd + tlymphocytes and c-kit expression together with vegf, microvessel density (mvd) and clinicopathological characteristics of tumor aggressiveness. cd + t cells and high c-kit immunoexpression revealed a positive and statistically significant correlation with vegf (r = . , p < . ; r = . , p = . for cd and c-kit respectively) and cd (r = . , p < . ; r = . , p = . for cd and c-kit respectively). a statistically significant association (p = . ) and a positive correlation (r = . , p = . ) between cd + t-lymphocytes and c-kit was also observed. tumors with high c-kit expression showed higher counts of cd + t-cells. the mvd of high cd /vegf tumors was significantly more elevated (p < . ). a similar association was observed for high c-kit/vegf tumors (p < . ). in this study high cd /vegf, high c-kit/vegf and high cd /c-kit tumors were statistically significantly associated with elevated grade of malignancy (p < . for cd /vegf, c-kit/vegf and cd /ckit), presence of neoplastic intravascular emboli (p < . for cd /vegf and cd /c-kit; p = . for c-kit/vegf) and presence of lymph node metastasis (p < . for cd /vegf, c-kit/ vegf and cd /c-kit). tumors with high cd /vegf (p = . ), high c-kit/vegf (p < . ) and high cd /c-kit (p = . ) expression were associated with shorter os time. interestingly the group of tumors with high c-kit/vegf retained their significance by multivariate analysis arising as independent predictor of os. results of this study suggest that t-lymphocytes may share common signaling pathways with c-kit and vegf in cmt progression and may contribute to increased angiogenesis, aggression and shorter os in these tumors. disclosures: no disclosures to report. few epidemiological data have been published on feline large granular lymphocyte (lgl) lymphoma. a recent study (krick et al. ) described clinicopathologic features in cats with lgl lymphoma, but no comparisons were made with cats with other diseases or other forms of feline lymphoma. therefore, the objective of this study was to assess differences in prevalence, signalment (breed, sex, and age), physical exam findings (body weight, body condition score, body temperature, heart rate, respiratory rate, and systolic blood pressure), and felv/ fiv status between cats with lgl lymphoma (group ) and all other type of feline lymphoma (group ). the electronic data-base of the san marco veterinary clinic was searched between january- and december- for cats with a cytological or histopathological diagnosis of lymphoma. differences between groups were assessed by t-test, mann whitney, pearson-chi square, pearson chi square yates corrected, and fisher's exact test. during the study period out of sick cats seen at the clinic were diagnosed with lymphoma (group : n = ; group : n = ). the prevalence of all type of feline lymphoma between and compared to sick cats did not change over time ranging from . % to . % per year (p = . ; overall prevalence . %, % ci . - . ). the lymphoma lgl prevalence between and compared to all types of lymphoma did not change over time ranging from . % to . % per year (p = . ; overall prevalence . %, % ci . - . ). among the variables studied, only sex (group : [ . %] females, [ . %] males; group : [ . %] females, [ . %] males; p = . ) and age (group : ae months; group : ae months; p = . ) were significantly different between groups. sixteen out of cats with lgl lymphoma were tested for their felv/fiv status resulting all felv-and one ( . %) fiv+. seventy-four out of cats with all other types of lymphoma were tested for their felv/fiv status resulting ( . %) felv+ and ( . %) fiv+. five cats ( . %) were both felv+/fiv+. prevalence of felv infection was significantly lower (p = . ) in group compared to group . there was no difference in prevalence of fiv infection between groups. lymphoma lgl affects more females and older cats compared to all other type of feline lymphoma. opposite to all other type of lymphoma, and in accordance to previous litterature information, felv+ status does not play a role in the pathogenesis of feline lgl lymphoma. disclosures: no disclosures to report. the activity of t regulatory cells (tregs) is known to be closely associated with the expression of foxp transcription factor. foxp regulatory t cells (foxp treg) are a distinct group of t lymphocytes that have immunosuppressive properties. normally this cells work for prevention of harmful autoimmune responses, however can also interfere with beneficial immune responses such as anti-tumor immunity. in human breast cancer these cells play a crucial role in tumor progression. in canine mammary tumors (cmt) there are only a few studies and this topic are not welldocumented. in this study we included malignant cmt and studied, by immunohistochemistry, the intratumoral foxp expression together with vascular endothelial growth factor (vegf), microvessel density (mvd, by cd antibody) and several clinicopathological characteristics. abundant foxp treg cells was statistically associated with presence of tumor necrosis (p = . ), nuclear grade (p = . ), poor differentiation of tumors (p < . ), high mitotic grade (p < . ), high histological grade of malignancy (p < . ), presence of neoplastic intravascular emboli (p < . ) and presence of lymph node metastasis (p < . ). intratumoral foxp levels were correlated with the levels of vegf (r = . ; p < . ) and intratumoral mvd (r = . ; p < . ). additionally tumors with abundant foxp treg cells were associated with shorter overall survival (os) time (p = . ). results suggest that treg cells play a role in cmt progression and may contribute to increased angiogenesis and aggression in these tumors. the association of intratumoral foxp expression with shorter os of animals suggests a utility of treg cells activity as a prognostic marker. disclosures: no disclosures to report. clinical manifestations of canine visceral leishmaniasis (canl) are non-specific and include progressive weight loss, anemia, lymphadenomegaly, hepatosplenomegaly, dermatological, renal and ocular alterations. cardiac lesions resulting in clinical signs has been scarcely described in dogs with vl, and the presence of the parasite in the cardiac tissue has been involved in few reports. accordingly, the present study aimed to evaluate histopathological abnormalities in cardiac tissue from dogs naturally infected by leishmania infantum chagasi. a total of dogs were evaluated. all dogs were symptomatic but no one presented clinical signs of cardiac involvement. in compliance with a federal law and under the owners' signed consent, all dogs were submitted to euthanasia and comprehensive post-mortem evaluation. samples from right atrium free wall (ra), right ventricle free wall (rv), interventricular septum (ivs) and left ventricle free wall (lv) were collected and evaluated. tissue samples were fixed in formalin, embedded in paraffin, sectioned at mm, and stained with hematoxylin and eosin (he) and anti-leishmania immunohistochemistry was also performed. the study was approved by the ethics committee in animal experimentation and animal welfare (protocol number / ). histopathological changes were observed in at least one of the four evaluated cardiac regions in % ( / ) of the dogs. the most frequent cardiac injury was an inflammatory reaction, characterized by the presence of mononuclear cell infiltrate in different degrees. of the evaluated regions, ra was the one with the highest incidence of histopathological changes, observed in % ( / ) of the animals, followed by rv, lv and siv, affected in . % ( / ), . % ( / ) and . % ( / ) of the dogs, respectively. immunohistochemistry revealed amastigotes in the cardiac tissue in % ( / ) of the dogs. a positive correlation was found between cardiac lesions and the presence of amastigotes in the myocardium (p < . ). disclosures: no disclosures to report. canine leishmaniosis is a life threatening zoonotic disease. the combination of meglumine antimoniate and allopurinol is considered the most effective therapy for canine leishmaniosis and constitutes the first line protocol against this disease. allopurinol is a parasitostatic drug used in long-term to maintain low parasite loads and to avoid clinical relapses. traditionally, allopurinol is considered a very safe drug in the dog. however, some reports indicate that xanthinuria and xanthine urolithiasis is produced after prolonged therapy with allopurinol in the dog. the aim of this prospective study was to evaluate the prevalence of urinary adverse effects of allopurinol treatment ( mg/kg/bid/po) in dogs with leishmaniosis. diagnosis was made by compatible clini-copathological abnormalitites with leishmaniosis and high leishmania infantum-specific antibody levels assessed by quantitative elisa. once leishmaniosis was diagnosed, a close follow-up (day , , , and during treatment) including physical examination, baseline laboratory tests (cbc, biochemistry profile, serum electrophoresis, urinalysis, urinary protein/creatinine ratio) and abdominal ultrasound was performed. in our preliminary results, dogs were included. dogs did not present any urinary abnormalities based on biochemistry profile, urinalysis and abdominal ultrasound at the time of diagnosis. four out of presented xanthinuria (day- [n = ], day- [n = ], and day- [n = ]). two out of dogs presented renal mineralization at day- of treatment. two out of dogs presented bladder urolithiasis since day- of treatment. xanthinuria was presented initially in all dogs that developed renal mineralization or bladder urolithiasis. dogs with renal mineralization and urolithiasis were treated with a restricted protein diet and, so far, they did not develop renal disease. the present study describes early xanthinuria, renal mineralization and urolithiasis as adverse effects due to chronic allopurinol treatment in dogs with leishmaniosis. neither mineral analysis nor renal biopsy was performed to confirm the origin of these lesions, but no urinary abnormality was present before allopurinol treatment was instituted. a thorough monitoring of dogs treated against leishmaniosis combined with urinalysis and abdominal ultrasound should be performed to evaluate urinary adverse effects and to help in the clinical management of these adverse effects. disclosures: no disclosures to report. giardia duodenalis is one of the most important gastrointestinal parasites in dogs and cats with a zoonotic potential. in germany the prevalence in dogs and cats reaches up to % and %, respectively. genotypes of two genetic assemblages of the parasites infect humans (assemblages a and b) and other mammals including small animals. in contrast, parasites of the assemblages c and d are specific for dogs, assemblage f for cats. objectives of the study were to analyse the prevalence, potential epidemiological risk factors and symptoms of g. duodenalis infections in dogs and cats. to detect g. duodenalis, feces from dogs and cats was analysed with an elisa technique. after dna extraction real time pcr as well as multi-locus sequence typing was performed for the following gene loci: triosephosphate isomerase-, glutamate dehydrogenase-, beta-giardin-gene, ssu rrna. with a questionnaire possible epidemiological risk factors were evaluated. statistical analyses were performed using spss (odds ratio, kolmogorow-smirnow test, spearman correlation). fecal samples of dogs and cats were collected over a time period of months. the elisa test was positive in / dogs and / cats. sixty-seven of giardia positive dogs and of positive cats had gastrointestinal signs. genotyping was successful in of dog samples and were assigned to assemblages as follows: assemblage a (n = ), a/c (n = ), a/d (n = ), b (n = ), b/d (n = ), c (n = ), c/d (n = ), d (n = ). only one of positive cat samples could be genotyped and was atypically identified as assemblage d. significant correlations between giardia infection and age, clinical signs, deworming status and staying abroad were found. in this monocenter study a prevalence rate of . % in dogs and . % in cats was detected, which is in good accordance with previous studies. the study further highlights a high rate ( %) of asymptomatically g. duodenalis infected animals. as potential zoonotic assemblages were detected, transmission of giardia from small animals to humans (and vice versa) cannot be excluded. especially young and not dewormed animals had a higher prevalence. disclosures: no disclosures to report. canine parvoviral enteritis remains a common cause of morbidity and mortality in young dogs. the goal of this study was to document a large cohort of affected dogs and analyze several factors as possible predictors of fatal outcome. medical records were retrospectively searched for dogs with parvoviral enteritis diagnosed with a positive fecal antigenic test or a fecal pcr. dogs were included only if the medical records were complete. the population was compared to the reference population of the hospital on the same time period with chi square tests and several factors were analyzed as possible predictors of death with a logistic regression. one hundred and forty seven cases were included. seventy percent of the dogs were non vaccinated puppies under the age of months. intact females and rottweiler, american staffordshire terrier and french beauce shepherd dogs were over-represented. clinical signs such as vomiting, diarrhea and dehydration were present in . %, . % and . % of the dogs respectively. hyperthermia, anemia and leucopenia were observed in . %, . % and . % of dogs respectively. the majority of the affected dogs were hospitalized for - days and the mortality rate was . % ( / dogs). hypoglycemia at admission was observed in / ( . %) dogs in which blood glucose was measured and was the only risk factor associated with death (p < . ). in this study, a predisposition of rottweiler, american staffordshire terrier and french beauce shepherd dogs was observed and hypoglycemia at admission was the only predictor of fatal outcome. disclosures: no disclosures to report. canine parvovirus (cpv) infections in dogs remain widespread around the world and still represent a major health threat in puppies. all vaccine manufacturers include this component in their core vaccination package, recommending two injections at - weeks interval from to weeks of age. despite broad vaccination coverage, number of reports suggesting lack of efficacy in vaccinated dogs have been reported, which implicate vaccines belonging to all major manufacturers. these cases are usually considered as being linked to the interference with maternal antibodies (matab), able to persist beyond weeks of age, which has led most expert groups to recommend a third vaccination around weeks of age. persistence of matab actually represents a major issue when immunizing puppies against parvovirosis. indeed, matab titres higher than / in the haemaglutination inhibition (hi) test can still inhibit vaccine uptake whereas such titres do not prevent field virus infection. in contrast, hi titres higher than / to / are usually considered as protective against disease and virus excretion. this "immunity gap"is therefore a critical period for the puppy and the outcome of the vaccination. in order to evaluate the impact of residual mda on the efficacy of a standard primary vaccination protocol, we performed a vaccination field trial with serological follow-up. eighty-eight puppies from to weeks of age presented at veterinary practice received injections at weeks interval. serology was performed by elisa before (at d /v ), during (at d /v ) and after (d ) vaccination. average maternal antibodies titres were strongly correlated with the age of the puppy at primary vaccination, remaining at vaccine inhibiting level until~ week of age. average titres increased significantly after st injection of primary vaccination in most groups and in all groups after the nd injection of primary vaccination. individual variability remained significant: vaccine uptake was inversely and strongly correlated to the pre-vaccinal matab titre at vaccination. seven out of puppies ( %) didn't seroconvert, despite vaccination complying to the recommended schedule. vaccination was started in such dogs between . and . weeks and completed between . and . weeks and average initial matab titre was . log compared to . for the general population. in conclusion, this trial supports the recommendation of an additional injection of primary vaccination at weeks, especially in areas of high parvovirus prevalence / pressure, where high levels of matab are likely to be transferred to puppies. disclosures: all authors are employees of merial. ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . . % of the dogs were pcr positive. the aim of our study was to evaluate the usefulness of determining serum level of c-reactive protein (crp) in dogs naturally infected with bacterium a. phagocytophilum as a possible indicator of the clinical phase of the disease. pcr and/or ifa positive dogs with clinical presentation and/or thrombocytopenia were included in the study. based on the results, the dogs were divided into groups: pcr positive dogs; ifa positive (subdivided according to titer level from : to > : ) and pcr negative dogs; positive control group -pcr and ifa negative dogs with clinical signs and/or thrombocytopenia; negative control groupclinically healthy, pcr and ifa negative dogs. serum level of crp was determined using lifeassays Ò canine crp (lifeassays, lund, sweden). an elevated concentration of crp (> mg/l) was determined in pcr positive and ifa positive dogs with an ifa titer ≥ : and coincides with the presence of clinical signs (most commonly general clinical signs, elevated body temperature, gastrointestinal problems) and/or mild ( . %) or severe ( %) thrombocytopenia. the assessment of crp concentration, in correlation with certain clinical alterations and thrombocytopenia, suggests that crp concentration is elevated in the acute phase of the disease and is in correlation with the aforementioned changes therefore can serve as an additional diagnostic parameter. the crp concentration in ifa positive dogs, regardless of ifa titer levels, and with present clinical signs and thrombocytopenia is higher (above detectable level, > mg/l) than in dogs without clinical signs or laboratory alterations, which may speak in favor of reinfection or reactivation of a persistent infection at least in cases when no other cause of inflammation can be found. specific treatment would therefore be reasonable in such cases, especially in cases of rising crp concentration. disclosures: no disclosures to report. gallbladder agenesis is a very rare cause of elevated liver enzymes in dogs. in this study, we evaluated the features of dogs [six males (three castrated) and nine females (two spayed)] with suspected gallbladder agenesis on ultrasonography. five different breeds were included: chihuahua (n = ), toy poodle ( ), german shepherd ( ), jack russell terrier ( ), and shiba dog ( ). the median age was . ( . - . ) years. ten dogs were asymptomatic, while the other five dogs showed decreased appetite ( ), vomiting ( ), ascites ( ), seizure ( ), and diarrhea ( ). all dogs showed elevated liver enzymes, with high alanine aminotransferase levels (median, u/l; - u/l) in dogs and high gamma-glutamyl transpeptidase levels (median, u/l; - u/l) in . gallbladder agenesis was confirmed using laparoscopy in dogs and laparotomy in three. liver biopsy samples were obtained from all dogs. additional computed tomography cholangiography was performed for dogs using a -slice multidetector computed tomography (mdct) scanner following the intravenous administration of contrast medium (meglumine iotroxate). the obtained images were analyzed on a workstation, and they revealed an absent gallbladder in nine dogs and a vestigial gallbladder in three. the common bile duct was dilated in five dogs. for all dogs, laparotomy or laparoscopy was used to visualize the gallbladder and liver abnormalities, including malformed lobes and surface irregularities. acquired portal systemic collaterals were visually confirmed in five dogs, who also exhibited hypoplasia of the portal vein on histological examination. in conclusion, most animals with gallbladder agenesis were asymptomatic in our study, indicating a good long-term prognosis. however, symptoms associated with portal hypertension must be monitored in animals with primary portal vein hypoplasia. disclosures: no disclosures to report. assessment of systolic arterial blood pressure (sap) is an important tool in small animal internal medicine practice, especially with diseases or clinical conditions that can cause hypertension or hypotension. the doppler method is noninvasive and has several advantages compared to oscillometric method. there are few studies about the effect of body position on sap in conscious dogs. the hypothesis was that animal positioning during measurement alters sap values. the study design was prospective and randomized regarding order of positioning measurements. one hundred and twenty client-owned, conscious, healthy or sick adult dogs, weighing up to kg were included. sap was recorded by doppler ultrasonography following american college of veterinary internal medicine consensus statement with animals positioned in sternal recumbency, right lateral recumbency and with the dog laying down on owner s lap. the order of body position was raffled at the time of measurement. five consecutive measurements on each body position were performed always on left forelimb and the average was calculated. sap values were higher in sternal recumbency ( mmhg, p %- % = - . ; p < . ) compared to those obtained on the owner s lap ( mmhg, p %- % = . - . ), and both were similar to right lateral recumbency ( mmhg, p %- % = - ). these results suggest that sap measurement obtained on owner s lap or right lateral recumbency can be used on clinic routine, but sap measurement obtained on sternal recumbency should be avoided, because such measures may be overestimated. disclosures: no disclosures to report. canine patients may be presented for blood pressure (bp) assessment when clinical diseases associated with systemic hypertension (ht) are suspected but not confirmed; this population may encompass patients that have normal bp, true ht or situational ht. the clinician's aim is to identify animals with ht reliably, while minimizing false positives. this prospective study investigated the repeatability of duplicate within-visit systolic bp assessments (sbp and sbp ) in consecutive canine patients presented for bp assessment in the small animal clinic ( duplicate sbp recorded from dogs) and in a control group of healthy dogs ( duplicate sbp obtained from control dogs), resulting in duplicate measurements for analysis. doppler methods were used for duplicate assessments and oscillometric methods were used for duplicate assessments; cuff size/location were consistent within any dog. sbp ≤ mmhg was considered normal (nml); sbp > mmhg was considered abnormal (abn). median (range) elapsed time between duplicate readings was ( - ) minutes; % of sbp were obtained within minutes of sbp . there was no correlation between elapsed time and change in sbp (p = . ). % of sbp were equal to or lower than sbp ; median decrease was ( - no dog with sbp > mmhg (n = ) had nml sbp . more dogs with abn sbp were panting ( / scored, %) compared to the group with dogs with nml sbp ( / scored, %, p = . ). sbp of dogs that stopped panting ( / , %) tended to decrease (p = . ). within-visit repeatability of bp diagnosis was good in dogs with nml sbp , but apparent false positive diagnoses of ht occurred in % of dogs with abn sbp . sbp > mmhg was repeatable in all dogs. panting may be associated with increased measured sbp by these methods. duplicate within-day measurements may help identify false positive ht diagnoses in dogs with initial sbp measurements > mmhg. disclosures: no disclosures to report. median age at presentation was years. ess, cocker spaniels, ckcs and border collies presented at . , . , . and . years respectively. females were over-represented with / females ( / fn and / fe) and males ( / mn and / me). % of the ess cases presented were fn dogs. clinical presentation varied between dogs. clinical signs of pyrexia ( %), lethargy ( %) and anorexia ( . %) were the most common. other signs included cough, tachypnoea, dyspnoea, dysphagia, vomiting, diarrhoea, neck or spinal pain, abdominal pain, joint effusion or dermatologic signs thirty-one animals presented with peripheral lymphadenomegaly, but five animals displayed only internal lymph node enlargement. cytology was performed in / cases pyogranulomatous ( ) or granulomatous ( ) lymphadenitis. lymph node culture or staining (gram, pas, zn) was performed in and animals respectively, all of which were negative twenty-four animals were initiated therapy at mg/kg q hours. mean treatment length was weeks median relapse time was weeks. this study documents dogs with cssrl in the uk suggesting an over-representation in spaniel breeds (particularly ess), with females and young dogs typically affected. cytologic and histopathologic examination confirmed sterile lymphadenitis with animals showing marked and rapid clinical improvement pastor . hospital clinic veterinari animal medicine and surgery department, faculty of veterinary medicine of murcia, murcia, spain if only blood is available. therefore, if no effusion is present, diagnosis of fip still remains challenging. disclosures: dr. elisabeth mueller is the managing director of laboklin gmbh & co.kg. dr. karola weider is employed at laboklin gmbh & co.kg. this laboratory offered the "combined rt-npcr and sequencing approach which samples are the most relevant for analysis? a retrospective study of cases from maisons alfort, france, umr virologie inra-enva-anses cyprus is an island state in the eastern mediterranean basin. no epidemiological studies have yet been performed on infectious agents in cats from cyprus. the aim of this study was to determine the prevalence of several infectious agents, including some vector-borne infections, in cats from cyprus.surplus edta-blood and serum samples were recruited from cypriot cats, from which signalment and lifestyle characteristics were recorded. dna was extracted and real-time quantitative polymerase chain reaction (qpcr) assays were used to detect haemplasmas (mycoplasma haemofelis, "candidatus mycoplasma haemominutum" and "candidatus mycoplasma turicensis"), leishmania spp.and bartonella henselae. conventional pcr assays were used to detect ehrlichia/anaplasma spp. samples yielding positive results for leishmania spp. or ehrlichia/anaplasma spp. underwent further characterisation (sequencing). elisas were performed for the detection of l. infantum antibodies, feline leukaemia virus (felv) antigen and feline immunodeficiency virus (fiv) antibodies. statistical analysis was performed using spss for the assessment of any associations between variables and infectious agents.of the samples extracted, were excluded due to failure of ≥one internal control pcr. of the remaining samples, ( . %) were positive by pcr for haemoplasma including ( . %) for m. haemofelis, ( . %) for "ca. m. haemominutum" and ( . %) for "ca. m. turicensis". nineteen ( . %) were positive for b. henselae. one cat ( . %) was pcr positive for ehrlichia/anaplasma spp. and sequencing revealed identity with anaplasma platys. leishmania spp. dna was detected in of the ( . %) cats; sequencing revealed l. infantum in of these cases. l. infantum serology was positive in of the cats tested ( . %). only one cat was positive for both leishmania pcr and serology. of the cats that underwent retroviral serology, ( . %) were felv and ( . %) were fiv positive.statistical analysis identified several significant associations (p < . ) including the following; haemoplasma infection and both outdoor access and feral-shelter cat origin, felv or fiv infection and both anaemia and feral-shelter cat origin.this study documents, for the first time, the presence of haemoplasmas, l. infantum, b. henselae, a. platys, felv and fiv in the feline population of cyprus. the prevalence of haemoplasma, fiv and b. henselae infections were among the highest reported in cats from mediterranean countries, while that of leishmania spp. was similar. this is the second report of a. platys infection in a cat from a mediterranean country.disclosures: no disclosures to report. bartonella species (spp.) are zoonotic pathogens, and infections in cats are common. prevalence in cats from southern germany is still unknown. the aim of this study was to determine the prevalence of bartonella spp. dna in blood of cats in southern germany and to evaluate associations between bartonella bacteremia, housing conditions, feline immunodeficiency virus (fiv), and feline leukemia virus (felv) status, including progressive, regressive, and abortive felv infection.blood samples of cats that were presented to different veterinary clinics in southern germany for various reasons were tested for bartonella spp. dna using a previously published conventional polymerase chain reaction (pcr) targeting a fragment of the s- s rrna intergenic spacer region. for statistical analysis, fisher's exact test was used.prevalence rate of bartonella spp. bacteremia was . % ( / ; ci: . %- . %). b. henselae was amplified in eleven of these cats. one cat was positive for b. clarridgeiae dna. most of the infected cats were clinically healthy, but half of the cats had thrombocytopenia, potentially caused by their bartonella spp. infection. there was no significantly higher risk to be infected with bartonella spp. when living mainly outdoors or being fiv-or felv-infected.prevalence of bartonella spp. bacteremia is low in southern german cats, but there is still a risk of human bartonella infection associated with cat ownership. most clinical changes of the bartonella spp.-infected cats were related to other diseases. however, thrombocytopenia was common and further studies are required to define its potential clinical relevance.disclosures: no disclosures to report. ante-mortem diagnosis of feline infectious peritonitis (fip) is still challenging. the aim of this study was to evaluate sensitivity and specificity of a "combined reverse transcription nested polymerase chain reaction (rt-npcr) and sequencing approach", detecting mutations at two different nucleotide positions within the spike gene, that previously were shown to correlate with the fip phenotype. the study population consisted of cats with confirmed fip and a defined control group of cats for which fip was considered an important differential diagnosis, but that were definitively diagnosed with other diseases. blood and/or effusion samples were examined for feline coronavirus (fcov) rna by rt-npcr and, if positive, nucleotide positions and were sequenced for nucleotide transitions. sensitivity, specificity, negative and positive predictive values were determined and % confidence intervals ( % ci) calculated.rt-npcr detected fcov in cats in blood (n = ) and/or effusion (n = ); all of them had fip. one of the mutations of interest was found in / of the pcr-positive blood samples and in / of the pcr-positive effusion samples. diagnostic specificity of the "combined rt-npcr and sequencing approach" was % in blood ( % ci . - . ) and effusion ( % ci . - . ). diagnostic sensitivity was . % ( % ci . - . ) in blood and . % ( % ci . - . ) in effusion.a positive test result therefore confirms a suspicion of fip. a negative result, however, cannot be used to rule out fip, especially feline infectious peritonitis (fip) is a viral disease caused by the virulent strain of feline coronavirus (fipv). the disease can appear under two clinical forms, dry or effusive, both leading to a fatal outcome. diagnosis was based on histopathologic lesions on necropsy until the recent discovery of mutations associated with the fipv strain in the c and spike (s) genes. our main goal was to detail the distribution of c or s gene mutations in different biological samples of cats suffering from fip.this was a retrospective, observational study of cats showing clinical signs compatible with fip. ten out of cats were of pure breed. . % were males and . % females. median age was . months at presentation. the clinical presentation, pathologic findings and virologic data were reviewed. according to clinical signs, cats were classified with a dry form and with a wet form of fip. the main clinical signs included dehydration, hyperthermia, icterus, abnormal abdominal palpation, neurological and ocular disorders.when possible blood, fecal material, effusion, fine needle aspiration (fna) from relevant organs or a combination of these, was recovered from each cat. feline coronavirus (fcov) was first researched by rt-pcr, then the c and part of the s genes were sequenced to determine the eventual presence of mutations.among the dry cases, fcov was detected in / blood samples, / fecal samples and fna ( / ). among the wet cases, / blood samples, / fecal samples and all effusion samples ( / ) were positive for the presence of fcov. c mutations were never found in fecal samples but were found in / effusion samples and in / fna. s mutations were detected in / fecal samples, / fna and / effusion samples. for three cats, no mutation, neither in c or s genes was identified despite the confirmation of fip by necropsy.s gene mutation is more frequently observed than c gene despite in two cases where only c mutations were identified. moreover the presence of strains harbouring s mutation in feces has never been described before and could suggest the possible diffusion of fipv among feline population.in conclusion, viral diagnosis of fip based on rt-pcr sequencing in effusion and fna samples is essential. rt-pcr resulting from blood samples should be carefully interpreted because of high risk of missing fipv.finally, searching for mutations in both s and c genes is recommended.disclosures: no disclosures to report. methicillin resistant staphylococcus aureus (mrsa) has recently become a great concern for pet animals' disease and zoonotic infection. mrsa strains transfer between pet animals and humans could occur. the aim of the present study was to determine the occurrence of mrsa in household dogs.from january to june , clinical samples were collected from dogs, patients of the veterinary teaching hospital of the department of veterinary sciences of messina (italy), affected by several diseases of various origins. all samples were processed by bacteriological conventional methods for isolation and identification. all strains were tested for phenotypic susceptibility to oxacillin and were subjected to a pcr protocol for the detection of meca gene. strains carrying the gene were considered methicillin resistant (mrs). lastly, on both mrs and methicillin sensitive (mss) strains, kirby-bauer disk diffusion susceptibility testing were performed to highlight resistance profiles using molecules belonging to the main classes of antimicrobials used in veterinary practice. strains resistant to at least one molecule of three or more classes of antibiotics were considered multidrug-resistant (mdr). the statistical analysis of the results was made using the z-test by a primer Ò software.forty staphylococcus spp. strains were isolated, belonging to species. the most frequently isolated microorganisms were staphylococcus aureus with isolations ( %) and staphylococcus pseudintermedius with isolations ( . %), followed by staphylococcus epidermidis with isolations ( %) and staphylococcus cohnii and staphylococcus warneri, both with isolations ( %). a single isolation ( . %) was obtained for each of the species staphylococcus chromogenes, staphylococcus haemolyticus, staphylococcus lentus, staphylococcus lugdunensis, staphylococcus saprophyticus, staphylococcus simulans and staphylococcus sciuri. thirteen ( . %) strains of staphylococcus spp. were phenotypically resistant to oxacillin and three staphylococcus aureus ( . %; n. from pyoderma, n. from exudative pleural effusion) were positive for the meca gene. all strains of staphylococcus spp. were mdr. our results showed the presence of mrsa and multidrug-resistant staphylococcal strains in household dogs. a lack of correspondence between antimicrobial susceptibility tests and molecular methods was found in the present study.disclosures: no disclosures to report. haemotropic mycoplasmas (haemoplasmas) are bacteria that infect domestic cats. approximately % of ill cats have haemoplasma infection. three main feline haemoplasma species have been detected worldwide; mycoplasma haemofelis (mhf), "candidatus m. haemominutum" (cmhm) and "candidatus m. turicensis" (cmt). a fourth haemoplasma called "candidatus m. haematoparvun-like" (cmhp) was latter identified in cats. the only published study in chile was carried out in cats, with prevalences by pcr of . % to mhf, and % to cmhm.the aim of this study was to perform molecular detection of haemoplasmas in cats from valdivia, southern chile. blood samples were taken from cats and used for haemoplasmas dna detection by quantitative real time pcr (qpcr) at universidad austral de chile. qpcr protocol was based on detection of feline dna targeting s housekeeping gene and mycoplasma spp. s rrna gene (universal primers, my sf forward, my sr y my sr , both reverse) by sybr green method. the melting temperature (tm) analysis allowed identifying the infecting mycoplasma species (mhf, cmhm, cmt). it was not posible to identify haemoplasmas species on co infected cats, so a second qpcr specie specific protocol was applied on these samples. second qpcr protocol was based on s rrna gene, with specific primers to detect mhf, cmhm, cmt and cmhp. all samples ( / ) were positive to s gene, proving presence of cat dna. from the cats, . % ( / ) were positive to haemoplasmas, where . % ( / ) corresponded to cmhm (tm . - . °c), . % ( / ) to mhf (tm . - . °c), % ( / ) to cmt (tm . - . °c) and . % ( / ) to co infections. associations between cmhm+mhf, cmhm+cmt, cmhm+cmhp and cmhm+mhf+cmhp were detected on co infected animals. these results agree with those found in previous